WO2005056782A2

WO2005056782A2 - Perhydrolase

Info

Publication number: WO2005056782A2
Application number: PCT/US2004/040438
Authority: WO
Inventors: Neelam S. Amin; Matthew G. Boston; Richard R. Bott; Marguerite A. Cervin; Edward M. Concar; Marc E. Gustwiller; Brian Edward Jones; Klaus Liebeton; Gregory S. Miracle; Hiroshi Oh; Ayrookaran J. Poulose; Sandra W. Ramer; Jeffrey J. Scheibel; Walter Weyler; Gregory M. Whited
Original assignee: Genencor International, Inc.; The Procter & Gamble Company
Priority date: 2003-12-03
Filing date: 2004-12-03
Publication date: 2005-06-23
Also published as: HK1105428A1; DK2295554T3; CN103333870A; EP2295554A3; EP2292743B1; ATE500322T1; WO2005056782A3; EP2664670A1; CN1981035A; US20140302003A1; EP2295554B1; US9282746B2; MXPA06005652A; PT1689859E; DK2664670T3; EP1689859A2; CN1981035B; EP1689859B1; EP2292743A3; US8772007B2

Abstract

The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

Description

PERHYDROLASE

The present application claims priority under 35 U.S.C. §119, to co-pending U.S. Provisional Patent Application Serial Number 60/526,764, filed December 3, 2003.

FIELD OF THE INVENTION The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

BACKGROUND OF THE INVENTION Detergent and other cleaning compositions typically include a complex combination of active ingredients. For example, most cleaning products include a surfactant system, enzymes for cleaning, bleaching agents, builders, suds suppressors, soil-suspending agents, soil-release agents, optical brighteners, softening agents, dispersants, dye transfer inhibition compounds, abrasives, bactericides, and perfumes. Despite the complexity of current detergents, there are many stains that are difficult to completely remove. Furthermore, there is often residue build-up, which results in discoloration (e.g., yellowing) and diminished aesthetics due to incomplete cleaning. These problems are compounded by the increased use of low (e.g., cold water) wash temperatures and shorter washing cycles. Moreover, many stains are composed of complex mixtures of fibrous material, mainly incorporating carbohydrates and carbohydrate derivatives, fiber, and cell wall components (e.g., plant material, wood, mud/clay based soil, and fruit). These stains present difficult challenges to the formulation and use of cleaning compositions. In addition, colored garments tend to wear and show appearance losses. A portion of this color loss is due to abrasion in the laundering process, particularly in automated washing and drying machines. Moreover, tensile strength loss of fabric appears to be an unavoidable result of mechanical and chemical action due to use, wearing, and/or washing and drying. Thus, a means to efficiently and effectively wash colored garments so that these appearance losses are minimized is needed. Cleaning compositions that comprise esterases, lipases and cutinases are well- known in the art. However, these enzymes have a very low ratio of perhydrolysis to hydrolysis. This results in the conversion of most of the ester substrate into acid, instead of the more desirable peracid. This is a serious drawback, since formula space and cost considerations render it feasible to include only a limited amount of substrate. In sum, despite improvements in the capabilities of cleaning compositions, there remains a need in the art for detergents that remove stains, maintain fabric color and appearance, and prevent dye transfer. In addition, there remains a need for detergent and/or fabric care compositions that provide and/or restore tensile strength, as well as provide anti- wrinkle, anti-bobbling, and/or anti-shrinkage properties to fabrics, as well as provide static control, fabric softness, maintain the desired color appearance, and fabric anti-wear properties and benefits. In particular, there remains a need for the inclusion of compositions that are capable of removing the colored components of stains, which often remain attached to the fabric being laundered. In addition, there remains a need for

improved methods and compositions suitable for textile bleaching. In addition to the fabric and garment cleaning area, bleaching is commonly used in the pulp and paper industry. Prior to production of paper, pulp is typically treated to remove undesirable colored contaminants. This provides pulp that is suitable for 5 production of paper of higher quality than pulp that is not treated to remove colored contaminants and other undesirable components present in pulp. For example, in the paper recycling industry, removal of ink is necessary. Although standard methods are suitable for deinking paper with oil or water-based inks, the increased use of electrostatic inks has made deinking problematic, as these inks are much more difficult to remove.

10 There are various methods available for deinking paper, including the use of enzymes (See e.g., U.S. Patent No. 5,370,770). However, there remains a need in the art for efficient, cost-effective methods for treatment of pulp for paper (recycled and new) product production. Bleaching is also commonly used in the personal care market (e.g., dental

15 whiteners, hair bleachers, etc.). Although personal care bleaching products have improved over the years, there remains a need for mild, easy to use, cost-effective bleaching methods for this setting.

20 SUMMARY OF THE INVENTION The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, 25 bleaching and disinfecting. In some embodiments, the present invention provides compositions comprising at least one perhydrolase, wherein the perhydrolase exhibits a perhydrolysis to hydrolysis GC821-2 ^ ^

ratio that is greater than 1. The present invention also provides isolated perhydrolases, wherein the perhydrolases exhibit a perhydrolysis to hydrolysis ratio that is greater than 1. In some preferred embodiments, the perhydrolase is M. smegmatis perhydrolase. In alternative preferred embodiments, the perhydrolase is at least approximately about 35% homologous to M. smegmatis perhydrolase. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2. In some preferred embodiments, the perhydrolases have immunological cross- reactivity with M. smegmatis perhydrolase. In still further embodiments, the perhydrolase is at least a portion of M. smegmatis perhydrolase, wherein the perhydrolase has a perhydrolysis to hydrolysis ration that is greater than 1. In alternative embodiments, the perhydrolase is a structural homologue of M. smegmatis perhydrolase, in which the active site is homologous to at least one amino acid selected from the group consisting of S 11 , D192, and HI 95 of the smegmatis perhydrolase. The present invention also provides isolated perhydrolase variants having amino acid sequences comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, at least one modification is made at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein the modified amino acid is selected from the group consisting of Cys7, AsplO, Serl 1, Leul2, Thrl3, Trρl4, Trpl6, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Vall25, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, Ilel53, Phel54, Thr 159, Thr 186, Ilel92, Ilel94, and Phel96. In further embodiments, the modification comprises at least one substitution at an amino acid position equivalent to a GC821-2

position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of Ml, K3, R4, 15, L6, C7, D10, Sl l, L12, T13, W14, W16, G15, V17, P18, V19, D21, G22, A23, P24, T25, E26, R27, F28, A29, P30, D31, V32, R33, W34, T35, G36, L38, 5 Q40, Q41, D45, L42, G43, A44, F46, E47, V48, 149, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, 160, D61, D62, P63, T64, D65, P66, R67, L68, N69, G70, A71, S72, Y73, S76, C77, L78, A79, T80, L82, P83, L84, D85, L86, V87, N94, D95, T96, K97, Y99F100, R101, R102, P104, LI 05, D106, 1107, A108, L109, G110, Mi l l, SI 12, VI 13, LI 14, VI 15, Tl 16, Ql 17, VI 18, LI 19, T120, S121, A122, G124, V125, G126,

10 T127, T128, Y129, P146, P148, W149, F150, 1153, F154, 1194, and F196. In some preferred embodiments, the variant perhydrolase exhibits a change in peracid hydrolysis compared to the wild-type perhydrolase. In some embodiments, the change in peracid hydrolysis is a decrease, while in other embodiments, the change in peracid hydrolysis is an increase.

15 In some alternative preferred embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.1 or less, in comparison with wild-type perhydrolase. In alternative preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid

20 sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of R4, L12, G15, P18, R27, W34L38, A44, E51, G52, L53, S54, T58, R67, L68, S72, A79, T80, D85, L86, V87, N94, K97, R101, VI 18, LI 19, G124, G126, and 1194. In further alternative embodiments, the variant perhydrolase exhibits a ratio of

25 peracid hydrolysis of about 0.2 or less, in comparison with wild-type perhydrolase. In yet additional embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in

M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of R4, 15, D10, L12, W14, G15, P18, V19, T25, R27, W34, L38, A44, 149, E50, E51, G52, L53, S54, A55, R56, T58, N59, D62, T64, D65, R67, L68, N69, S72, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, T96, K97, Rl 01 , L82, P83, L86, V87, N94, T96, K97,

F100, R101, LI 09, Ml 11, LI 14, VI 18, LI 19, A122, G124, G126, T127, Y129, W149, and 1194. In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.3 or less, in comparison with wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of R4, 15, D10, LI 2, W14, G15, L12, P18, V19, G22, A23, T25, E26, R27, W34, G36, L38, Q41, L42, G43, A44, 149, E50, E51, G52, L53, S54, A55, R56, T57, N59, T58, D62, T64, D65, R67, L68,

N69, G70, S72, Y73, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, T96, K97, Y99, F100, R101, R102, P104, L109, G110, Mi l l, L114, VI 18, L119, A122, G124, V125, G126, T127, Y129, W149, F154, and 1194. In yet further embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.4 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of R4, 15, L6, D10, Sll, L12, W14, G15, W16, P18, V19, G22, A23, T25, E26, R27, F28, W34,

T35, G36, L38, Q41, L42, G43, A44, D45, E47, 149, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, T58, 160, D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76, „ „ . GC821-2

C77, A79, T80, L82, P83, D85, L86, V87, N94, P66, T96, K97, Y99, FlOO, RlOl, R102, P104, 1107, L109, G110, Mill, S112, L114, V118, L119, S121, A122, G124, V125, G126, T127, Y129, W149, F150, F154, 1194, and F196. In some embodiments, the variant perhydrolase exhibits a ratio of peracid 5 hydrolysis of about 0.5 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122,

10 A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GI 10, G124, G43, L109, Ll 19, L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, Tl 16, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, Ll 19, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54,

15 A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, FlOO, RlOl, L109, Ml 11, Ll 14, Ll 19, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, GI 10, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, Ll 14, Ll 19, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI 18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85,

20 E26, E47, E51, F150, F196, F28, GI 10, G124, G36, G43, G52, G70, 1107, 15, 160, L109, L119, L53, L6, L68, L82, Mi l l, P104, P66, R102, R67, Sll, SI 12, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216. In additional embodiments, the variant perhydrolase exhibits a ratio of peracid

25 hydrolysis of about 0.6 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in .

GC821-2 ^)

M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GI 10, G124, G43, L109, Ll 19, L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, Ll 19, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, FlOO, RlOl, L109, Ml 11, Ll 14, Ll 19, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, GI 10, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, Ll 14, Ll 19, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI 18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, 1107, 15, 160, L109, L119, L53, L6, L68, L82, Mil l, P104, P66, R102, R67, Sll, SI 12, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106,

D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, 1107, 1194, K97, L105, L109, Ll 14, Ll 19, L38, L68, L78, L82, L84, Ml 11, N69, N94, P104, P63, P66, R102, R27, SI 1, SI 12, S54, S72, Tl 16, T120, T127, T13, T25, T57, T80, T96, VI 13, V125, V19, W16, Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216. In yet additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.7 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GI 10, G124, G43, L109, Ll 19, GC821-2 ^

L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, T116, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, Ll 19, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, 5 A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, FlOO, RlOl, L109, Ml 11, Ll 14, Ll 19, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, GI 10, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI 18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85,

10 E26, E47, E51, F150, F196, F28, GI 10, G124, G36, G43, G52, G70, 1107, 15, 160, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, Sll, SI 12, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, 1107, 1194, K97, L105, L109, Ll 14, Ll 19, L38, L68, L78, L82, L84, Ml 11, N69, N94, P104, P63,

15 P66, R102, R27, SI 1, SI 12, S54, S72, Tl 16, T120, T127, T13, T25, T57, T80, T96, VI 13, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, 149, 15, 160, L105, L109, L12, L38, L42, L53, L84, L86, Ml 11, N59, P146, P24, P66, Q41, R102, R27, R56, SI 12, S121, S54, S72, Tl 16, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129,

20 Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216. In still further embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.8 or less, in comparison with wild-type perhydrolase. In some preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in

25 M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, G110, G124, G43, L109, L119, GC821-2 "^ ^

L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, Tl 16, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, Ll 19, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87,

N94, T96, FlOO, RlOl, L109, Ml 11, Ll 14, Ll 19, W149, Yld29, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI 18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, GI 10, G124, G36, G43, G52, G70, 1107, 15, 160, L109, L119, L53, L6, L68, L82, Mil l, P104, P66, R102, R67, Sl l, SI 12, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, 1107, 1194, K97, L105, L109, Ll 14, Ll 19, L38, L68, L78, L82, L84, Ml 11, N69, N94, P104, P63, P66, R102, R27, SI 1, SI 12, S54, S72, Tl 16, T120, T127, T13, T25, T57, T80, T96,

VI 13, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, 149, 15, 160, L105, L109, L12, L38, L42, L53, L84, L86, Mi l l, N59, P146, P24, P66, Q41, R102, R27, R56, SI 12, S121, S54, S72, Tl 16, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61, D65, D85, D95, E47, E51, F150, F196, F46, G110, G126, G36, G43, G52, 1107, 1194, 149, 15, 160, 189, Ll 14, L42, L53, L68, L78, L84, Mi l l, N59, N94, P146, P24, P30, P63, P66, P83, Ql 17, RlOl, R4, SI 12, S121, S72, Tl 16, T120, T127, T13, T57, T96, VI 13, V125, V17, V19, V32, V87, W149, Y129, Y73, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216. In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis of about 1.5 or greater, in comparison with wild-type perhydrolase. In some i'Jt H„ !S " <!* ■>^•

GC821-2

preferred embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GI 10, G124, G43, L109, Ll 19, L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, Tl 16, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, Ll 19, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87,

N94, T96, FlOO, RlOl, L109, Ml11, Ll14, Ll19, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, Ll14, Ll19, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, G110, G124, G36, G43, G52, G70, 1107, 15, 160, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, Sll, SI12, S121, S54, S72, T25, T35, T57, T58, VI18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52J107, 1194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, Mill, N69, N94, P104, P63, P66, R102, R27, SI1, SI12, S54, S72, Tl16, T120, T127, T13, T25, T57, T80, T96, VI13, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, 149, 15, 160, L105, L109, L12, L38, L42, L53, L84, L86, Mill, N59, P146, P24, P66, Q41, R102, R27, R56, SI12, S121, S54, S72, Tl 16, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61, D65, D85, D95, E47, E51, F150, F196, F46, G110, G126, G36, G43, G52, 1107, 1194, 149, 15, 160, 189, L114, L42, L53, L68, L78, L84, Mill, N59, N94, P146, P24, P30, P63, P66, P83, Q117, it.. GC821-2

RlOl, R4, SI 12, S121, S72, Tl 16, T120, T127, T13, T57, T96, VI 13, V125, V17, V19, V32, V87, W149, Y129, and Y73, Y99, A108, A44, C7, D10, D106, D31, D61, D85, E26, E51, FlOO, F28, F46, GI 10, G22, G36, G43, G52, G70, 1107, 1153, 149, 15, 189, K3, L105, L53, L6, L78, L86, Ml, N69, P104, P146, P18, P24, P30, P83, Q117, Q40, Q41, 5 R102, R27, R33, R4, S121, S72, S76, T120, T128, T13, T35, T80, T96, VI 15, VI 18, V32V48, V87, W34, G190, V191, G193, T197, E198, A199, R202, D203, G205, V206, A209, E210, Q211, S214, and L215. In additional embodiments, the variant perhydrolase exhibits a ratio of peracid hydrolysis between about 1.2 and about 1.5, in comparison with wild-type perhydrolase. 10 In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, C7, D106, D31, D61, D85, E26, E50, E51, FlOO, F150, F28, F46, G110, G126, 15 G22, G70, 1107, K3, L105, L42, L6, L78, Mill, N59, N69, P104, P146, P148, P18, P30, P63, Ql 17, Q40, Q41, R102, R27, R33, R4, S54, S76, Tl 16, T120, T128, T64, T80, T96, V113, V115, V118, W34, and Y73. In yet further embodiments, the present invention provides variant perhydrolases in which the variant perhydrolases exhibit a change in perhydrolysis, such that the ratio of 20 variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is at least about 1.2. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of 25 C7, D10, L12, G15, P18, V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, 149, E51, L53, S54, A55, T57, D61, P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80, L82, P83, D85, L86, D95, GC821-2 ^

K97, RlOl, T103, P104, L105, D106, 1107, L109, Mil 1, VI 13, Ql 17, VI 18, S121, G124, V125, G126, T127, P148, F150, 1153, F154, and F196. In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type 5 perhydrolase perhydrolysis is about 0.8 or less. In some embodiments, the variant perhydrolase comprising at least one modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A108, A122, A23, A29, A44, A55, A71, A79, C7,

10 C77, D10, D106, D21, D45, D61, D62, D65, D85, E26, E47, E50, E51, FlOO, F150, F154, F196, F28, F46, G110, G124, G126, G15, G22, G36, G52, G70, 1107, 1153, 1194, 149, 15, 160, 189, K3, K97, L105, L109, Ll 14, Ll 19, L12, L38, L42, L53, L6, L68, L78, L82, L84, K86, Ml, Mill, N59N94, P146, P18, P24, P30, P66, P83, Q40, Q41, RlOl, R102, R27, R33, R4, R56, R67, Sl l, SI 12, S54, S72, S76, T103, T116, T120, T127,

15 T128, T13, T25, T35, T57, T64, T80, T96, VI 13, VI 15, VI 18, V125, V17, V19, V32, V48, V87, W13, W149, W16, W34, Y129, Y73, and Y99. In alternative embodiments, the present invention provides variant perhydrolases comprising at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino

20 acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A108, A122, A23, A29, A44, A55, A71, A79, C7, C77, D10, D106, D21, D31, D45, D61, D62, D65, D85, E26, E47, E50, E51, FlOO, F150, F154F196, F28, F46, G110, G124, G126, G15, G22, G36, G43, G52, G70, 1107, 1153, 1194, 149, 15, 160, 189, K3, K97, L105, L109, L114, L119, L12, L38, L42, L53, L6, L68,

25 L78, L82, L84, L86, Ml, Ml 11, N59, N69, N94, P104, P146, P148, P18, P24, P30, P63, P66, P83, Q117, Q40, Q41, RlOl, R102, R27, R33, R4, R56, R67, Sl l, S112, S121, S54, S72, S76, T103, Tl 16, T120, T127, T128, T13, T25, T35, T57, T58, T64, T80, T96, GC821-2 . ^

VI 13, VI 15, VI 18, V125, V17, V19, V32, V48, V87, W14, W149, W16, W34, Y129, Y73, and Y99. In yet additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type 5 perhydrolase perhydrolysis is between about 1.2 and about 2. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of C7, D10, L12, G15, PI 8,

10 V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, 149, E51, L53, S54, A55, T57, D61, P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80, L82, P83, D85, L86, D95, K97, RlOl, T103, P104, L105, D106, 1107, L109, Mill, VI13, Q117, VI18, S121, G124, V125, G126, T127, P148, F150, 1153, F154, F196, G190, E198, A199, R202, D203, V206, A209, E210, Q211, and

15 V212. In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 2 and about 2.5. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one

20 substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A44, C7, D10, D85, D95, E26, E47, 1107, L12, L42, P104, P148, S54, Q40, Ql 17, D203, V206, E210. In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis,

25 such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 2.5 and about 3. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at GC821-2

an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A44, C7, 1107, K97, L12, L78, P104, Q40, and V125. 5 In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 3.0 and about 5. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis

10 perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of D10, D85, L53, L78, and S54. In still further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type

15 perhydrolase perhydrolysis is about 0.1 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, G110, G52,

20 G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, SI 1, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, and W34. In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.2 or less. In some embodiments, the variant perhydrolase

25 comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from GC821-2 "^"

the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GI 10, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, 5 P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, and Y73. In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.3 or less. In some embodiments, the variant

10 perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sl l, S54, T120,

15 T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, GI 10, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Mi l l, 0 N94, P146, Q41, R102, R27, R4, R56, SI 1, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, and Y129. In yet additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.4 or less. In some embodiments, the variant 5 perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is GC821-2 O. "^ ^• ;.i

selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GI 10, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, 5 N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, G110, G22, G70, 1153, 1194, 160, 189, K97, L114, L119, L12, L38, L6, L68, L82, Mi l l, N94, P146, Q41, R102, R27, R4, R56, Sl l, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, G110, G126, G22, 1194,0 189, K97, Ll 14, L84, N59, P146, P83, R102, R27, R33, R4, R56, SI 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18, V125, V19, and V87. In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.5 or less. In some embodiments, the variant 5 perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120,0 T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, G110, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, GI 10, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Ml 11,5 N94, P146, Q41, R102, R27, R4, R56, Sl l, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, G110, G126, G22, 1194, 189, K97, Ll 14, L84, N59, P146, P83, R102, R27, R33, R4, R56, SI 12, S54, S72, S76, , , GC821-2

T120, T127, T13, T25, T57, T96, VI 18, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, Gl lO, G126, G15, G36, 1107, 149, 15, K97, L109 ,L119, L12 L38, L6, L68, L84, L86, Mi l l, N59, P146, P24, Q40, RlOl, R102, R27, R33, R4, R56, SI 12, S72, S76, T127, T25, T35, T80, T96, VI 15, V32, V87, W34, and Y129. 5 In further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.6 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising t least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising 10 the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GllO, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GI 10, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, 15 N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, GI 10, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Ml 11, N94, P146, Q41, R102, R27, R4, R56, Sll, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, Gl lO, G126, G22, 1194, 20 189, K97, Ll 14, L84, N59, P146, P83, R102, R27, R33, R4, R56, SI 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, Gl lO, G126, G15, G36, 1107, 149, 15, K97, L109 ,L119, L12 L38, L6, L68, L84, L86, Mil 1, N59, P146, P24, Q40, RlOl, R102, R27, R33, R4, R56, SI 12, S72, S76, T127, T25, T35, T80, T96, VI 15, V32, V87, W34, Y129, A108, A44, A55, 25 D21, D62, F150, gl26, G36, G52, 1107, 15, 189, L109, Ll 14, Ll 19, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, SI 12, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, VI 15,V118, V32, V48, V87, W149, and Y73. GC821-2 ^ ^

In yet further embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.7 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, SI 1, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GI 10, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, GI 10, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Ml 11, N94, P146, Q41, R102, R27, R4, R56, Sl l, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, GllO, G126, G22, 1194, 189, K97, Ll 14, L84, N59, P146, P83, R102, R27, R33, R4, R56, SI 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, GllO, G126, G15, G36, 1107, 149, 15, K97, L109 ,L119, L12 L38, L6, L68, L84, L86, Ml 11, N59, P146, P24, Q40, RlOl, R102, R27, R33, R4, R56, SI 12, S72, S76, T127, T25, T35, T80, T96, VI 15, V32, V87, W34, Y129, A108, A44, A55,

D21, D62, F150, gl26, G36, G52, 1107, 15, 189, L109, Ll 14, Ll 19, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, SI 12, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115,V118, V32, V48, V87, W149,Y73, A122, A23, A29, A71, A79, C7, D61, D62, D85, E26, E51, FlOO, F28, F46, GI 10, G126, G52, G70, 1107, 149, 15, 160, 189, L109, Ll 14, L12, L38, L68, L82, L86, Ml 11, N59, N94, P83, R102, R33, R4,

SI 12, S72, S76, T103, Tl 16, T128, T25, T35, T57, T58, T64, V19, V32, V48, V87, Y129, Y73, and Y99. _, , , , , GC821-2 ^

In additional embodiments, the variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.8 or less. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at 5 an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55,

10 D62, F150, F154, GI 10, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, Gl lO, G22, G70, 1153, 1194, 160, 189, K97, L114, L119, L12, L38, L6, L68, L82, Mill, N94, P146, Q41, R102, R27, R4, R56, Sll, S54, T120, T13, T25, T35, T80, V48, W14,

15 W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, GllO, G126, G22, 1194, 189, K97, Ll 14, L84, N59, P146, P83, R102, R27, R33, R4, R56, SI 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, GI 10, G126, G15, G36, 1107, 149, 15, K97, L109 ,L119, L12 L38, L6, L68, L84, L86, Mill, N59, P146, P24, Q40, RlOl, R102, R27, R33, R4, R56, SI 12,

20 S72, S76, T127, T25, T35, T80, T96, VI 15, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, gl26, G36, G52, 1107, 15, 189, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, SI 12, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115N118, V32, V48, V87, W149,Y73, A122, A23, A29, A71, A79, C7, D61, D62, D85, E26, E51, FlOO, F28, F46, Gl lO, G126, G52, G70, 1107, 149, 15,

25 160, 189, L109, Ll 14, L12, L38, L68, L82, L86, Ml 11, N59, N94, P83, R102, R33, R4, SI 12, S72, S76, T103, T116, T128, T25, T35, T57, T58, T64, V19, V32, V48, V87, Y129, Y73, Y99, A108, A122, A29, A55, C77, D10, D106, D45, D61, D62, D65, D85, GC821-2 ^ ^

E47, E50, FlOO, F150, F28, F46, GI 10, G124, G126, G15, G36, 1153, 1194, 15, 160, 189, K3, K97, L105, L109, Ll 14, Ll 19, L38, L42, L68, L84, L86, Ml, N59, P24, P30, P83, RlOl, R27, R4, R56, SI 12, S54, S76, T103, Tl 16, T120, T127, T128, T13, T35, T64, VI 13, V17, V19, V32, V48, V87, Y129, Y73, and Y99. 5 The present invention also provides perhydrolase variants, wherein the perhydrolase variants exhibit greater perhydrolysis activity and decreased peracid hydrolysis activity as compared to wild-type perhydrolase. In some embodiments, the variant perhydrolases exhibit perhydrolysis activity ratio of at least about 1.2, and peracid hydrolysis activity ratio of about 0.8 or less, as compared to wild-type perhydrolase. In

10 alternative embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ED NO:2, wherein at least one substitution is selected from the group consisting of A29, A44, A55, A71, A79, C7, D10, D106, D31, D85, E26, E47, F150, F154, F196, F28,

15 G124, G126, G36, G43, 1153, L109, L42, L53, L109, L42, L53, L109, L42, L53, L68, L82, L86, Ml 11, N69, P104, P148, P18, P63, P66, P83, Ql 17, Q40, RlOl, R67, S54, S121, S72, S76, T25, T64, VI 15, and V19. In additional embodiments, the perhydrolase exhibits perhydrolysis activity ratio of at least about 1.2, a peracid hydrolysis activity ratio of about 0.8 or less, and a protein

20 concentration ratio of at least 0.5, as compared to wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A29, A44, A71, A79, C7,

25 D85, E26, E47, E51, F150, F154, F196, F28, G124, G126, G36, 1153, L109, L12, L53, L68, L82, Mi l l, N69, P104, P148, P18, P63, P66, P83, Ql 17, Q40, RlOl, R67, S121, S54, S72, S76, T25, T64, V125, and V19. , „ if«''- !f^ iE -■.' ' ¹ i ' !'' ■•^• ιl' lⁱ--t ^,r"ιp_^ι ιw GC821-2

The present invention provides variant perhydrolases that exhibit an increase in expression of the perhydrolase variants, as compared to the expression of wild-type perhydrolase. In some embodiments, the variant perhydrolase comprises at least one modification comprising at least one substitution at an amino acid position equivalent to a 5 position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein at least one substitution is selected from the group consisting of A2, 15, C7, F8, Sll, L12, T13, W14, W16, V17, P18, V19, E20, G22, A23, P24, T25, A29, P30, V32, T35, G36, V37, A39, F46, E47, S54, A55, R56, T58, 160, D61, D62, P63, T64, P66, R67, L68, N69, G70, S72, Y73, L74, P75, S76, C77, L78, A79, T80, L82, 10 P83, L84, L86, 189, T93, T96, K97, A98, Y99, FlOO, RlOl, R102, T103, P104, L105, D106, 1107, A108, L109, Gl lO, SI 12, VI 13, L114, VI 15, T116, Q117, VI 18, L119, T120, S121, A122, G124, V125, G126, T127, T128, Y129, P130, P132, K133, L135, V136, S138, P141, L142, A143, M145, H147, W149, F150, Q151, 1153, G157, Q159, T161, T162, L164, A165, R166, V167, Y168, A170, L171, A172, M175, K176, P178, 15 A182, G183, S184, V185, 1186, T188, 1194, F196, V191, N201, L208, A209, Q211, Q213, S214, L215, and L216. The present invention also provides isolated proteins comprising homologs of M. smegmatis perhydrolase, wherein the homologs are proteins within the SGNH-hydrolase family of proteins. In alternative preferred embodiments, the isolated proteins have at 20 least about 35% identity with the amino acid sequence of M. smegmatis perhydrolase, in which the protein comprises at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, GI 10, Ll 14, L135, F180, G205, SI 1, D192, and H195. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 25 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2. , ,

I i ϊ GC821-2 C Ω

The present invention also provides isolated proteins having at least about 38% identity with the amino acid sequence of M. smegmatis perhydrolase, wherein the protein exhibits perhydrolysis activity. In further embodiments, the perhydrolase is at least approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 5 95%, or 99% homologous to M. smegmatis perhydrolase. In additional preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2. The present invention also provides homologs of M. smegmatis perhydrolase, wherein the homologs are perhydrolases comprising at least one motif selected from the 10 group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In preferred embodiments, the homologs exhibit perhydrolysis. In some particularly preferred embodiments, the homologs exhibit a perhydrolysis to hydrolysis ratio that is great than about 1. In still further embodiments, the homologs are immunologically cross-reactive with antibodies raised against M. smegmatis 15 perhydrolase. In yet additional embodiments, antibodies raised against the homolog cross-react with M. smegmatis perhydrolase. The present invention also provides isolated proteins having at least- about 35% identity with the amino acid sequence of at least one M. smegmatis perhydrolase homolog, wherein the proteins exhibit perhydrolysis activity. 20 In some particularly preferred embodiments, the present invention provides proteins having perhydrolase activity, wherein the proteins are in the form of a multimer in solution. In some more preferred embodiments, the protein is a perhydrolase that comprises a dimer. In alternative particularly preferred embodiments, the protein is a perhydrolase that comprises an octamer. In still further embodiments, the protein is in the 25 form of a multimer in solution and the protein is selected from the group consisting of M. smegmatis perhydrolase, M. smegmatis perhydrolase homologs, and M. smegmatis perhydrolase variants. In yet further embodiments, the protein is selected from the group

consisting of modified serine hydrolases and modified cysteine hydrolases, wherein the modified serine hydrolases or modified cysteine hydrolases comprise increased perhydrolase activity as compared to unmodified serine hydrolases or unmodified cysteine hydrolases 5 The present invention also provides proteins having perhydrolase activity, wherein the protein comprises at least one motif selected from the group consisting of GDSL- GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In some embodiments, the protein is obtained from a member of the Rhizobiales. In some preferred embodiments, the protein is obtained from a member of the genus

10 Mycobacterium. The present invention also provides isolated genes identified using at least one primer selected from the group consisting of SEQ ID NOS :21-69. The present invention also provides methods for identifying a perhydrolase, comprising the steps of: identifying source of the perhydrolase; analyzing the source to

15 identify sequences comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT; expressing the sequences identified in step b) to produce the perhydrolase; and testing the perhydrolase for perhydrolysis activity. In some embodiments, the analyzing step is an amplification step wherein the primer

20 sequences set forth in SEQ ID NOS :21-69 are used to amplifying the sequences comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL- ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In still further embodiments, the source is selected from the group consisting of environmental sources and metagenomic sources. The present invention also provides proteins identified using the

25 methods set forth herein. The present invention further provides isolated nucleic acid sequences encoding the proteins identified using the methods set forth herein. In some particularly preferred embodiments, the proteins exhibit a perhydrolysis to hydrolysis jV ! » ft w >™P- « ^" »^{•■ ■}" GC821-2 f

ratio that is greater than about 1. In still further embodiments, the proteins exhibit a perhydrolysis activity that is at least about 0.2, compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase. In yet additional embodiments, the proteins comprise at least three residues selected from the group consisting of L6, W14, W34, 5 L38, R56, D62, L74, L78, H81, P83, M90, K97, GllO, L114, L135, F180, G205, Sl l, D192, and H195. In further embodiments, the analyzing step comprises searching at least one amino acid database. In yet further embodiments, the analyzing step comprises searching at least one nucleic acid database to identify nucleic acid sequences encoding the amino acid 10 sequences of the perhydrolase. In still further embodiments, the source is selected from the group consisting of environmental sources and metagenomic sources. The present invention further provides isolated nucleic acid sequences encoding the proteins identified using the methods set forth herein. In some particularly preferred embodiments, the proteins exhibit a perhydrolysis to hydrolysis ratio that is greater than 15 about 1. In still further embodiments, the proteins exhibit a perhydrolysis activity that is at least about 0.2, compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase. In yet additional embodiments, the proteins comprise at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, Gl lO, L114, L135, F180, G205, Sl l, D192, and H195, as set forth

20 in SEQ ID NO:2. The present invention also provides variant perhydrolases having altered substrate specificities as compared to wild-type M. smegmatis perhydrolase. In some embodiments, the variant perhydrolases have altered para nitrophenyl caproate (PNC) activity, as compared to wild-type M. smegmatis perhydrolase.

25 The present invention also provides variant perhydrolases having altered pi values as compared to wild-type M. smegmatis perhydrolase. In some embodiments, the variant perhydrolases comprise at least one positively charged mutation, while in alternative . . „ , a * if..- if -- "»» ^•«-^»- 'i' *^v GC821-2

embodiments, the variant perhydrolases comprise at least one negatively charged mutation. The present invention also provides variant perhydrolases that have increased stability, as compared to wild-type M. smegmatis perhydrolase. In some preferred 5 embodiments, the stability of the variant perhydrolase is selected from the group consisting of thermostability, enzymatic stability, and chemical stability. The present invention also provides variant perhydrolases, wherein the variant perhydrolase exhibits at least one altered surface property. In some preferred embodiments, the variants comprise at least one mutation comprising at least one

10 substitution at sites selected from the group consisting of the residues set forth in Table 15-1. The present invention also provides perhydrolase variants having at least one improved property as compared to wild-type perhydrolase. The present invention also provides expression vectors comprising a

15 polynucleotide sequence encoding at least one perhydrolase variant. The present invention further provides host cells comprising at least one such expression vector. In some preferred embodiments, a host cell is selected from the group consisting of Bacillus sp., Streptomyces sp., Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by the host cells.

20 The present invention also provides compositions comprising at least a portion of at least one perhydrolase. In some preferred embodiments, the perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2. In further embodiments, the perhydrolase is encoded by a polynucleotide sequence comprises SEQ ID NO: 1. In additional embodiments, the sequence comprises at least a portion of SEQ ID NO:l. In

25 further embodiments, the present invention provides expression vectors comprising the polynucleotide sequence encoding at least a portion of at least one perhydrolase. The present invention also provides host comprising at least one expression vectors. In some , , GC821-2 ^'"^"^

embodiments, the host cells are selected from the group consisting of Bacillus sp., Streptomyces sp., Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by these host cells. The present invention also provides variant perhydrolases, wherein the 5 perhydrolases comprise at least one substitution corresponding to the amino acid positions in SEQ ID NO:2, and wherein the variant perhydrolase has better performance in at least one property, compared to wild-type M. smegmatis perhydrolase. The present invention further provides isolated polynucleotides comprising a nucleotide sequence (i) having at least about 70% identity to SEQ ID NO:l, or (ii) being 10 capable of hybridizing to a probe derived from the nucleotide sequence set forth in SEQ ID NO:l, under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence set forth in SEQ ID NO:l. In some embodiments, the present invention also provides vectors comprising these polynucleotide sequences. In additional embodiments, the present invention also 15 provides host comprising at least one expression vectors. In some embodiments, the host cells are selected from the group consisting of Bacillus sp., Streptomyces sp., Escherichia, and Pantoea sp. The present invention also provides perhydrolases produced by these host cells. The present invention also provides polynucleotides comprising a sequence 20 complementary to at least a portion of the sequence set forth in SEQ ID NO: 1. The present invention also provides methods of producing enzymes having perhydrolase activity, comprising: transforming a host cell with an expression vector comprising a polynucleotide having at least 70% sequence identity to SEQ ID NO: 1 ; cultivating the transformed host cell under conditions suitable for the host cell to produce 25 the perhydrolase; and recovering the perhydrolase. In some preferred embodiments, the host cell is selected from the group consisting of Streptomyces, Pantoea, Escherichia, and Bacillus species. GC821-2

The present invention also provides probes comprising a 4 to 150 polynucleotide sequence substantially identical to a corresponding fragment of SEQ ID NO:l, wherein the probe is used to detect a nucleic acid sequence coding for an enzyme having perhydrolase activity. 5 The present invention also provides cleaning compositions comprising: a) at least 0.0001 weight percent of a perhydrolase that exhibits a perhydrolysis to hydrolysis ratio that is greater than 1; b) a molecule comprising an ester moiety; and c) optionally, an adjunct ingredient. The present invention further provides cleaning compositions comprising: a) at

10 least 0.0001 weight percent of a perhydrolase that exhibits a perhydrolysis to hydrolysis ratio that is greater than 1; b) a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, the peroxygen source being selected from the group consisting of: a per-salt; an organic peroxyacid; urea hydrogen peroxide; a carbohydrate and carbohydrate oxidase mixture, and mixtures thereof; c)

15 from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety; and d) optionally, an adjunct ingredient. The present invention also provides cleaning compositions comprising: a) from about 0.0001 to about 1 weight percent of a variant perhydrolase having an amino acid sequence comprising at least one modification of an amino acid made at a position

20 equivalent to a position in M smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2; b) a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, the peroxygen source being selected from the group consisting of: a per-salt; an organic peroxyacid; urea hydrogen peroxide; a carbohydrate and carbohydrate oxidase mixture; and mixtures thereof; c)

25 from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety; and d) optionally, an adjunct ingredient. In some preferred embodiments, the cleaning compositions further comprise at least one adjunct ingredient. In some particularly , . GC821-2

preferred embodiments, the adjunct ingredient is selected from the group consisting of surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removal/anti- 5 redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, pigments and mixtures thereof. In additional embodiments, the present invention provides cleaning compositions wherein: the perhydrolase exhibits a perhydrolysis to hydrolysis molar ratio that is greater

10 than about 0.1 ; the per-salt is selected from the group consisting of alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphates, alkalimetal persulphates and mixtures thereof; the carbohydrate is selected from the group consisting of mono- carbohydrates, di- carbohydrates, tri- carbohydrates, oligo- carbohydrates and mixtures thereof; the carbohydrate oxidase is selected from the group consisting of aldose oxidase

15 (IUPAC classification EC 1.1.3.9), galactose oxidase (IUP AC classification EC 1.1.3.9), cellobiose oxidase (IUPAC classification EC 1.1.3.25), pyranose oxidase (IUPAC classification EC 1.1.3.10), sorbose oxidase (IUPAC classification EC 1.1.3.11) hexose oxidase (IUPAC classification EC1.1.3.5). glucose oxidase (IUPAC classification EC 1.1.3.4) and mixtures thereof; and the molecule comprising an ester moiety has the

20 formula:

(i) wherein R¹ is a moiety selected from the group consisting of H, substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and 25 heteroaryl; (ii) each R is an alkoxylate moiety; (iii) R is an ester-forming moiety having the formula: _. GC821-2

R CO- wherein R is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; (iv) x is 1 when R is H; when R is not H, x is an integer that is equal to or less than the number of carbons in R¹; 5 (v) p is an integer that is equal to or less than x; (vi) m is an integer from 0 to 50; and (vii) n is at least 1

In alternative embodiments, the present invention provides cleaning compositions 10 wherein: a) R¹ is an C₂-C₃₂ substituted or unsubstituted alkyl or heteroalkyl moiety; b) each R is independently an ethoxylate or propoxylate moiety; and c) m is an integer from 1 to 12. In some embodiments, R is an ester-forming moiety having the formula: R⁴CO- wherein R is: a) a substituted or unsubstituted alkyl, alkenyl or alkynyl moiety comprising from 1 to 22 carbon atoms; or b) a substituted or unsubstituted aryl, alkylaryl, 15 alkylheteroaryl or heteroaryl moiety comprising from 4 to 22 carbon atoms. In still further embodiments of the cleaning compositions, the molecule comprising the ester moiety has the formula:

20 wherein: a) R is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, the R moiety that comprises an amine moiety being selected from the group consisting of substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; b) each R is an alkoxylate moiety; c) R is an ester-forming moiety having the formula: R CO- wherein R may be 25 H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; d) x is 1 when R is H; when R¹ is not H, x is an integer that is equal to or less than the number of carbons in R ; e) p is an integer that is equal to or less than x; f) m is .

GC821-2 -^' )

an integer from 0 to 12; and g) n is at least 1.

In still further embodiments of the present cleaning compositions, the molecule comprising an ester moiety has a weight average molecular weight of less than 600,000 Daltons. In yet additional embodiments, an adjunct ingredient is selected .from the group consisting of surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, pigments and mixtures thereof. The present invention further provides methods of cleaning comprising the steps of: a) contacting a surface and/or an article comprising a fabric with any of the cleaning compositions provided above and/or a composition comprising any of the cleaning compositions provided above; and b) optionally washing and/or rinsing the surface or material. In alternative embodiments, the present invention provides methods of cleaning, the method comprising the steps of: a) contacting a surface and/or an article comprising a fabric with any suitable cleaning composition provided above and/or a composition comprising any suitable cleaning provided above; and b) optionally washing and/or rinsing the surface or material. The present invention also provides bleaching compositions comprising at least one perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, Upases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. _, GC821-2 ^

The present invention also provides bleaching compositions comprising at least one perhydrolase variant having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2. . 5 In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. 10 The present invention also provides bleaching compositions comprising at least one perhydrolase variant having at least one improved property as compared to wild-type perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme 15 derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. The present invention also provides bleaching compositions comprising at least one perhydrolase variant comprising at least one substitution corresponding to the amino acid positions in SEQ ID NO:2, and wherein the variant perhydrolase has better 20 performance in at least one property compared to wild-type M. smegmatis perhydrolase. . In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, 25 oxidoreductases, hemicellulases, and cellulases. The present invention also provides bleaching compositions comprising at least one perhydrolase that is at least approximately about 35% homologous to M. smegmatis GC821-2 ^{' "})

perhydrolase. . In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. The present invention also provides disinfecting compositions comprising at least one perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. The present invention also provides disinfecting compositions comprising at least one perhydrolase variant having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ED NO:2. . In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. The present invention also provides disinfecting compositions comprising at least one perhydrolase variant having at least one improved property as compared to wild-type perhydrolase. In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. GC821-2 " 1 ^^

The present invention also provides disinfecting compositions comprising at least one perhydrolase variant comprising at least one substitution corresponding to the amino acid positions in SEQ ID NO:2, and wherein the variant perhydrolase has better performance in at least one property compared to wild-type M. smegmatis perhydrolase. . In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. The present invention also provides disinfecting compositions comprising at least one perhydrolase that is at least approximately about 35% homologous to M. smegmatis perhydrolase. . In some particularly preferred embodiments, the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1. In some embodiments, the bleaching compositions further comprise at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases. In some preferred embodiments, the perhydrolase is at least approximately 70% homologous to M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the present invention provides perhydrolases that cross react with antibody generated against M. smegmatis perhydrolase, particularly that comprising the amino acid sequence set forth in SEQ ID NO:2. In further embodiments, the present invention provides perhydrolases that are structural homologs of the M. smegmatis perhydrolase, in which active site comprises sites homologous to SI 1, D 192, and HI 95 of the M. smegmatis perhydrolase. In yet additional embodiments, the present invention provides perhydrolases comprising one or more modifications at the following residues: Cys7, AsplO, Serl 1, Leul2, Thrl3, Trpl4, Trplό, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, _,

GC821-2 ^"^ ^""

Vall25, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, Del 53, Phel54, Thrl59, Thrl 86, Hel92, ϋel94, and Phel96. However, it is not intended that the present invention be limited to perhydrolases with these modifications only at these residues, as perhydrolases with other modifications also find use with the present invention. In some embodiments, at least one perhydrolase of the present invention is used in a cleaning process wherein an article to be cleaned is exposed to a sufficient amount of the at least one perhydrolase under conditions such that the perhydrolase cleans and/or bleaches, and/or decolorizes any/all stains present on the article (e.g., laundry and dish detergents). In some embodiments, the cleaning further comprises disinfecting. In some embodiments, the article cleaned, bleached and/or disinfected using at least one perhydrolase of the present invention comprises textiles and/or hard surfaces, while in other embodiments, the article is paper or pulp, and in still further embodiments, at least one perhydrolase is used as a personal care product to whiten or bleach hair, teeth, skin, etc. Thus, in some embodiments, the present invention provides compositions for use in various cleaning, bleaching, and/or disinfecting applications. Indeed, it is not intended that the present invention be limited to any particular application. In some preferred embodiments, the perhydrolase comprises SEQ ED NO:2. In some preferred alternative embodiments, the perhydrolase is encoded by the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, the present invention provides enzymes with activities that result in high peracid/acid ratios. In alternative embodiments, the present invention provides the perhydrolase of Mycobacterium smegmatis, as well as sequence and/or structural homologs of this protein. In additional embodiments, the present invention provides enzymes that have been modified so as to express perhydrolase activity with a high perhydrolysis to hydrolase ratio either in addition to or instead of the enzyme's original activity. In additional embodiments, the present invention provides modified enzymes with altered substrate specificity, Km, kcat, perhydrolase activity, and/or peracid , , GC821-2 ^

degradation activity. In additional embodiments, the present invention provides means to identify, produce, and characterize enzymes that comprise the perhydrolysis activity of the present invention. The present invention further provides methods and compositions comprising 5 at least one perhydrolase for cleaning, disinfecting, bleaching, and other applications, including but not limited to paper and pulp bleaching, fabric and garment cleaning, hard surface cleaning, and personal care applications (e.g., oral care, hair care, and skin care). In some preferred embodiments, the present invention provides methods and compositions for bleaching cotton and other fabrics. Indeed, the present invention finds 10 use in the bleaching and cleaning of various textiles. It is not intended that the present invention be limited to any particular setting, application or use, as it is contemplated that it will find use in numerous areas where an enzymatic generation of peracids is desired over the use of preformed peracids or hydrogen peroxide or other bleaching chemicals, under conditions including but not limited to a wide range of pHs and temperatures. The 15 present invention also finds use in applications where peracid hydrolysis is useful, such as in the clean up of peracids. Furthermore, the present invention provides means to produce perhydrolase enzymes suitable for cleaning, disinfecting, bleaching, and other applications, including personal care. 20 DESCRIPTION OF THE FIGURES Figure 1 provides a phylogenetic tree of M. smegmatis perhydrolase and other related sequences. Figure 2 provides an overview phylogenetic tree, showing the major branches of 25 the bacteria and the origin of the active clones/sequences compared to M. smegmatis. Figure 3 provides a schematic of four structural families of serine hydrolases, including perhydrolase (SGNH-hydrolase family), chymotrypsin, subtilisin, and α/β GC821-2 '"

hydrolase. Figure 4 provides a diagram of the structure of the perhydrolase fold. Figure 5 provides a map of plasmid pET26-M4aEl 1. Figure 6 provides a purification table showing the enzyme activity of the enzyme of the present invention through various steps in the purification process. Figure 7 provides a graph which shows the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 40 minutes. Figure 8 provides a graph showing the peracid production by 30 mM acetate equivalents and 29 mM hydrogen peroxide, tested at various pHs. These results show that using the perhydrolase composition of the present invention, there is peracid generation over a wide pH range. In contrast, with TAED and hydrogen peroxide, peracid generation is limited to alkaline conditions. Figure 9 provides a graph showing the peracid production by 0.1 ppm perhydrolase enzyme in 30 mM ethyl acetate and 20 mM hydrogen peroxide at various temperatures. These results show that the perhydrolase of the present invention works at a wide range of temperatures, including low temperatures. Figure 10 provides a graph showing the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 4, 10, and 30 minutes. Figure 11 provides a graph showing the ratio of peracetic acid to acetic acid generated by various enzymes from 10 mM triacetin and 29 mM hydrogen peroxide in 4 and 10 minutes. Figure 12 provides a map of plasmid pMSATNcoI. Figure 13 provides a map of plasmid pMS ATNco 1 - 1. Figure 14 provides a map of plasmid pAH505. Figure 15 provides a map of plasmid pSFNASally. Hr--' r. GC821-2 ^ ^

Figure 16 provides a map of plasmid pCP606. Figure 17 provides a map of plasmid pCP649. Figure 18 provides a map of plasmid pSECGT-MSAT. Figure 19 provides a map of plasmid pSEGT-phdA4. 5 Figure 20 provides a map of plasmid pMC355rbs. Figure 21 provides a graph showing the degree of bleaching by three detergents tested alone and in comparison with the M. smegmatis perhydrolase of the present invention. Figure 22 provides a graph showing the bleaching ability of the M. smegmatis 0 perhydrolase tested on cotton. Figure 23 provides a graph showing the bleaching ability of the M. smegmatis perhydrolase tested on linen.

5 DESCRIPTION OF THE INVENTION The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. In particular, the present invention provides improved methods and0 compositions comprising perhydrolysis enzymes with high peracid/acid ratios for cleaning, bleaching, disinfecting and other applications. In some preferred embodiments, the present invention provides improved methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting. 5 Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA , „

GC821-2 @

fields, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual", Second Edition (Cold Spring Harbor), [1989]); and Ausubel et al., "Current Protocols in Molecular Biology" [1987]). All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference. Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.

Definitions Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionaries of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not GC821-2 O

limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical 5 limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly

10 written herein. As used herein, the term "bleaching" refers to the treatment of a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient length of time and under appropriate pH and temperature conditions to effect a brightening (i.e., whitening) and/or cleaning of the material. Examples of chemicals suitable for bleaching include but are not limited to

15 ClO₂, H₂O₂, peracids, NO₂, etc. As used herein, the term "disinfecting" refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items. It is not intended that the present invention be limited to any particular surface, item, or contaminant(s) or microbes to be removed.

20 As used herein, the term "perhydrolase" refers to an enzyme that is capable of catalyzing a reaction that results in the formation of sufficiently high amounts of peracid suitable for applications such as cleaning, bleaching, and disinfecting. In particularly preferred embodiments, the perhydrolase enzymes of the present invention produce very high perhydrolysis to hydrolysis ratios. The high perhydrolysis to hydrolysis ratios of

25 these distinct enzymes makes these enzymes suitable for use in a very wide variety of applications. In additional preferred embodiments, the perhydrolases of the present invention are characterized by having distinct tertiary structure and primary sequence. In GC821-2 H-i

particularly preferred embodiments, the perhydrolases of the present invention comprises distinct primary and tertiary structures. In some particularly preferred embodiments, the perhydrolases of the present invention comprise distinct quaternary structure. In some preferred embodiments, the perhydrolase of the present invention is the smegmatis 5 perhydrolase, while in alternative embodiments, the perhydrolase is a variant of this perhydrolase, while in still further embodiments, the perhydrolase is a homolog of this perhydrolase. In further preferred embodiments, a monomeric hydrolase is engineered to produce a multimeric enzyme that has better perhydrolase activity than the monomer. However, it is not intended that the present invention be limited to this specific M.

10 smegmatis perhydrolase, specific variants of this perhydrolase, nor specific homologs of this perhydrolase. As used herein, the term "multimer" refers to two or more proteins or peptides that are covalently or non-covalently associated and exist as a complex in solution. A "dimer" is a multimer that contains two proteins or peptides; a "rrimer" contains three

15 proteins or peptides, etc. As used herein, "octamer" refers to a multimer of eight proteins or peptides. As used herein, the phrase "perhydrolysis to hydrolysis ratio" is the ratio of the amount of enzymatically produced peracid to that of enzymatically produced acid by the perhydrolase, under defined conditions and within a defined time. In some preferred

20 embodiments, the assays provided herein are used to determine the amounts of peracid and acid produced by the enzyme. As used herein, "personal care products" means products used in the cleaning, bleaching and/or disinfecting of hair, skin, scalp, and teeth, including, but not limited to shampoos, body lotions, shower gels, topical moisturizers, toothpaste, and/or other

25 topical cleansers. In some particularly preferred embodiments, these products are utilized on humans, while in other embodiments, these products find use with non-human animals (e.g., in veterinary applications). GC821-2

As used herein, "pharmaceutically-acceptable" means that drugs, medicaments and/or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and other animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit risk ratio. As used herein, "cleaning compositions" and "cleaning formulations" refer to compositions that find use in the removal of undesired compounds from items to be cleaned, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes) etc. The term encompasses any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, granule, or spray composition), as long as the composition is compatible with the perhydrolase and other enzyme(s) used in the composition. The specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use. The terms further refer to any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any object and/or surface. It is intended that the terms include, but are not limited to detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents). Indeed, the term "cleaning composition" as used herein, includes unless otherwise indicated, granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high- foaming type; _

GC821-2 ^

machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries such as bleach additives and "stain-stick" or pre-treat types. As used herein, the terms "detergent composition" and "detergent formulation" are used in reference to mixtures which are intended for use in a wash medium for the cleaning of soiled objects. In some preferred embodiments, the term is used in reference to laundering fabrics and/or garments (e.g., "laundry detergents"). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e.g., "dishwashing detergents"). It is not intended that the present invention be limited to any particular detergent formulation or composition. Indeed, it is intended that in addition to perhydrolase, the term encompasses detergents that contain surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and solubilizers. As used herein, "enhanced performance" in a detergent is defined as increasing cleaning of bleach-sensitive stains (e.g., grass, tea, wine, blood, dingy, etc.), as determined by usual evaluation after a standard wash cycle. In particular embodiments, the perhydrolase of the present invention provides enhanced performance in the oxidation and removal of colored stains and soils. In further embodiments, the perhydrolase of the present invention provides enhanced performance in the removal and/or decolorization of stains. In yet additional embodiments, the perhydrolase of the present invention provides enhanced performance in the removal of lipid-based stains and soils. In still further embodiments, the perhydrolase of the present invention provides enhanced performance in removing soils and stains from dishes and other items. .

As used herein the term "hard surface cleaning composition," refers to detergent compositions for cleaning hard surfaces such as floors, walls, tile, bath and kitchen fixtures, and the like. Such compositions are provided in any form, including but not limited to solids, liquids, emulsions, etc. 5 As used herein, "dishwashing composition" refers to all forms for compositions for cleaning dishes, including but not limited to granular and liquid forms. As used herein, "fabric cleaning composition" refers to all forms of detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. 10 As used herein, "textile" refers to woven fabrics, as well as staple fibers and filaments suitable for conversion to or use as yams, woven, knit, and non-woven fabrics. The term encompasses yams made from natural, as well as synthetic (e.g., manufactured) fibers. As used herein, "textile materials" is a general term for fibers, yam intermediates, 15 yam, fabrics, and products made from fabrics (e.g., garments and other articles). As used herein, "fabric" encompasses any textile material. Thus, it is intended that the term encompass garments, as well as fabrics, yams, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material. As used herein, the term "compatible," means that the cleaning composition 20 materials do not reduce the enzymatic activity of the perhydrolase to such an extent that the perhydrolase is not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter. As used herein, "effective amount of perhydrolase enzyme" refers to the quantity of perhydrolase enzyme necessary to achieve the enzymatic activity required in the 25 specific application (e.g., personal care product, cleaning composition, etc.). Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme variant used, the cleaning application, the GC821-2

specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like. As used herein, "non-fabric cleaning compositions" encompass hard surface cleaning compositions, dishwashing compositions, personal care cleaning compositions (e.g., oral cleaning compositions, denture cleaning compositions, personal cleansing compositions, etc.), and compositions suitable for use in the pulp and paper industry. As used herein, "oral cleaning compositions" refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like. Oral care compositions that find use in conjunction with the perhydrolases of the present invention are well known in the art (See e.g., U.S. Patent Nos 5,601,750, 6,379,653, and 5,989,526, all of which are incorporated herein by reference). As used herein, "pulp treatment compositions" refers to the use of the present perhydrolase enzymes in compositions suitable for use in papermaking. It is intended that the term encompass compositions suitable for the treatment of any pulp material, including wood, as well as non-wood materials, such as "agricultural residues" and "fiber crops," including but not limited to wheat straw, rice straw, com stalks, bagasse (sugar cane), rye grass straw, seed flax straw, flax straw, kenaf, industrial hemp, sisal, textile flat straw, hesperaloe, etc. Thus, the present invention also encompasses the use of the perhydrolases of the present invention in pulp treatment methods. As used herein, "oxidizing chemical" refers to a chemical that has the capability of bleaching pulp or any other material. The oxidizing chemical is present at an amount, pH and temperature suitable for bleaching. The term includes, but is not limited to hydrogen peroxide and peracids. As used herein, "acyl" is the general name for organic acid groups, which are the residues of carboxylic acids after removal of the -OH group (e.g., ethanoyl chloride, GC821-2 ^

CH₃CO-Cl, is the acyl chloride formed from ethanoic acid, CH₃COO-H). The names of the individual acyl groups are formed by replacing the "-ic" of the acid by "~yl." As used herein, the term "acylation" refers to the chemical transformation which substitutes the acyl (RCO-) group into a molecule, generally for an active hydrogen of an 5 -OH group. As used herein, the term "transferase" refers to an enzyme that catalyzes the transfer of functional compounds to a range of substrates. As used herein, "leaving group" refers to the nucleophile which is cleaved from the acyl donor upon substitution by another nucleophile. 10 As used herein, the term "enzymatic conversion" refers to the modification of a substrate to an intermediate or the modification of an intermediate to an end-product by contacting the substrate or intermediate with an enzyme. In some embodiments, contact is made by directly exposing the substrate or intermediate to the appropriate enzyme. In other embodiments, contacting comprises exposing the substrate or intermediate to an 15 organism that expresses and/or excretes the enzyme, and/or metabolizes the desired substrate and/or intermediate to the desired intermediate and/or end-product, respectively. As used herein, the phrase "detergent stability" refers to the stability of a detergent composition. In some embodiments, the stability is assessed during the use of the detergent, while in other embodiments, the term refers to the stability of a detergent 20 composition during storage. As used herein, the phrase, "stability to proteolysis" refers to the ability of a protein (e.g., an enzyme) to withstand proteolysis. It is not intended that the term be limited to the use of any particular protease to assess the stability of a protein. As used herein, "oxidative stability" refers to the ability of a protein to function 25 under oxidative conditions. In particular, the term refers to the ability of a protein to function in the presence of various concentrations of H2O2 and/or peracid. Stability under various oxidative conditions can be measured either by standard procedures known to . . GC821-2

those in the art and/or by the methods described herein. A substantial change in oxidative stability is evidenced by at least about a 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the enzymatic activity, as compared to the enzymatic activity present in the absence of oxidative compounds. 5 As used herein, "pH stability" refers to the ability of a protein to function at a particular pH. In general, most enzymes have a finite pH range at which they will function. In addition to enzymes that function in mid-range pHs (i.e., around pH 7), there are enzymes that are capable of working under conditions with very high or very low pHs. Stability at various pHs can be measured either by standard procedures known to those in 10 the art and/or by the methods described herein. A substantial change in pH stability is evidenced by at least about 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the enzymatic activity, as compared to the enzymatic activity at the enzyme's optimum pH. However, it is not intended that the present invention be limited to any pH stability level nor pH range. 15 As used herein, "thermal stability" refers to the ability of a protein to function at a particular temperature, hi general, most enzymes have a finite range of temperatures at which they will function. In addition to enzymes that work in mid-range temperatures (e.g., room temperature), there are enzymes that are capable of working in very high or very low temperatures. Thermal stability can be measured either by known procedures or 20 by the methods described herein. A substantial change in thermal stability is evidenced by at least about 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the catalytic activity of a mutant when exposed to a different temperature (i.e., higher or lower) than optimum temperature for enzymatic activity. However, it is not intended that the present invention be limited to any 25 temperature stability level nor temperature range. As used herein, the term "chemical stability" refers to the stability of a protein (e.g., an enzyme) towards chemicals that adversely affect its activity. In some GC821-2 ;

embodiments, such chemicals include, but are not limited to hydrogen peroxide, peracids, anionic detergents, cationic detergents, non-ionic detergents, chelants, etc. However, it is not intended that the present invention be limited to any particular chemical stability level nor range of chemical stability.

As used herein, the phrase "perhydrolase activity improvement" refers to the relative improvement of perhydrolase activity, in comparison with a standard enzyme. In some embodiments, the term refers to an improved rate of perhydrolysis product, while in other embodiments, the term encompasses perhydrolase compositions that produce less hydrolysis product. In additional embodiments, the term refers to perhydrolase compositions with altered substrate specificity. As used herein, the phrase "alteration in substrate specificity" refers to changes in the substrate specificity of an enzyme. In some embodiments, a change in substrate specificity is defined as a difference between the Kcat/K_m ratio observed with an enzyme compared to enzyme variants or other enzyme compositions. Enzyme substrate specificities vary, depending upon the substrate tested. The substrate specificity of an enzyme is determined by comparing the catalytic efficiencies it exhibits with different substrates. These determinations find particular use in assessing the efficiency of mutant enzymes, as it is generally desired to produce variant enzymes that exhibit greater ratios for particular substrates of interest. For example, the perhydrolase enzymes of the present invention are more efficient in producing peracid from an ester substrate than enzymes currently being used in cleaning, bleaching and disinfecting applications. Another example of the present invention is a perhydrolase with a lower activity on peracid degradation compared to the wild type. Another example of the present invention is a perhydrolase with higher activity on more hydrophobic acyl groups than acetic acid. However, it is not intended that the present invention be limited to any particular substrate composition nor any specific substrate specificity.

As used herein, "surface property" is used in reference to an electrostatic charge, as well as properties such as the hydrophobicity and/or hydrophilicity exhibited by the surface of a protein. As used herein, the phrase "is independently selected from the group consisting of " means that moieties or elements that are selected from the referenced Markush group can be the same, can be different or any mixture of elements as indicated in the following example: A molecule having 3 R groups wherein each R group is independently selected from the group consisting of A, B and C. Here the three R groups may be: AAA, BBB, CCC, AAB, AAC, BBA, BBC, CCA, CCB, or ABC. In reference to chemical compositions, the term "substituted" as used herein, means that the organic composition or radical to which the term is applied is: (a) made unsaturated by the elimination of at least one element or radical; or (b) at least one hydrogen in the compound or radical is replaced with a moiety containing one or more (i) carbon, (ii) oxygen, (iii) sulfur, (iv) nitrogen or (v) halogen atoms; or (c) both (a) and (b).

Moieties which may replace hydrogen as described in (b) immediately above, that contain only carbon and hydrogen atoms, are hydrocarbon moieties including, but not limited to, alkyl, alkenyl, alkynyl, alkyldienyl, cycloalkyl, phenyl, alkyl phenyl, naphthyl, anthryl, phenanthryl, fluoryl, steroid groups, and combinations of these groups with each other and with polyvalent hydrocarbon groups such as alkylene, alkylidene and alkylidyne groups. Moieties containing oxygen atoms that may replace hydrogen as described in (b) immediately above include, but are not limited to, hydroxy, acyl or keto, ether, epoxy, carboxy, and ester containing groups. Moieties containing sulfur atoms that may replace hydrogen as described in (b) immediately above include, but are not limited to, the sulfur- containing acids and acid ester groups, thioether groups, mercapto groups and thioketo -.. f GC821-2 ^S

groups. Moieties containing nitrogen atoms that may replace hydrogen as described in (b) immediately above include, but are not limited to, amino groups, the nitro group, azo groups, ammonium groups, amide groups, azido groups, isocyanate groups, cyano groups and nitrile groups. Moieties containing halogen atoms that may replace hydrogen as 5 described in (b) immediately above include chloro, bromo, fluoro, iodo groups and any of the moieties previously described where a hydrogen or a pendant alkyl group is substituted by a halo group to form a stable substituted moiety. It is understood that any of the above moieties (b)(i) through (b)(v) can be substituted into each other in either a monovalent substitution or by loss of hydrogen in a 10 polyvalent substitution to form another monovalent moiety that can replace hydrogen in the organic compound or radical. As used herein, the terms "purified" and "isolated" refer to the removal of • contaminants from a sample. For example, perhydrolases are purified by removal of contaminating proteins and other compounds within a solution or preparation that are not 15 perhydrolases. In some embodiments, recombinant perhydrolases are expressed in bacterial or fungal host cells and these recombinant perhydrolases are purified by the removal of other host cell constituents; the percent of recombinant perhydrolase polypeptides is thereby increased in the sample. As used herein, "protein of interest," refers to a protein (e.g., an enzyme or 20 "enzyme of interest") which is being analyzed, identified and/or modified. Naturally- occurring, as well as recombinant proteins find use in the present invention. As used herein, "protein" refers to any composition comprised of amino acids and recognized as a protein by those of skill in the art. The terms "protein," "peptide" and polypeptide are used interchangeably herein. Wherein a peptide is a portion of a protein, 25 those skilled in the art understand the use of the term in context. As used herein, functionally and/or structurally similar proteins are considered to be "related proteins." In some embodiments, these proteins are derived from a different GC821-2 ^ ^

genus and/or species, including differences between classes of organisms (e.g., a bacterial protein and a fungal protein). In some embodiments, these proteins are derived from a different genus and/or species, including differences between classes of organisms (e.g., a bacterial enzyme and a fungal enzyme). In additional embodiments, related proteins are provided from the same species. Indeed, it is not intended that the present invention be limited to related proteins from any particular source(s). In addition, the term "related proteins" encompasses tertiary structural homologs and primary sequence homologs (e.g., the perhydrolase of the present invention). In further embodiments, the term encompasses proteins that are immunologically cross-reactive. In most particularly preferred embodiments, the related proteins of the present invention very high ratios of perhydrolysis to hydrolysis. As used herein, the term "derivative" refers to a protein which is derived from a protein by addition of one or more amino acids to either or both the C- and N-terminal end(s), substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, and/or deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence, and/or insertion of one or more amino acids at one or more sites in the amino acid sequence. The preparation of a protein derivative is preferably achieved by modifying a DNA sequence which encodes for the native protein, transformation of that DNA sequence into a suitable host, and expression of the modified DNA sequence to form the derivative protein. Related (and derivative) proteins comprise "variant proteins." In some preferred embodiments, variant proteins differ from a parent protein and one another by a small number of amino acid residues. The number of differing amino acid residues may be one or more, preferably 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In some preferred embodiments, the number of different amino acids between variants is between 1 and 10. In some particularly preferred embodiments, related proteins and particularly variant proteins comprise at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, . „ GC821-2 ^'1

70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity. Additionally, a related protein or a variant protein as used herein, refers to a protein that differs from another related protein or a parent protein in the number of prominent regions. For example, in some embodiments, variant proteins have 1, 2, 3, 4, 5, or 10 5 corresponding prominent regions that differ from the parent protein. Several methods are known in the art that are suitable for generating variants of the perhydrolase enzymes of the present invention, including but not limited to site- saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other

10 recombinatorial approaches. In particularly preferred embodiments, homologous proteins are engineered to produce enzymes with the desired activity(ies). In some particularly preferred embodiments, the engineered proteins are included within the SGNH-hydrolase family of proteins. In some most preferred embodiments, the engineered proteins comprise at least

15 one or a combination of the following conserved residues: L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, GI 10, Ll 14, L135, F180, G205. In alternative embodiments, these engineered proteins comprise the GDSL-GRTT and/or ARTT motifs. In further embodiments, the enzymes are multimers, including but not limited to dimers, octamers, and tetramers. In yet additional preferred embodiments, the engineered 0 proteins exhibit a perhydrolysis to hydrolysis ratio that is greater than 1. An amino acid residue of a perhydrolase is equivalent to a residue of M. smegmatis perhydrolase if it is either homologous (t.e., having a corresponding position in either the primary and/or tertiary stracture) or analogous to a specific residue or portion of that residue in M. smegmatis perhydrolase (i.e., having the same or similar functional 5 capacity to combine, react, and/or chemically interact). In some embodiments, in order to establish homology to primary structure, the amino acid sequence of a perhydrolase is directly compared to the M. smegmatis _. ' >, GC821-2 ^""^

perhydrolase primary sequence and particularly to a set of residues known to be invariant in all perhydrolases for which sequence is known. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), 5 the residues equivalent to particular amino acids in the primary sequence of M. smegmatis perhydrolase are defined. In preferred embodiments, alignment of conserved residues conserves 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues are also adequate to define equivalent residues. In preferred embodiments, conservation of the catalytic serine and histidine residues are maintained.

10 Conserved residues are used to define the corresponding equivalent amino acid residues of M. smegmatis perhydrolase in other perhydrolases (e.g., perhydrolases from other Mycobacterium species, as well as any other organisms). In some embodiments of the present invention, the DNA sequence encoding M. smegmatis perhydrolase is modified. In some embodiments, the following residues are

15 modified: Cys7, AsplO, Serl 1, Leul2, Thrl3, Trpl4, Trpl6, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Vall25, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, ϋel53, Phel54, Thrl59, Thrl86, Ilel92, Ilel94, and Phel 96. However, it is not intended that the present invention be limited to sequence that are modified at these positions. Indeed, it is intended that the

20 present invention encompass various modifications and combinations of modifications. In additional embodiments, equivalent residues are defined by determining homology at the level of tertiary structure for a perhydrolase whose tertiary structure has been determined by x-ray crystallography. In this context, "equivalent residues" are defined as those for which the atomic coordinates of two or more of the main chain atoms

25 of a particular amino acid residue of the carbonyl hydrolase and M. smegmatis perhydrolase (N on N, CA on CA, C on C, and O on O) are within 0.13nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and ,- . GC821-2 '^" ,

positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the perhydrolase in question to the M. smegmatis perhydrolase. As known in the art, the best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available. Equivalent residues 5 which are functionally and/or structurally analogous to a specific residue of M. smegmatis perhydrolase are defined as those amino acids of the perhydrolases that preferentially adopt a conformation such that they either alter, modify or modulate the protein structure, to effect changes in substrate binding and/or catalysis in a manner defined and attributed to a specific residue of the M. smegmatis perhydrolase. Further, they are those

10 residues of the perhydrolase (in cases where a tertiary structure has been obtained by x- ray crystallography), which occupy an analogous position to the extent that although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of

15 M. smegmatis perhydrolase. The coordinates of the three dimensional structure of M. smegmatis perhydrolase were determined and are set forth herein (See e.g., Example 14) and find use as outlined above to determine equivalent residues on the level of tertiary structure. In some embodiments, some of the residues identified for substitution, insertion or

20 deletion are conserved residues whereas others are not. The perhydrolase mutants of the present invention include various mutants, including those encoded by nucleic acid that comprises a signal sequence. In some embodiments of perhydrolase mutants that are encoded by such a sequence are secreted by an expression host. In some further embodiments, the nucleic acid sequence comprises a homolog having a secretion signal.

25 Characterization of wild-type and mutant proteins is accomplished via any means suitable and is preferably based on the assessment of properties of interest. For example, pH and/or temperature, as well as detergent and /or oxidative stability is/are determined GC821-2 .

in some embodiments of the present invention. Indeed, it is contemplated that enzymes having various degrees of stability in one or more of these characteristics (pH, temperature, proteolytic stability, detergent stability, and/or oxidative stability) will find use. In still other embodiments, perhydrolases with low peracid degradation activity are 5 selected. As used herein, "expression vector" refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a

10 sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid,"

15 "expression plasmid," and "vector" are often used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art. In some preferred embodiments, the perhydrolase gene is ligated into an

20 appropriate expression plasmid. The cloned perhydrolase gene is then used to transform or transfect a host cell in order to express the perhydrolase gene. This plasmid may replicate in hosts in the sense that it contains the well-known elements necessary for plasmid replication or the plasmid may be designed to integrate into the host chromosome. The necessary elements are provided for efficient gene expression (e.g.. a

25 promoter operably linked to the gene of interest). In some embodiments, these necessary elements are supplied as the gene's own homologous promoter if it is recognized, (t.e., transcribed, by the host), a transcription terminator (a polyadenylation region for , „

GC821-2 - v

eukaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the perhydrolase gene. In some embodiments, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid- infected host cells by growth in antimicrobial-containing media is also included. The following cassette mutagenesis method may be used to facilitate the constraction of the perhydrolase variants of the present invention, although other methods may be used. First, as described herein, a naturally-occurring gene encoding the perhydrolase is obtained and sequenced in whole or in part. Then, the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded perhydrolase. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the protein gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the perhydrolase gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by Ml 3 primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region ,,,;, , . to ,. , , GC821-2 ©I

which does not contain a site. Once the naturally-occurring DNA and/or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are 5 ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites. As used herein, "corresponding to," refers to a residue at the enumerated position in a protein or peptide, or a residue that is analogous, homologous, or equivalent to an 10 enumerated residue in a protein or peptide. As used herein, "corresponding region," generally refers to an analogous position along related proteins or a parent protein. The terms "nucleic acid molecule encoding," "nucleic acid sequence encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of 15 deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence. As used herein, the term "analogous sequence" refers to a sequence within a protein that provides similar function, tertiary stracture, and/or conserved residues as the 20 protein of interest (i.e., typically the original protein of interest). For example, in epitope regions that contain an alpha helix or a beta sheet stracture, the replacement amino acids in the analogous sequence preferably maintain the same specific structure. The term also refers to nucleotide sequences, as well as amino acid sequences. In some embodiments, analogous sequences are developed such that the replacement amino acids result in a 25 variant enzyme showing a similar or improved function. In some preferred embodiments, the tertiary structure and/or conserved residues of the amino acids in the protein of interest are located at or near the segment or fragment of interest. Thus, where the GC821-2

segment or fragment of interest contains, for example, an alpha-helix or a beta-sheet structure, the replacement amino acids preferably maintain that specific structure. As used herein, "homologous protein" refers to a protein (e.g., perhydrolase) that has similar action and/or structure, as a protein of interest (e.g., an perhydrolase from another source). It is not intended that homologs be necessarily related evolutionarily. Thus, it is intended that the term encompass the same or similar enzyme(s) (i.e., in terms of structure and function) obtained from different species. In some preferred embodiments, it is desirable to identify a homolog that has a quaternary, tertiary and/or primary stracture similar to the protein of interest, as replacement for the segment or fragment in the protein of interest with an analogous segment from the homolog will reduce the disraptiveness of the change. In some embodiments, homologous proteins have induce similar immunological response(s) as a protein of interest. As used herein, "homologous genes" refers to at least a pair of genes from different species, which genes correspond to each other and which are identical or very similar to each other. The term encompasses genes that are separated by speciation (i.e., the development of new species) (e.g., orthologous genes), as well as genes that have been separated by genetic duplication (e.g., paralogous genes). These genes encode "homologous proteins." As used herein, "ortholog" and "orthologous genes" refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typically, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes. As used herein, "paralog" and "paralogous genes" refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paralogs evolve new functions, even though some functions are often related to the original one. Examples of paralogous genes include, but are not limited to

genes encoding trypsin, chymotrypsin, elastase, and thrombin, which are all serine proteinases and occur together within the same species. As used herein, "wild-type" and "native" proteins are those found in nature. The terms "wild-type sequence," and "wild-type gene" are used interchangeably herein, to 5 refer to a sequence that is native or naturally occurring in a host cell. In some embodiments, the wild-type sequence refers to a sequence of interest that is the starting point of a protein engineering project. The genes encoding the naturally-occurring protein may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative

10 sequences encoding regions of the protein of interest, preparing genomic libraries from organisms expressing the protein, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced. The term "recombinant DNA molecule" as used herein refers to a DNA molecule

15 that is comprised of segments of DNA joined together by means of molecular biological techniques. The term "recombinant oligonucleotide" refers to an oligonucleotide created using molecular biological manipulations, including but not limited to, the ligation of two or more oligonucleotide sequences generated by restriction enzyme digestion of a

20 polynucleotide sequence, the synthesis of oligonucleotides (e.g., the synthesis of primers or oligonucleotides) and the like. The degree of homology between sequences may be determined using any suitable method known in the art (See e.g., Smith and Waterman, Adv. Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443 [1970]; Pearson and Lipman, Proc. Natl.

25 Acad. Sci. USA 85:2444 [1988]; programs such as GAP, BESTFIT, FAST A, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., Nucl. Acid Res., 12:387-395 [1984]). GC821-2

For example, PILEUP is a useful program to determine sequence homology levels. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the 5 progressive alignment method of Feng and Doolittle, (Feng and Doolittle, J. Mol. Evol., 35:351-360 [1987]). The method is similar to that described by Higgins and Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. Another example of a useful algorithm is the BLAST algorithm, described by Altschul et

10 al, (Altschul et al, J. Mol. Biol., 215:403-410, [1990]; and Karlin et al, Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). One particularly useful BLAST program is the WU-BLAST-2 program (See, Altschul et al, Meth. Enzymol.,, 266:460-480 [1996]). parameters "W," "T," and "X" determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring

15 matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989]) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands. As used herein, "percent (%) nucleic acid sequence identity" is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues of the sequence.

20 As used herein, the term "hybridization" refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, as known in the art. As used herein, the phrase "hybridization conditions" refers to the conditions under which hybridization reactions are conducted. These conditions are typically classified by degree of "stringency" of the conditions under which hybridization is

25 measured. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5°C (5° below the Tm of the probe); "high GC821-2 O

stringency" at about 5-10° below the Tm; "intermediate stringency" at about 10-20° below the Tm of the probe; and "low stringency" at about 20-25° below the Tm. Alternatively, or in addition, hybridization conditions can be based upon the salt or ionic strength conditions of hybridization and/or one or more stringency washes. For example, 6xSSC = very low stringency; 3xSSC = low to medium stringency; lxSSC = medium stringency; and 0.5xSSC = high stringency. Functionally, maximum stringency conditions may be used to identify nucleic acid sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify nucleic acid sequences having about 80% or more sequence identity with the probe. For applications requiring high selectivity, it is typically desireable to use relatively stringent conditions to form the hybrids (e.g., relatively low salt and/or high temperature conditions are used). The phrases "substantially similar and "substantially identical" in the context of at least two nucleic acids or polypeptides typically means that a polynucleotide or polypeptide comprises a sequence that has at least about 40% identity, more preferable at least about 50% identity, yet more preferably at least about 60% identity, preferably at least about 75% identity, more preferably at least about 80% identity, yet more preferably at least about 90%, still more preferably about 95%, most preferably about 97% identity, sometimes as much as about 98% and about 99% sequence identity, compared to the reference (i.e., wild-type) sequence. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See e.g., Altschul, etal, J. Mol. Biol. 215:403-410 [1990]; Henikoff et al, Proc. Natl. Acad. Sci. USA 89:10915 [1989]; Karin et al, Proc. Natl. Acad. Sci USA 90:5873 [1993]; and Higgins et al, Gene 73:237 - 244 [1988]). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Pearson et al, Proc. Natl. Acad. Sci. USA 85:2444-2448 [1988]). One indication that two polypeptides are substantially identical is GC821-2 .^'"^") 1

that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative 5 substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency). As used herein, "equivalent residues" refers to proteins that share particular amino acid residues. For example, equivalent resides may be identified by determining

10 homology at the level of tertiary structure for a protein (e.g., perhydrolase) whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the protein having putative equivalent residues and the protein of interest (N on N, CA on CA, C on C and O on O) are within 0.13 nm and

15 preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non- hydrogen protein atoms of the proteins analyzed. The preferred model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available, determined using methods known to those skilled in the art

20 of crystallography and protein characterization/analysis. As used herein, the terms "hybrid perhydrolases" and "fusion perhydrolases" refer to proteins that are engineered from at least two different or "parental" proteins. In preferred embodiments, these parental proteins are homologs of one another. For example, in some embodiments, a preferred hybrid perhydrolase or fusion protein

25 contains the N-terminus of a protein and the C-terminus of a homolog of the protein. In some preferred embodiment, the two terminal ends are combined to correspond to the full-length active protein. ... ., -,, ,« » , , GC821-2 'T

The term "regulatory element" as used herein refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Additional regulatory elements include splicing signals, 5 polyadenylation signals and termination signals. As used herein, "host cells" are generally prokaryotic or eukaryotic hosts which are transformed or transfected with vectors constructed using recombinant DNA techniques known in the art. Transformed host cells are capable of either replicating vectors encoding the protein variants or expressing the desired protein variant. In the 10 case of vectors which encode the pre- or prepro-form of the protein variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium. The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means transformation, transduction or transfection. Means of transformation include protoplast transformation, calcium chloride precipitation, electroporation, naked DNA 15 and the like as known in the art. (See, Chang and Cohen, Mol. Gen. Genet., 168:111 - 115 [1979]; Smith et al, Appl. Env. Microbiol., 51:634 [1986]; and the review article by Ferrari et al, in Harwood, Bacillus, Plenum Publishing Corporation, pp. 57-72 [1989]). The term "promoter/enhancer" denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (for example, the 20 long terminal repeats of retrovirases contain both promoter and enhancer functions). The enhancer/promoter may be "endogenous" or "exogenous" or "heterologous." An endogenous enhancer/promoter is one which is naturally linked with a given gene in the genome. An exogenous (heterologous) enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (t.e., molecular biological 25 techniques). The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal GC821-2 'T ^'

of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [1989], pp. 16.7-16.8). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40. The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell which has stably integrated foreign or exogenous DNA into the genomic DNA of the transfected cell. The terms "selectable marker" or "selectable gene product" as used herein refer to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drag upon the cell in which the selectable marker is expressed. As used herein, the terms "amplification" and "gene amplification" refer to a process by which specific DNA sequences are disproportionately replicated such that the amplified gene becomes present in a higher copy number than was initially present in the genome. In some embodiments, selection of cells by growth in the presence of a drag

(e.g., an inhibitor of an inhibitable enzyme) results in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drag or by amplification of exogenous (i.e., input) sequences encoding this gene product, or both. Selection of cells by growth in the presence of a drug (e.g. , an inhibitor of an inhibitable enzyme) may result in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drag or by amplification of exogenous (i.e., input) sequences encoding this gene product, or both. "Amplification" is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (t.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template ,, GC821-2

specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out. As used herein, the term "co-amplification" refers to the introduction into a single 5 cell of an amplifiable marker in conjunction with other gene sequences (i.e., comprising one or more non-selectable genes such as those contained within an expression vector) and the application of appropriate selective pressure such that the cell amplifies both the amplifiable marker and the other, non-selectable gene sequences. The amplifiable marker may be physically linked to the other gene sequences or alternatively two separate pieces

10 of DNA, one containing the amplifiable marker and the other containing the non- selectable marker, may be introduced into the same cell. As used herein, the terms "amplifiable marker," "amplifiable gene," and "amplification vector" refer to a marker, gene or a vector encoding a gene which pepnits the amplification of that gene under appropriate growth conditions.

15 As used herein, the term "amplifiable nucleic acid" refers to nucleic acids which may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid" will usually comprise "sample template." As used herein, the term "sample template" refers to nucleic acid originating from a sample which is analyzed for the presence of "target" (defined below). In contrast,

20 "background template" is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a

25 test sample. "Template specificity" is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will _„ GC821-2 ^ "^'

process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Qβ replicase, MDV-1 RNA is the specific template for the replicase (See e.g., Kacian et al, Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acids are not replicated by this amplification enzyme. Similarly, in the case of T7 5 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (See, Chamberlin et al, Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (See, Wu and Wallace, Genomics 4:560 [1989]). Finally,

10 Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences. As used herein, the term "primer" refers to an oligonucleotide, whether occurring

15 naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single

20 stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the

25 primers will depend on many factors, including temperature, source of primer and the use of the method. As used herein, the term "probe" refers to an oligonucleotide (t.e., a sequence of - GC821-2 ^

nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene 5 sequences. It is contemplated that any probe used in the present invention will be labeled with any "reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

10 As used herein, the term "target," when used in reference to amplification methods (e.g., the polymerase chain reaction), refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the "target" is sought to be sorted out from other nucleic acid sequences. A "segment" is defined as a region of nucleic acid within the target sequence.

15 As used herein, the term "polymerase chain reaction" ("PCR") refers to the methods of U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference, which include methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two

20 oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following

25 annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension GC821-2 -"^ -.^

constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of 5 the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified". As used herein, the term "amplification reagents" refers to those reagents

10 (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.). With PCR, it is possible to amplify a single copy of a specific target sequence in

15 genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of

20 primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications. As used herein, the terms "PCR product," "PCR fragment," and "amplification product" refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the

25 case where there has been amplification of one or more segments of one or more target sequences. As used herein, the terms "restriction endonucleases" and "restriction enzymes" ...», » ,' , : „ ,,

GC821-2 . ^

refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

The Present Invention - -In some most particularly preferred embodiments, the present invention finds use in the enzymatic generation of peracids from ester substrates and hydrogen peroxide. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Importantly, the present invention provides means for effective cleaning, bleaching, and disinfecting over broad pH and temperature ranges. In some embodiments, the pH range utilized in this generation is 4-12. In alternative embodiments, the temperature range utilized is between 5° and 90°C. The present invention provides advantages over the presently used systems (See e.g., EP Appln, 87- 304933.9) in that bleaching is possible at the optimum pH of peracid oxidation, as well as providing bleaching at neutral pH, acidic pHs, and at low temperatures. While the present invention is described herein most fully in regard to laundry and fabric care, it is not intended that the present invention be limited to these applications. Indeed, the present invention finds use in various settings, particularly those in which bleaching by peracids and/or hydrogen peroxide are desired, including but not limited to laundry, fabric treatment, pulp and paper processing, personal care applications, disinfection and cleaning of hard surfaces. For example, it is contemplated that the compositions of the present invention will find use in bleaching of pulp, including use in methods such as those set forth in U.S. Patent Nos. 6,569,286, 5,785,812, 6,165,318, and 4,400,237, all of which are herein incorporated by reference. Historically, sodium perborate, and more recently, sodium percarbonate, have been used as bleaching compounds, particularly in European laundry detergents. This GC821-2 ^)

compound decomposes rapidly in aqueous solution to yield hydrogen peroxide (H₂O₂), which is the active bleaching species. As sodium perborate is more active at temperatures above 80°C, and less active in the temperature range of 40-60°C (i.e., wash temperatures that have become most commonly preferred as of the 1950s), bleaching activators have been incorporated into laundry detergents that contain sodium perborate. Indeed, most laundry detergents contain bleaching activators. These activators are compounds with O- or N-bounded acetyl groups that are able to react with the strongly nucleophilic hydroperoxy anion to yield peroxyacetic acid. Since the reacting species is hydroperoxy anion, alkaline pHs are essential for the efficient conversion of these activators to peracids. The peroxyacetic acid is decomposed in weakly basic media to form singlet oxygen (See, Hofmann et al, J. Prakt. Chem., 334:293-297 [1992]). Hydrogen peroxide is a particularly effective bleach at high temperatures (e.g., >40"C) and pH (>10), conditions that are typically used in washing fabrics in some settings. However, as indicated above, cold water washing is becoming more commonly used and results in less effective bleaching by H₂O₂ than use of hot water. To overcome this low temperature disadvantage, detergent formulations typically include bleach boosters, such as TAED (N,N,N'N'-tetraacetylethylenediamine), NOBS (nonanoyloxybenzene sulfonate), etc. These boosters combine with H₂O₂ to form peracetic acid, a peracid species that is more effective than H₂O₂ alone. Although it helps the bleaching capability of detergent, the TAED reaction is only approximately 50% efficient, as only two out of the four acetyl groups in TAED are converted to peracids. Additionally, conversion of TAED into peracetic acid by hydrogen peroxide is efficient only at alkaline pHs and high temperatures. Thus, the TAED reaction is not optimized for use in all bleaching applications (e.g., those involving neutral or acidic pHs, and cold water). The present invention provides means to overcome the disadvantages of TAED use. For example, the present invention finds use in cold water applications, as well as those involving neutral or acidic pH levels. Furthermore, the present invention provides GC821-2 . . ^

means for peracid generation from hydrogen peroxide, with a high perhydrolysis to hydrolysis ratio. The present invention further provides advantages over compositions that contain enzymes such as esterases and lipases) which have very low perhydrolysis to hydrolysis ratios. 5 In addition to its applications in detergents, the present invention provides methods and compositions for the use of peracids in textile bleaching and in various other applications. In some embodiments, the present invention provides one-step methods for textile processing applications, including but not limited to one-step desizing, scouring and bleaching processes (See e.g., EP WO 03002810, EP 1255888, WO 0164993, and US

10 20020007516, all of which are hereby incorporated by reference). As described in greater detail herein, in some embodiments, bleaching involves processing textile material before it is dyed and/or after it is incorporated into textile goods. However, it is not intended that the present invention be limited to any particular regimen of use nor any particular textile material.

15 Furthermore, the peracetic technology of the present invention finds use as an effective bactericide (See, Baldry, J. Appl. Bacteriol., 54:417-423 [1983]). Thus, the present invention provides compositions and methods for the sterilization/disinfection of various objects, including but not limited to medical devices, medical equipment, industrial equipment, and fermenters, as well as any additional object that needs to be

20 sterilized or disinfected. As discussed in greater detail below, during the development of the present invention, the enzyme of the present invention was used in a standard cell kill experiment to demonstrate this suitability. In additional embodiments, the present invention provides compositions and methods suitable for use in biofilm control, such as in cooling towers.

25 Also as described in more detail in the Examples below, the present invention provides many advantages for cleaning and/or sterilization of a wide range of objects, including but not limited to clothing, fabrics, medical devices, etc. In addition, the GC821-2

present invention provides compositions that are effective in cleaning, bleaching, and disinfecting, over a range of wash temperatures and pHs. In additional embodiments, the present invention finds use in degradation of peracids through the perhydrolase peracid degradation activity. In some preferred embodiments, this activity is used in peracid waste clean up applications. Furthermore, the perhydrolase enzymes of the present invention are active on various acyl donor substrates, as well as being active at low substrate concentrations, and provide means for efficient perhydrolysis due to the high peracid:acid ratio. Indeed, it has been recognized that higher perhydrolysis to hydrolysis ratios are preferred for bleaching applications (See e.g., U.S. Patent No. 5,352,594, 5,108,457, 5,030,240, 3974,082, and

5,296,616, all of which are herein incorporated by reference). In preferred embodiments, the perhydrolase enzymes of the present invention provide perhydrolysis to hydrolysis ratios that are greater than 1. In particularly preferred embodiments, the perhydrolase enzymes provide a perhydrolysis to hydrolysis ratio greater than 1 and are find use in bleaching. In addition, it has been shown to be active in commonly used detergent formulations (e.g., Ariel Futur, WOB, etc.). Thus, the present invention provides many advantages in various cleaning settings. As indicated above, key components to peracid production by enzymatic perhydrolysis are enzyme, ester substrate, and hydrogen peroxide. Hydrogen peroxide can be either added directly in batch, or generated continuously "in situ." Current washing powders use batch additions of H₂O₂, in the form of percarbonate or perborate salts that spontaneously decompose to H₂O₂. The perhydrolase enzymes of the present invention find use in the same washing powder batch method as the H₂O₂ source. However, these enzymes also find use with any other suitable source of H₂O₂, including that generated by chemical, electro-chemical, and/or enzymatic means. Examples of chemical sources are the percarbonates and perborates mentioned above, while an - -* f v , ,, GC821-2

example of an electrochemical source is a fuel cell fed oxygen and hydrogen gas, and an enzymatic example includes production of H₂O₂ from the reaction of glucose with glucose oxidase. The following equation provides an example of a coupled system that finds use with the present invention.

Glucose oxidase Glucose + H₂O -- gluconic acid + H2O2 +

10 Perhydrolase H₂O₂ + ester substrate • •-> alcohol + peracid

It is not intended that the present invention be limited to any specific enzyme, as 15 any enzyme that generates H₂O₂ with a suitable substrate finds use in the methods of the present invention. For example, lactate oxidases from Lactobacillus species which are known to create H₂O₂ from lactic acid and oxygen find use with the present invention. Indeed, one advantage of the methods of the present invention is that the generation of acid (e.g., gluconic acid in the above example) reduces the pH of a basic solution to the 20 pH range in which the peracid is most effective in bleaching (i.e., at or below the pKa). Other enzymes (e.g., alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, amino acid oxidase, etc.) that can generate hydrogen peroxide also find use with ester substrates in combination with the perhydrolase enzymes of the present invention to generate peracids. In some preferred embodiments, the ester substrates are selected from one or 25 more of the following acids: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Thus, as described herein, the present ?»-"i ,. f '-

invention provides definite advantages over the currently used methods and compositions for detergent formulation and use, as well as various other applications.

DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting. 10 Cloning and Characterization of M. smegmatis Perhydrolase The cloning of the M. smegmatis perhydrolase (i.e., referred to herein as the "ph T ' gene, which encodes the "Phd" protein; this perhydrolase gene is sometimes herein referred to as the "acf gene and the protein is sometimes referred to as the "Act" protein) 15 of the present invention was based on peptide sequence data from the acyltransferase purified from Mycobacterium parafortuitum (previously known as Corynebacterium oxydans) and published information regarding the 7-aminocephalosporanic acid (7-ACA) arylesterase gene of Agrobacterium radiobacter (Sakai et al, J. Ferment. Bioengineer., 85: 138-143 [1998]). Two peptide sequences from purified M. parafortuitum 20 acyltransferase were found to be similar to internal N- and C-terminal regions of the A. radiobacter 7-ACA-arylesterase (47% and 42% identity respectively). A set of PCR primers was designed based on the amino acid sequence of these internal peptides (designated "AtintF" and "AtintR"). Another set of primers was developed based on the 5' and 3' ends ("ATNcoI" and "ATBamHl") of the A. 25 radiobacter 7-ACA DNA sequence. A single product of the expected size was amplified from M. parafortuitum chromosomal DNA using both sets of primers. The full length product, amplified by the ATNcoI/ ATBamHl primer pair, was cloned into pET16b and . - _ι GC821-2

transformed into BL21 cells (Novagen, Madison,- WI). This clone had a sequence identical to that of the A. radiobacter 7-ACA gene. As it was determined that purified M. parafortuitum perhydrolase was not the 7-ACA acyl esterase, it was concluded that this was not the gene encoding the perhydrolase of the present invention. 5 Thus, efforts were further focused on M. smegmatis for cloning and expression of the perhydrolase of the present invention. To identify the M. parafortuitum gene based on enzyme activity screening, a plasmid library of M. parafortuitum DNA in M. smegmatis was constracted using a plasmid with a promoter to drive expression of cloned genes. Surprisingly, M. smegmatis itself was found to be positive for perhydrolase and

10 acyltransferase activity. Thus, in some instances herein, the perhydrolase is referred to as "ACT" (or "Act"). A protein BLAST search of the M. smegmatis unfinished genome using the sequence of the A. radiobacter 7-ACA identified a 2 kb contig containing an ORF (open reading frame) that encoded a hypothetical protein that was similar but not identical to the 7-ACA protein. Based on this sequence, primers were designed and used

15 to amplify the gene from M. smegmatis (ATCC 10143). By adding an E. coli ribosome binding site upstream of the start codon, a clone that expressed active enzyme was obtained. The vector used was either pCR2.1TOPO or pBluntllTOPO (Invitrogen, Carlsbad, CA), in E. coli Top 10 cells. The gene was expressed constitutively from the plasmid-encoded lac promoter. This enzyme carried out the same reactions as the

20 originally described M. parafortuitum acyltransferase. During the characterization of the perhydrolase of the present invention, standard protein BLAST searches identified a few proteins (<20) with sequence similarity of 30- 80%. This group included the 7-ACA arylesterases from A. radiobacter and other organisms, which have 43% identity with M. smegmatis perhydrolase. All of the

25 identified homologs with at least 40% similarity have a GDS motif very near the N- terminal end. All of the proteins also contain most of the conserved residues which could place them within the suggested GDSL family of lipolytic enzymes (See e.g., Upton and GC821-2 ^'^) f^

Buckley, Trends Biochem. Sci., 20:178 [1995]). However, enzymes mentioned in this paper do not appear on homology searches with the perhydrolase protein. Indeed these proteins have less than 20% similarity with the perhydrolase and its homologs, suggesting that the acyltransferase-related (and perhydrolase of the present invention) enzymes form a subfamily. The natural function of the enzyme of the present invention and the closely related proteins, apart from the 7-ACA arylesterase, have not been biochemically determined. M. smegmatis appears to be the only organism with the acyltransferase/perhydrolase in an operon with a putative penicillin binding protein (PBP). While it is not intended that the present invention be limited to any particular mechanism, this suggests that the enzyme may be involved in cell wall synthesis/structure or modification of molecules taken up from the environment. There are no homologues of the perhydrolase of the present invention that have been identified in M. tuberculosis or M. leprae to date. However, some organisms were determined to have multiple homologues (e.g., S. meliloti). During the development of the present invention, various mutations were made in the M. smegmatis perhydrolase in order to assess its activity. This enzyme contains two cysteine residues, which were hypothesized as potentially forming disulfide bonds, both of which were changed to alanine, in order to determine whether or not the C residues had any effect on the activity of the enzyme. Activity assay results obtained using the transesterification (in aqueous solution) assay described herein indicated that C7A, as well as C77A, and a double mutant (C7A and C77A) were of the same size and specific activity. Many enzymes have the amino acid serine as part of their active site and are therefore referred to, among other designations, as "serine hydrolases." The active site may consist of a catalytic triad of S (serine), D (aspartic acid) and H (histidine).

Examples of such enzymes include, but are not limited to subtilisin (D32-H64-S215), chymotrypsin (H57-D102-S195) and lipases in the alpha/beta hydrolase family (e.g.,

16 GC821-2 -

S126-D176-H206). A typical motif for lipases is the GDSL motif (Upton and Buckley, supra [1995]) in which the S is the active site serine. Since the perhydrolase of the present invention was determined to have a GDSL (amino acids 9-12) motif, the SI 1 was mutated to an A, in order to confirm the involvement of this S in the active site. As indicated in the Examples, the activity assay results indicated that S 11 A had only 1 % of the activity of the wild-type enzyme. Deletion of the C-terminal 25 amino acids also resulted in abrogation of the activity, suggesting that these amino acids either contained a residue involved directly in the active site, and/or that the structure of the protein was affected such that the active site was no longer able to catalyze the reactions. In addition, the predicted active site residues, D192 and HI 95 were mutated to A. Neither mutant had activity, confirming that the active site residues of the perhydrolase of the present invention consist of S 11 , D 192 and H 195. However, it is not intended that the present invention be limited to any particular mechanism, nor is the present invention limited to mutation(s) at any particular active site residues.

Cloning of M. parafortuitum Perhydrolase There were some differences between the N-terminal peptide sequence obtained from the M. parafortuitum enzyme and the N-terminal sequence of M. smegmatis perhydrolase. However, there was a sequence in the C-terminal region of the M. smegmatis perhydrolase identical to the C-terminal peptide sequence of the M. parafortuitum enzyme. Two primers were designed to amplify a partial sequence of the M. parafortuitum perhydrolase gene; the sequence of the reverse primer was identical to the sequence of the corresponding region in M. smegmatis perhydrolase gene, and the sequence of the forward primer was based on smegmatis codon usage. The forward primer, MP5: 5'-

ATGGGTACCCGACGAATTCTGTCCTTCGGTGATTCCCTGACCT-3' (SEQ ID NO: 11) and the reverse primer MPC-intR 5'- <" i,„^ ft _> ι~-u . -i' ,. ,„ .,- GC821-2

GATTCCGTCGACGCCGTCGGTGCTGATCACCGAACCCGCGTCGAAGAACGG- 3 ' (SEQ ID NO: 12). The partial gene was amplified from the chromosome of M. parafortuitum and cloned into pCR2.1TOPO (Invitrogen, Carlsbad, CA). Sequence analysis showed that the enzyme is very similar, but not identical to the M. smegmatis 5 perhydrolase (77% identity). Based on the molecular weights of the monomers of the perhydrolases determined by SDS-PAGE (MP AT: 26 kDa, MSAT: 24 kDa, MP cloned AT: ~18 kDa), the clone from primers made to the internal fragment was determined to be missing approximately 70 amino acids (~8 kDa). The remaining sequence at the 5'- end of the M. parafortuitum gene can be obtained by any of several methods suitable and 10 familiar to those skilled in the art of molecular biology, including, but not limited to, inverse PCR, probing of plasmid/cosmid libraries of M. parafortuitum chromosomal DNA, sequencing of the gene directly from chromosomal DNA (e.g., as performed by Fidelity Systems, Bethesda Maryland).

15 Expression of the M. smegmatis Perhydrolase The perhydrolase is an intracellular protein in its native host. Production of the perhydrolase in non-native hosts may also be done intracellularly. However, in some embodiments, a signal sequence is added to the perhydrolase, which facilitates expression of the perhydrolase by secretion into the periplasm (i.e., in Gram-negative organisms, 20 such as E. coli), or into the extracellular space (i.e., in Gram-positive organisms, such as Bacillus and Actinomycetes), or eukaryotic hosts (e.g., Trichoderma, Aspergillus, Saccharomyces, and Pichia). Of course, these are just a few examples of possible prokaryotic and eukaryotic hosts. It is not intended that the present invention be limited to these specific hosts, as various other organisms find use as expression hosts in the 25 present invention. A variety of commercially available expression systems, including but not limited to pBAD, plac, T7, find use in the expression of the perhydrolase in Gram-negative hosts GC821-2 ^ .,? ^

(e.g., E. co/t). In some embodiments, the same types of promoters find use in another Gram-negative host, Pantoea citrea. To test expression in E. coli two strategies were used: 1) adding an RBS (ribosome binding site) to the 5' end of ϋ ephd gene and cloning the gene into 5 pCRBLUNTϋTOPO (Invitrogen), thus allowing expression directly from the pLac promoter available in that vector; and 2) cloning ύiephd gene under control of the T7 promoter in the plasmid pΕT16b (Novagen). In the latter system, expression of the gene is inducible by addition of IPTG to the growing culture and use of a specific host cell (e.g., BL21(λDE3)pLysS (Novagen)) that contains the λDE3 lysogen encoding the T7

10 RNA polymerase. The first strategy produces a plasmid capable of allowing expression of the perhydrolase protein in other Gram-negative hosts (e.g., P. citrea): To express protein in E. coli or P. citrea using the first strategy, cultures were grown from single, purified colonies at 37°C overnight in L broth plus the appropriate antibiotic (example, kanamycin 50 μg/ml). Expression of the protein was determined by

15 the pNB assay (See, Example 1) after lysis of the cells. Expression of the perhydrolase using the T7 expression system requires induction of the culture with the addition of IPTG (e.g., 100 mmole IPTG added at an OD550 of 0.4). Overnight cultures, inoculated from a single colony, are used to inoculate the expression culture of the desired volume (25 mis to several liters) at an OD550 of 0.1. The

20 expression culture was then grown at the desired temperature (e.g., 25°C, 30°C, 37°C) until an OD550 of 0.4 was reached, after which IPTG was added. Expression was allowed to continue for 3 hours to overnight. Protein expression was monitored by pNB activity assay as described in Example 1. Usually, expression from the T7 system gives a high titer of protein, sufficient for further analysis such as crystallography.

25 Bacillus species are well-known as suitable hosts for expression of extracellular proteins (e.g., proteases). Intracellular expression of proteins is less well known. Expression of the perhydrolase protein intracellularly in Bacillus subtilis can be done --. ,y

using a variety of promoters, including, but not limited to pVeg, pSPAC, pAprE, or pAmyE in the absence of a signal sequence on the 5' end of the gene. In some embodiments, expression is achieved from a replicating plasmid (high or low copy number), while in alternative embodiments, expression is achieved by integrating the 5 desired construct into the chromosome. Integration can be done at any locus, including but not limited to the aprE, amyE, or pps locus. In some embodiments, the perhydrolase is expressed from one or more copies of the integrated construct. In alternative embodiments, multiple integrated copies are obtained by the integration of a construct capable of amplification (e.g., linked to an antibiotic cassette and flanked by direct repeat

10 sequences), or by ligation of multiple copies and subsequent integration into the chromosome. In some embodiments, expression of the perhydrolase with either the replicating plasmid or the integrated construct is monitored using the pNB activity assay (described herein) in an appropriate culture. As with Bacillus, in some embodiments, expression of the perhydrolase in the

15 Gram-positive host Streptomyces is done using a replicating plasmid, while in other embodiments, expression of the perhydrolase is accomplished via integration of the vector into the Streptomyces chromosome. Any promoter capable of being recognized in Streptomyces finds use in driving transcription of the perhydrolase gene (e.g., glucose isomerase promoter, A4 promoter). Replicating plasmids, either shuttle vectors or

20 Streptomyces only, also find use in the present invention for expression (e.g., pSECGT).

Structure of M. smegmatis Perhydrolase The crystal structure of the M. smegmatis perhydrolase was determined to 2.2 Angstroms. The structure confirmed findings with gel filtration sizing columns, that 25 indicated this enzyme is an octamer. The structure of the monomer places the enzyme in the class known as SGNH-hydrolases (See e.g., Molgaard et al, Structure 8: 373-383 [2000]). The active site residues were identified as SI 1-D192-H195, based on GC821-2 ^

homology, confirming the identification of the catalytic triad based on loss of activity in the SI 1 A, D192A, and HI 95 A mutations described above. Figure 3 provides schematics showing the stracture of the M. smegmatis perhydrolase, as well as other serine hydrolases. As indicated, this enzyme has a different stracture than the enzymes shown here (chymotrypsin, subtilisin, and α/β hydrolase). Indeed, the structural analysis of the perhydrolases of the present invention indicates that this group of enzymes has a different form and active site than do these other enzymes. A schematic diagram of the structure of the monomer is illustrated in Figure 4. The structures of four other enzymes in the SGNH-hydrolase family have been solved, namely Aspergillus aculeatus rhamnogalucturonan acetylesterase (RGAE), Bos taurus platelet activating factor (PAF- AH(lb)a), Streptomyces scabies esterase (SsEst) and the thioesterase/Protease I/Phospholipase Li (TAP or Tes) from E. coli. Very little sequence or functional homology is present in these enzymes. Basically, the sequence identity is reserved for the residues involved in the active site and those defining the family. While the overall folding of the enzymes is similar (See e.g., Molgaard et al, supra [2000], for overlaying of structures), there are structural differences. For example, there is a loop covering the active site in SsEst, compared to RGAE and TAP which have active sites that are surface- exposed. The M. smegmatis perhydrolase has an active site that is somewhat buried. The binding residues of the M. smegmatis perhydrolase were identified as Cys7, Asp 10, Serl 1, Leul2, Thrl3, Trpl4, Trpl6, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65,

Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Vall25, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, Ilel53, Phel54, Thrl59, Thrl86, Uel92, Ilel94, and Phel96. These sites were derived from direct observation and by modeling studies to model substrate binding to the enzyme, using methods known in the art. As indicated above, the M. smegmatis perhydrolase was found to be an octamer in the crystalline state. However, it is contemplated to be either a hexamer or octamer in solution. The octamer is seen to be a tetramer of dimers, two molecules are much more H ^■ f„ . -u ι ,.' GC821-2 ^■-

closely and extensively interacting and these are termed the "act transferase" dimers. Several of the conserved sites are found along this dimer interface. For example, residues Trp 14, Arg 27, Arg 56, His 81 and Pro 83, were found to be conserved in natural isolates that have perhydrolase activity and are contemplated to be critical in forming the interface. In addition one other residue, Glu 51 , which is conserved in all but one of the natural isolates (and in that case it is a homologous enzyme) was identified. One additional feature of interest in that in the natural isolates showing perhydrolase activity, all share an insertion of residues 69-81. This region forms a loop that is at the dimer interface. Without this loop, it is believed that much of the dimer interface would be lost and it is likely that dimers and subsequent aggregation would not occur. Thus, there is a correlation of the insertion with the structural aggregation particularly dimer formations and the appearance of perhydrolase activity. However, it is not intended that the present invention be limited to any particular mechanisms. Key residues were found to be associated with desired activity in selected homologs. Indeed, there are several conserved residues that are contemplated to have importance for acyltransferase activity. These include Leu 6, Trp 14, Arg 27, Trp 34, Asp 62, Leu74, Leu 78 His 81, Pro83, Met 90, Lys 97, and Leu 114. In additional analyses, the association of the perhydrolase with carbamate was investigated. The native octamer was determined in space group P4 with unit cell dimensions: a= 98.184 b= 98.184 and c= 230.119 α=90.00 β=90.00 γ=90.00, this crystal diffracted to about 2.0 A. The carbamate-inhibited crystal grew in the space group PI with unit cell dimensions a=67.754, b=80.096, and c=85.974 =104.10° , β=l 12.10°, and γ=97.40° and these crystals diffract to a resolution exceeding 1.0 A. The carbamate was bound in a manner to exploit the interactions between the keto oxygen of the carbamate and residues forming the oxyanion hole, the amide N atoms of Ser 11 and Ala 55 and Asn 94 ND2. The hydrophobic side chain extends along the „. i ι , ι

GC821-2 r^~

hydrophobic surface of the binding site out into the surface opening between pairs of dimers in the octamer stracture. The carbamate moiety direction highlights the pivotal role of the S54V mutation. The hydrophobic moiety passes adjacent to the side chain of ser 54. Mutating the serine side to valine increased the hydrophobicity, and also served as a gatekeeper to prevent hydrophilic nucleophiles (e.g., water) for competing with desired deacylating nucleophiles. The residues surrounding the carbamate moiety on the same and neighboring molecules forming the extended entry are expected to influence the selection of the optimal de-acylating nucleophile. The stracture showed that each monomer was inhibited with carbamate covalently attached. Thus, all octamer active sites were found to be active and functional. The side chain of carbamate resembles the leaving groups of the substrates tested. Thus, the carbamate moiety indicates the access direction for substrate.

M. smegmatis Perhydrolase is an SGNH-Hydrolase The perhydrolase of the present invention has certain components that indicate it is in the SGNH-hydrolase family of enzymes. This family is defined by having the four conserved amino acids SGN and H in four blocks, similar to the blocks that describe the lipolytic family of enzymes (See, Upton and Buckley, supra). In the case of the M. smegmatis perhydrolase, these correspond to SI 1, G52, N94 and HI 95 which correspond to Blocks I II, III and V according to Upton and Buckley (Upton and Buckley, supra) and Molgaard et al. (Molgaard et al, supra). These amino acids are also conserved within the closest sequence homologs of the perhydrolase. As indicated herein, the sequences were aligned using the Alignment program in Vector NTi (Informax, Invitrogen) In the following alignment providing a comparison of homolog sequences, the double underline indicates the residues involved in the active site. AR: Agrobacterium rhizogenes Q9KWA6; RR: Rhizobium rhizogenes NF006; SM: Sinorhizobium meliloti RSM02162; MS: Mycobacterium smegmatis Act; MP: GC821-2

Mycobacterium parafortuitum Phd partial sequence; PD: Prosthecobacter dejongeii RVM04532. The amino acids within the blocks defining the SGNH-hydrolase family are indicated in bold letters. Block I Block II GDS β

AR ( 1) MAESRSI CFGDSIiT GWIPVPESSP TLRYPFEQR mSAMAAALGDGYSIIEEGLSARTTSVED- -PN

RR (1 ) MAESRSILCFGDSIiTWGWIPVPESSP TLRYPFEQRWTGAMAAALGDGYSIIEEGLSARTTSVED-PN

RM (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSP TIiRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSLDD-PN SM (1) -MVEKRSVLCFGDSLTWGWIPVKESSP TLRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSIiDD-PN

MS (1) MAKRILCFGDSLTWGWVPVEDGAP TERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDD-PT

MP GTRRILSFGDSLTWGWIPVEEGVP TERFPrøVRWTGVLADLLGDRYEVIEEGLSARTTTAED- PA

PD (1) MKTILCFGDSNTWGYDPASlOT'APFPlJRHGPEVRWTGVIύyCALGAGFRVIEEGQNGRTTVHED- -PL Block III GxND AR (67) DPRl-NGSAYLPMAI-MHLPLDLVIIIiGTliroTKSYli RTPYEIANGMGKIAGQVLTSAGGIGTPYPAPia^ RR (67) DPRIJSGSAYLPMALASHLPLDLVIILLGTimTKSYFRRTPYEIANGMGK]ΛGQVI,TSAGGIGTPYPAPKLLIVSPPPIAP RM ( 78 ) DARLNGSTYLPMAIASHIiPIiD VI IMLGTNDTKS YFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLWAPPPLAP SM (67) DAliU^GSWLPMAl^SHLPLDLVIIMLGT DTKSYFHRTPYEIANGMGiaVGQVLTCAGGVGTPYPAPKVLVVAPPPLAP

MS (65) DPlSLNGASYlPSCl^raLPLDLVIIMLGTNDTl YFJRR^^

MP (65) DPia-SGSQYLPSCI-WHl-PLDL-WLMLGTlrøTKANFGRTPFDIATGMGVIATQVLTSAGGVGTSYPAPQVLIVAPPP-.GE PD (65) NICRKGIΦYLPACLESHKPl-DLVILMLGTimϋKSTli^^ Block V DGIHF

AR U47) MPDPWFEGMFGGGYEKSLELAKQYKAIJ-SFLi >FLDAGEFVKTDGCppi 'SAETNIT^ ID NO : 14)

RR (147) MPDPWFEGMFGGGYEKSLELAKQYKAI-WFl_lCTroFLDAGEFVKTDGCDGIHFSAETNITIβHAIAAKV^ ID NO : 15)

RM ( 158 ) MPDPWFEGMFGGGYEKSraLSGLYKAI-yJFMlSVEFFAAGDCISTDGip iHlliSAETNIlϊIβltølADKVAALF (SEQ Tfl NO : 16 ) SM U47) MPDPHFEGMFGGGYEKSl^LSGLYKALADFMKVEFFAAGDCISTDGIKIHLSAETNIiaGHAIADKVAALF-- - (SEQ ID NO : 17)

MS (145) MPHPWFQLIFEGGEQj ΕIJUlVYSAl-ASFMlWPFFDAGSVISTDGV piHFT^^ (SEQ ID N0 : 18)

MP 145) LPHPWFDLVFSGGREKTAELARVYSALASFMKVPFFDAGSVISTDGVEGI-- (SEQ ID NO : 19)

PD (144 ) SAMPDLDAKIPHGAARSAEFPRHYKAQAVALKCEYFNSQEIVETSPVEGIttl-EASEHLKLGEALAEKVKVLLG (SEQ ID NO : 20) The primers used to identify homologs for each of the Blocks indicated above are provided below:

Block I (forward 5 '-3) le: acggtcctgtgctttggngaytcnyt (SEQ ID NO:21) If: acggtcctgtgctttggngayagyyt (SEQ ID NO:22) -j. •-•■•" '"- ■' GC821-2 Si

lg: gcggtcctgttctwnggngaytcnyt (SEQ ID NO:23) lh: gcggtcctgttctwnggngayagyyt (SEQ ID NO:24) li: gctcgaaccgtcctctgttttggngaytcnyt (SEQ ID NO:25) lj: gctcgaaccgtcctctgttttggngayagyyt (SEQ ID NO:26) Ik: gctcgaaccgtcctctgtttnggngaytc (SEQ ED NO:27) 11: gctcgaaccgtcctctgttttggngaytcnytn (SEQ ID NO:28) lm: gctcgaaccgtcctctgttttggngaytcnytg (SEQ ID NO:29) 1A: gccaagcgaattctgtgtttcggngaytcnyt (SEQ ID NO:30) IB: gccaagcgaattctgtgtttcggngayagyyt (SEQ ID NO:31)

10 Block III (reverse 5 '-3) 3c: attccgcgcttcagrtcrttnvtncc (SEQ ED NO:32) 3d: attccgcgcttcagrtcrttnwgncc (SEQ ED NO:33) 3e: attccgcgcttcagrtcrttnscncc (SEQ ED NO:34)

15 3f: attccgcgcttcagrtcrttnrancc (SEQ ID NO:35) 3k: attccgcgcttcagrtcrttnrtncc (SEQ ID NO:36) 31: attccgcgcttcagrtcrttnytncc (SEQ ID NO:37) 3m: attccgcgcttcagrtcrttnsgncc (SEQ ID NO:38) 3n: attccgcgcttcagrtcrttnwcncc (SEQ ID NO:39)

20 3o: attccgcgcttcagrtcrttnyancc (SEQ ED NO:40) 3ρ: attccgcgcttgrsrtcrttnrtncc (SEQ ED NO:41) 3q: attccgcgcttgrsrtcrttnytncc (SEQ ED NO:42) 3r: attccgcgcttgrsrtcrttnsgncc (SEQ ED NO:43) 3 s: attccgcgcttgrsrtcrttnwcnnn (SEQ ED NO:44)

25 3t: attccgcgcttgrsrtcrttnyancc (SEQ ID NO:45) 3A: gcgccggaagtaggccttggtrtcrttnvtncc (SEQ ED NO:46) 3B: gcgccggaagtaggccttggtrtcrttnwgncc (SEQ ED NO:47) 3C: gcgccggaagtaggccttggtrtcrttnscncc (SEQ ED NO:48) 3D: gcgccggaagtaggccttggtrtcrttnrancc (SEQ ED NO:49)

30 Block III (forward 5 -3) 3g: cggaattatcatgctgggnabnaayga (SEQ ED NO:50) 3h: cggaattatcatgctgggncwnaayga (SEQ ID NO:51) 3i: cggaattatcatgctgggngsnaayga (SEQ ID NO:52)

35 3j: cggaattatcatgctgggntynaayga (SEQ ED NO:53) 3u: ccggaattatcatgctnggnabnaayga (SEQ ED NO:54) 3v: ccggaattatcatgctnggncwnaayga (SEQ ED NO:55) 3w: ccggaattatcatgctnggngsnaayga (SEQ ED NO:56) 3x: ccggaattatcatgctnggntynaayga (SEQ ED NO:57) tt B^ i ÷!ι <Ui " I' •*' n M -« 'S ._™!^{! i}f - ⁱ GC821-2 ^ -^

Block V (reverse 5 '-3) 5c: acccttagcgtttggrtgnrtnccrtc (SEQ ED NO:58) 5d: atccttagcgtttggrtgnavnccrtc (SEQ ED NO:59) 5 5e: aatcttagccgtgrrrtgnrtnccrtc (SEQ ED NO:60) 5f: aatcttagccgtgrrrtgnrcnccrtc (SEQ ED NO:61) 5g: aatcttagccgtgrrrtgntmccrtc (SEQ ED NO:62) 5h: ccgctggtcctcatctggrtgnrtnccrtc (SEQ ED NO:63) 5i: ccgctggtcctcatctggrtgnrcnccrtc (SEQ ED NO:64) 10 5j: ccgctggtcctcatctggrtgntmccrtc (SEQ ED NO:65) 5k: ccgctggtcctcatcraartgnrtncc (SEQ ED NO:66) 5A: cgattgttcgcctcgtgtgaartgnrtnccrtc (SEQ ED NO:67) 5B: cgattgttcgcctcgtgtgaartgnrcnccrtc (SEQ ED NO:68) 5C: cgattgttcgcctcgtgtgaartgntmccrtc (SEQ ED NO:69) 15

As described in greater detail herein, the sequence and stracture results are supported by the activity data that indicate the perhydrolase enzymes of the present 20 invention differ from lipolytic enzymes known in the art.

Identification of Homologs As well known in the art, proteins with a desired activity may be identified in several ways, including but not limited to: 1) searching available databases for proteins 25 with sequence homology (30-100%); 2) screening environmental isolates for the desired activity; and 3) examining type strains from ATCC of the genus identified to have activities (e.g., Mycobacterium and Corynebacterium, as described herein in particular embodiments). By doing a standard protein-protein BLAST search, several homologs were 30 identified from fully or partially sequenced genomes. From the known gene sequence, several homologs were amplified by PCR from the chromosome of the parent organism

and cloned into a pET expression vector, essentially as described for the cloning ofphd from M. smegmatis into pET16b. Homologues identified by this BLAST search included: Agrobacterium rhizogenes Q9KWA6, A. rhizogenes Q9KWB1 A. tumefaciens Q8UFG4, A. tumefaciens Q8UAC0 (now AgrL, identical to 7-ACA arylesterase), A. tumefaciens Q9ZI09, A. tumefaciens (radiobacter)ACA, Prosthecobacter. dejongeii

RVM04532 , Rhizobium. loti Q98MY5 , R. meliloti Q92XZ1, R. meliloti Q9EV56, R. rhizogenes NF006, R. rhizogenes NF00602875, R. solanacerarum Q8XQI0, Sinorhizobium meliloti RSM02162, S. meliloti RSM05666, Mesorhizobium loti RMLO00301, ,4. rhizogenes Q9KWA6, and A rhizogenes Q9KWB1. Based on these results, a homology tree of proteins with sequence homology (20-

80%) to M. smegmatis perhydrolase was generated. As shown in Figure 2, an enzyme in the family of lipolytic enzymes described by Upton and Buckley (supra) is that of V. mimicus. This phylogenetic tree was generated using the alignment program in Vector NTi (Informax, Invitrogen). The green arrow indicates M. smegmatis perhydrolase, the red arrow indicates A. radiobacter 7-ACA arylesterase, the blue arrow indicates E. coli

TAP, and the black arrow indicates A. aculeatus RGAE. As further indicated in Figure 2, the perhydrolase is not closely related to this enzyme. The perhydrolase and its closest relatives, Prosthecobacter dejongeii RVM04532, R. rhizogenes NF006, A. rhizogenes Q9KWA6, R. meliloti Q92XZ1, S. meliloti RSM02162, A. rhizogenes Q9KWB 1 and R. rhizogenes NF00602875 come off their own branch (t.e., a branch that is different from the 7-ACA arylesterase-like proteins and the RGAE/TAP-like proteins). However, it is contemplated that some additional, more distantly related homologs will find use in the present invention due to perhydrolase activity or will serve as a suitable backbone for modification to the desired perhydrolase activity. In addition to the sequence and homology analysis, environmental isolates were grown on a rich medium (N-MISO: g 1: glucose 10 g, yeast extract 10 g, KNO₃ 1.5, GC821-2 ' ^ »

KH₂PO₄ 3.4 g, NaH₂PO₄.H₂0 3.4 g, Salt Solution C 10 ml [Salt Solution C: g/1: MgSO₄7H₂O 25, FeSO₄7H₂O 2.8, MnSO H₂O 1.7, NaCl 0.6, NaMoSO .2H₂O, ZnSO₄.7H₂O 0.06, in 0.1N HC1]), assayed and those positive for the transesterification reaction were purified as described in the Examples. This is one of the screening methods that can be used to identify perhydrolase These data show that the present invention finds use in identification of additional enzymes with the desired perhydrolase activity.

Additional Investigations of Homologues In addition to the above analyses, an enzyme library of novel "GDSL-type" esterases which are homologous to the prototype M. smegmatis perhydrolase was created. In order to identify new "GDSL"-type esterases, a sequence homology based screening procedure was established and used to screen libraries set up from complex metagenomic DNA (at BRAIN). An enzyme library comprising 19 "GDSL"-type esterases (See, below) was developed. The sequences in this library were:

S248_M2bBll (DNA) ATGTTCGCGCTTTGCACGGCCGCGTCAGCGGCCCCCGATCGCACCGTCGTCTT

TTTTGGGGACAGCCTGACCGCGGGGTACGGCCTCGATGACCCGCAGACCCAG TCCTACCCGGCCAGGATCCAGGAGAAGGTCGACGCCGCGGGCCTGCGCTGGA AGGTCGTGAATGCCGGCCTCTCGGGCGAGACGAGCGCCGGCGGCCTGCGGCG GGTCGACTGGGTGCTCGGCCAGCACATCGACGCCTTTGTCCTGGCGCTTGGCG CCAACGATGGCCTGCGGGGGATCGACCCCCAGGTCACGAGGGCCAATCTCCA

GGAGATCATCAACCGGGTCCGCTCCCGGTGGCCCCGCGCGGCGATCGTCATC GCCGGGATGAAAATGCCCCAGAGCATGGGACAGGACTACGCCGCGAATTTTG ACCGGATCTTCCCCGGTCTCGCCGCGAGGAATTCGGCCACGCTCATCCCCTTT CTATTAGAAGGGGTCGCCGCCCATCCTAGCCTCAACCAAGGCGACGGCATCC ACCCGACGGCCGCCGGGGACGCACTCGTTGCAGGGACCGTGTGGACGTACCT

GCTTCCGATCCTGCGGTCAGCACACTAA (SEQ ED NO:70) GC821-2 ^

S248_M2bBll (Amino Acid)

MFALCTAASAAPDRTWFFGDSLTAGYGLDDPQTQSYPARIQEKVDAAGLRWK WNAGLSGETSAGGLRRVDWVLGQHBDAFVLALGANDGLRGBDPQVTRANLQEE NRVRSRWPRAAIVIAGMKMPQSMGQDYAANFDRIFPGLAARNSATLE'FLLEGV AAHPSLNQGDGfflPTAAGDALVAGTVWTYLLPILRSAH (SEQ ED NO:71)

S248_M40cD4 (DNA) ATGCGCTTTGCTAAGCTCACTGCCGTCATCTTTGCCCTGATAGTCTTGCACAG CCCCCTTGCCGCCGCCGCGCCGCCCACCGTGATGGTGTTTGGCGACAGTCTGA CCGCCGGGTTGGGATTGCCGGCCGATGCTGCATTTCCGGCGCAGCTCCAGGC AAAGCTGCACGATATGGGTATCCTGCAGAAATCGCCGCGCGCGCCACCTCGG GGCAAACGACGGCCGGCGGGTTGGCGAGCCTTGCGGATGCGCTGGCCGCAA AGCCGGATTTGGTGATCCTCGAACTCGGCGCCAATGACATGCTGCGCGCGGT CGATCCGGCCAGCGTGCGCGCCAATCTCGATGCAATGATGACGAAAATCCAG

GCGAGCGGCGCTAAACTGCTGCTGACCGGAATGCAGGCGGCGCCCAATTGGG GCGAGGACTATAAGCACGATTTCGACCGCCTTTATCCCGAGCTTGCGAAGGC GCACGGGGTGACGCTTTATCCATTCTTTCTTGATGGGGTGGCGCTGGACCCGG CGCTGAACCAGGCGGATGGAATGCACCCGAACGCCAAGGGGGTCGCCGTGA TCGTCGACCGTATCGCGCCCGTCGTCGCCAAGATGCTGAGAGGCCAGTCATA A (SEQ ED NO:72)

S248_M40cD4 (Amino Acid)

MRFAKLTAVIFALIVLHSPLAAAAPPTVMVFGDSLTAGLGLPADAAFPAQLQAKL HDMGΠΆEIAARATSGQTTAGGLASLADALAAKPDLVILELGANDMLRAVDPAS VRANLDAMMTKIQASGAKLLLTGMQAAPNWGEDYKHDFDRLYPELAKAHGVT LYPFFLDGVALDPALNQADGMHPNAKGVAVIVDIΑAPWAKMLRGQS (SEQ ED

NO:73)

S248_M44aA5 (DNA)

ATGATCGCATGGCTTACCGGATGCGGCAGCGCAAAGACGCAACCGCAGCCCG CAAGTTCCATCCCGCCATCCAGTATTCCAGCAACCGCAAAACCTGCGACAAC GGATATCAGACCGATCATCGTTGCTTTCGGCGACAGCCTGACTGCAGGATAC GGCGTCAGTAGTGAACAAAGCTATCCGGCCAATCTTCAACGCGATCTGGATG

CGCGTGGATATCATGCCCACGTCATCAACGAAGGCATCAGCGGCAACACATC GAAAGACGGCGTTCTCAGGGCCCAGGCGATTGCGGCACTCCATCCGGCTGTC GTCATCGTTGCCTTCGGCGGCAACGACGGTCTGCGTGGCCTCCCCATCGGAG ACACGGAAATGAATCTGGCAACGATCATCTCAACCATGCAGCATGCCCATGC CAAGGTAATTTTAGGCGGAATTACTTTGCCTCCCAACTATGGCAGCGAATAC f ; .,_|t , , „, .. . - „

GC821-2

ATCGCCAAATTCAATGCGATCTATAAAAAGCAGGCAGCCGCGTATCATGTGC CCCTGCTGCCCTTCATGCTGAAGGGGGTGTATGGCGTGCCCGGTTCCATGCAG AGCGACGGCATCCATCCGACCGCCAAGGGCTGCCAGCAAGTGGCCAGAAACT TCCTGCCCTTGTTATTGCCGCTCCTGCACAAATCAGGGAAGAAATCCATGGAG TCGAAAGCATTGTCTCGACGTCATTAA (SEQ ED NO:74)

S248_M44aA5 (Amino Acid)

MIAWLTGCGSAKTQPQPASSPPSSffATAKPATTDIRPIIVAFGDSLTAGYGVSSEQ SYPANLQRDLDARGYHAHVINEGISGNTSϊαDGVLRAQAIAALHP AWIVAFGGN DGLRGLPIGDTEMNLATESTMQEϋ^AKVILGGITLPPNYGSEYIAKFNAIYKKQA AAYHVPLLPFMLKGVYGVPGSMQSDGIHPTAKGCQQVARNFLPLLLPLLHKSGK KSMESKALSRRH (SEQ BD NO:75)

S261 M2aA12 (DNA)

ATGAAAAACATCCTTGCATTTGGCGACAGTCTGACCTGGGGTTTTGTGGCCGG

ACAGGATGCGCGCCATCCGTTTGAAACCCGCTGGCCAAACGCATTGGCGGCC GGCCTTGGGGGCAAAGCCCGCGTAATTGAAGAGGGTCAGAACGGCCGCACT ACGGTGTTCGACGATGCCGCCACCTTCGAATCTCGAAATGGCTCGGTGGCATT GCCGCTGCTACTGATCAGCCACCAGCCGTTGGACCTGGTAATCATCATGCTCG GCACCAATGACATCAAGTTTGCCGCCCGCTGCCGCGCCTTTGATGCTTCAATG GGCATGGAACGGCTGATCCAGATCGTCAGAAGTGCCAACTACATGAAGGGCT ACAAGATACCTGAAATCCTCATCATATCGCCGCCCAGCCTCGTGCCGACGCA GGATGAATGGTTCAACGACCTCTGGGGCCATGCCATCGCCGAGTCAAAACTC TTCGCCAAGCACTACAAGCGCGTGGCCGAAGAACTGAAAGTGCATTTCTTTG ATGCAGGCACGGTGGCCGTCGCCGACAAGACCGACGGCGGACATCTCGATGC TGTGAATACTAAAGCCATTGGCGTCGCATTGGTGCCGGTGGTGAAATCAATA CTCGCTCTCTAA (SEQ ED NO:76)

S261_M2aA12 (Amino Acid)

MKNILAFGDSLTWGFVAGQDARHPFETRWPNALAAGLGGKARVIEEGQNGRTT VFDDAATFESRNGSVALPLLLISHQPLDLVIIMLGTNDπaFAARCRAFDASMGMER LIQIVRSANYMKGYKIPEILIISPPSLVPTQDEWFNDLWGHAIAESKLFAKHYKRVA EELKNHFFDAGTVANADKTDGGHLDAVNTKAIGVALVPWKSILAL (SEQ ED NO:77) %» i ,.''^u i ' 'V -,; GC821-2

S279_M70aE8 (DNA) ATGCCGAAAATAGCCAAACTCGCGCCGTCGGATGTGATCGTAGCTTTCGGCG ACAGTCTGACGTTCGGCACCGGCGCAACGGAAGCGGAGAGTTATCCCATCGT GCTCGCACAATTGATCGGTCGCACCGTGGTGCGCGCGGGTGTGCCGGGTGAG 5 GTAACCGAAGGCGGGCTTGCGCGCCTGACCGACGTTATCGAAGAACACAAGC CGAAGCTGATTATTGTTTGCCTGGGCGGCAACGACATGCTGCGCAAGGTCCA GGAAGACCAGACCCGCGCCAATTTGCGCGCCATTATTAAAACCATCAAGGCG CAAGGCATCGCCGTGGTACTGGTCGGTGTGCCGAAGCCCGCGCTGGTGACCA GTGCGCCGCCGTTCTACGAGGAGATCGCCAAAGAGTTCGGTATCCCTTACGA 10 AGGCAAGATTGTTACCGACGTGTTGTACCAACGCGATCAGAAATCCGATTCC ATACATCCCAATGCCAAAGGCTATCGGCGCATGGCCGAAGCGATAGCCACGC TGCTGAAAAAATCCGGAGCCATTTAA (SEQ ED NO:78)

15 S279:M70aE8 (Amino Acid) MPKIAKLAPSDVIVAFGDSLTFGTGATEAESYPIVLAQLIGRTVVRAGVPGEVTEG GLARLTDVIEEHKPKLΠVCLGGNDMLRKVQEDQTRANLRAΠKTD AQGIAWLV G .PALVTSAPPFYEEIAKEFGIPYEGKIVTDVLYQRDQKSDSIHPNAKGYRRMA EAIATLLKKSGAI (SEQ ED NO:79) 20

S279_M75bA2 (DNA) ATGGAACGGACCGGCCGCGCTGGCGATCGGTGTCGGCGTGGGGCTGGCGAGC 25 CTGAGCCCGGTCGCGCTGGCGACGCCGCCGCGGGGCACCGTGCCGGTGTTCA CCCGATCGGGGACAGCCTGACGGACGAGTATTTTGAGCCGTTCTTCCAGTGG GGGTTCTGCGGGAAGTCGTGGGCCGAGATTTTGGTGGAGACGGGGCGGGCGA GCATGGGCCCGACGGCGCAGCAGGCGGGGATCAGCGAGCCGGAGGGATGGT CGGATCCGCGGAACACGGGGTATCAGCACAACTGGGCGCGGTACTCGTGGAG 30 CTCCTCAGACGCGCTGACCGAGGAGTCGCCGGGGGCGACGCTGAGCGTGCTG CTTGGGGCGGAGTACGCGGTGGTGTTCATTGGGACCAACGACTTCAATCCGT CGTGGCCGGCGTATCAGAGCGTGTATCTGAGCCAGTGGAGCGACGAGCAGAT CGACACGTACGTGAACGGGGTGGTGCAGAACATCGCGCAGATGGTGGACTCG CTGAAGTCGGTCGGGGCGAAGGTGGTGCTTGCGCCGCCGGTGGATTTTCAGT 35 TCGCGGGGTTCCTGCGGAACTCATGCCCGGATCCGATGCTGCGCGAGCAGGC GGGTATTCTGACACGGAAGTGCCACGACCGGGTGCGGTCGATGGCGCGGCAG AAGCACGTGGTGTTCGTGGACATGTGGCGGCTGAACCGCGATTTGTTCGGCA ACGGGTTCGCGATCAGCTACGGCCTTCGGAACACGGTGCGCGTGGGGGACTC GGAGATCGGGCTGCAACTGGCCGGGCTGACGGGATCGGCGGGGCTGGTTCCG 40 GACGGGATCCATCCGCAGCGGGTGGTGCAGGGGATCTGGGCGAATGCGTTCA „, ₁, . .

I-_' ¹ li u aiu ' ^, 'ii"i.uι ""ii"^{, „};^;iι ^β GC821-2 ^

TCGTGGGTCTGAACGCGCATGGGGCGAACATCGCGCCCATCGGCGAGGCGGA GATGTGCGCGATGGGGGGGGTCGTGTACGGGGGAACGGACACGCTGGCGAA CTTCCTGCCGCCGGTCGCGGGCTACGTGGAGGACTTCCGCAACGCGGGGGAC TTCGTGTGCACGGCGGACTTCAACCATGACCTTGGCGTGACGCCGACGGACA TCTTCGCGTTCATCAACGCGTGGTTCATGAATGATCCCTCGGCGCGGATGAGC AACCCGGAGCACACGCAGATCGAGGACATCTTCGTGTTTCTGAATCTGTGGC TGGTGGGGTGCTAA (SEQ ED NO:80)

10 S279JM75bA2 (Amino Acid) MERTGRAGDRCRRGAGEPEPGRAGDAAAGHRAGVHPIGDSLTDEYFEPFFQWG FCGKSWAEILVETGRASMGPTAQQAGISEPEGWSDPRNTGYQHNWARYSWSSS DALTEESPGATLSVLLGAEYAWFIGTNDFNPSWPAYQSVYLSQWSDEQEDTYVN GWQNIAQMVDSLKSVGAKWLAPPVDFQFAGFLRNSCPDPMLREQAGILTRKC 15 HDRVRSMARQKHVVFVDMWRLNRDLFGNGFAISYGLRNTVRVGDSEIGLQLAG LTGSAGLVPDGIHPQRVVQGIWANAFEVGLNAHGANIAPIGEAEMCAMGGWYG GTDTLANFLPPVAGYVEDFRNAGDFVCTADFNHDLGVTPTDIFAFINAWFMNDP SARMSNPEHTQIEDEFVFLNLWLVGC (SEQ ED NO:81) 20 M091_M4aEl l (DNA) ATGAAGACCATTCTCGCCTATGGCGACAGCCTGACCTATGGGGCCAACCCGA TCCCGGGCGGGCCGCGGCATGCCTATGAGGATCGCTGGCCCACGGCGCTGGA 25 GCAGGGGCTGGGCGGCAAGGCGCGGGTGATTGCCGAGGGGCTGGGTGGTCG CACCACGGTGCATGACGACTGGTTTGCGAATGCGGACAGGAACGGTGCGCGG GTGCTGCCGACGCTGCTCGAGAGCCATTCGCCGCTCGACCTGATCGTCATCAT GCTCGGCACCAACGACATCAAGCCGCATCACGGGCGGACGGCCGGCGAGGC CGGGCGGGGCATGGCGCGGCTGGTGCAGATCATCCGCGGGCACTATGCCGGC 30 CGCATGCAGGACGAGCCGCAGATCATCCTCGTGTCGCCGCCGCCGATCATCC TCGGCGACTGGGCGGACATGATGGACCATTTCGGCCCGCACGAAGCGATCGC CACCTCGGTGGATTTCGCTCGCGAGTACAAGAAGCGGGCCGACGAGCAGAAG GTGCATTTCTTCGACGCCGGCACGGTGGCGACGACCAGCAAGGCCGATGGCA TCCACCTCGACCCGGCCAATACGCGCGCCATCGGGGCAGGGCTGGTGCCGCT 35 GGTGAAGCAGGTGCTCGGCCTGTAA (SEQ ED NO:82)

M091_M4aEl l (Amino Acid) MKTILAYGDSLTYGANP GGPRHAYEDRWPTALEQGLGGKARVIAEGLGGRTT 40 VHDDWFANADRNGARVLPTLLESHSPLDLIVIMLGTNDIKPHHGRTAGEAGRGM GC821-2

ARLVQIERGHYAGRMQDEPQELVSPPPΠLGDWADMMDHFGPHEAIATSVDFARE YKKRADEQKVHFFDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL

(SEQ ED NO:83)

Estl05 (DNA)

ATGCGCACGCTTCACCGAAGCCTGCTCGCAAGCGCGGCCGCGCTTTTTCTAGC

GGCATCCGGCAACGCAACGGCGCAGTTCTCGAACGTCTATTTCTTCGGCGAC AGCCTGACCGACGCGGGTTCCTTCAAGCCTGTGCTGCCTCCTGGTAC AGGATT ATTCACGACGAATCCCGGCCCGGTATGGCCGCAGGTATTCGGGGCGAACTAC GGCGTCGCGGTGACGCCCGCAAACCAGGGTGGGACCGATTATGCGCAGGGTG GCGCGCGCGTGACGAGCCTGCCTGGCGTTCCGACGTCGCAGCCGACCGGCAG CGCGGTACCGATCGCTACGCAGATTTCGCAGTTCCTCGGCTCGGGTCCGGCG GATCCGAACGCATTCTATTCGGTGTGGGGCGGCGCGAACGACATCTTTTTCCA

GCTGGGGTTGGCGCAGGCGGGCATGGCGACGCCGGCGCAGGTCCAGTCGGCC GTCGGCTTGGCCGCGGTCCAGCTGGCGCAGGCAACTGCGGCGCTCAACGCCA GCGGCGCGCGATTCATCACGGTTATCAACGTGCCGGACATCGGTAAAACGCC GTTCGGCGTCGGCTCCGGTCAAGGAGCGCAGATCACCGCTCTGTCGTCTTTCT TCAACAGCACGCTGTTCGGCGCGCTCGACGCCACGGGCATCCAGACGATGCG CGTGAACGGGTTCGCGGTGCTGAACGAGGTGGTCGCGGACCCGGCGGCTTAT GGCTTCGCGAATGCATCAACGCCAGCGTGCGGGGCCACGCCATCGCTCGTCT GCACGTCGGCGAACTTCGTCACGCCCTTGGCCGCGCAGACCTTCCTCTTCGCA GACGGCGTTCACCCCACCACGGCCGGGCACGCCCTCATCGCCCAAGCGGTCC AGGCGATGATCACCGGTCCCCAACAGATGGCGGCGTTGGGCGACGCCCCGCT CGCCGTCGAGCAGGCCAACTTCCGCGCGCTCGACAACCGCATGTGGTCGAGC CTCAATGCGCCGCGCAGCCCGGGCAAGCTCCAGGGTTGGGCGGCCTACGACT ACAGCCACACGGACCTGCAGGCGGGACCGACCAATGGCAGCGGACACATGA ACACCGTTGCGGTCGGGGTCGACATGAAAGTCTCCGATCATATGCTCGCCGG CGCGATGTTCGGCTATACCAACACCAAGGGCGACTTCGGCGGCCCCGGCGGC GGATACACACTGAAGCAGCCTGTGGGCACTGCCTATGCGGGTTACGGCGTGG GCCCTTGGTATGTCGGCGCGACGCTCGGCACAGGTGGCCTCGACTACTCGGA CGTCACGCGCGCCATCCCGCTTGGCTTGGCGGTTCGCACCGAGAGCGCCGAG GCCCGAGGCTACGAGTTCACGGGCCGGATCCTCGGCGGCTACTGGTTCACGA TGCGCGACCTGATGCACGGGCCGTACGCGCGTCTCGCGTGGACGAAGGCCGT

CGTCAAGCGGTTTTCCGAGGAGAGCACCGACAGCACGGCGTTGAACTACGAC AGGCAGGAGCGCAAGCAACTGCTGTGGAGCCTCGGATGGCAACTCGCCGGC AACGTCGGCAGCATCCGTCCCTACGCGCGGGCGACCTGGGAGATCGACTCCA AGGATCAGGACCGCAGCGTTGGCGCATCGTCGGTCACGCTGGGCGGCTTTTA CAGTGTTCCGGTCGCGAAGCCGGACAATAGCTATGCGCTCTTCAGCCTCGGC GC821-2 ^ 4

GCGAGTACCGAGCTCGGGAGCGTCACCGGGTTTGTCGCGGGCTCGGCCACCG CAGGCCGGGCGGATGCCAACTATTGGGCGGTCACGGTCGGCCTGCGGATGCC GTTGTAG (SEQ ED NO:84)

Estl05 (Amino Acid)

MRTLHRSLLASAAALFLAASGNATAQFSNVYFFGDSLTDAGSFKPVLPPGTGLFT TNPGPVWPQVFGANYGVAVTPANQGGTDYAQGGARVTSLPGVPTSQPTGSAVPI ATQISQFLGSGPADPNAFYSVWGGANDIFFQLGLAQAGMATPAQVQSAVGLAAV QLAQATAALNASGARFITVINVPDIGKTPFGVGSGQGAQITALSSFFNSTLFGALD ATGIQTMRVNGFAVLNEWADPAAYGFANASTPACGATPSLVCTSANFVTPLAA QTFLFADGVHPTTAGHALIAQAVQAMITGPQQMAALGDAPLAVEQANFRALDN RMWSSLNAPRSPGKLQGWAAYDYSHTDLQAGPTNGSGHMNTVAVGVDMKVS DHMLAGAMFGYTNTKGDFGGPGGGYTLKQPVGTAYAGYGVGPWYVGATLGT GGLDYSDVTRAE^»LGLAVRTES AEARGYEFTGRILGGYWFTMRDLMHGPYARLA WTKAWKRFSEESTDSTALNYDRQERKQLLWSLGWQLAGNVGSEIPYARATWE EDSKDQDRSVGASSVTLGGFYSVPVAKPDNSYALFSLGASTELGSVTGFVAGSAT AGRADANYWAVTVGLRMPL (SEQ ED NO:85)

Estll4 (DNA)

ATGGGGCGATCGAGAGTTCTGAAGGCTGTTTTCCTGGTGGCGTGCCTTGTGGG

TCGGCTCGCGGCGCATGCCGAGGCGTCGCCCATCGTGGTCTACGGCGATAGC CTCTCTGACAACGGCAATCTGTTTGCGCTCACCGGCGGTGTCGCGCCGCCCTC GCCGCCGTACTTCAACGGACGGTTTTCTAATGGCCCGGTGGCCGTGGAGTATC TCGCGGCCGCGCTGGGATCTCCGCTGATCGATTTCGCGGTCGGCGGGGCGAC GACCGGCCTCGGCGTCAACGGCGATCCCGGTGGTTCGCCGACGAGTCTCGGC GCGGCGGGATTGCCGGGGCTTCAGACGACATTCGCCGCCACGCAAGGCACGC TGGGTCCGTACGTTGGTGGTCTCTTCGTGGTGTGGGCGGGTCCGAACGACTTC TTGTCGCCCTCGCCGCTTGACACGAACGCTTTTCAGATTGCGAACCGGGCCGT GTCCAACATCCTCGGCGTGGTGGCATCACTTCAGGCACTCGGCGTCGAGCGC ATCCTCGTCCCCGGCATGCCCGATCTCGGTCTGACGCCCGCTCTTCAGCCCAT CGCAGGCGCAGCCACCGCGTTCACCGATTTGTTCAACTCGATGCTGCGCGCG GGCTTGCCGAACGACGTGCTGTACCTGGACACGGCGACAATCTTCCGATCGA TCGTGGCAGACCCTGGGGCCTACGGCTTGACCAACGTGACCACGCCGTGCCT GATTGGTGCGACCGTCTGCGCGAATCCGGATCAGTACCTGTTCTGGGATGGT ATTCATCCTACGACGGCGGGGCACGCGATCTTGGGCAATGCCCTCGTCGCCC AGGCAGTCCCCGAGCCCGCGACCATGGTGCTCGTGCTGACGGGTCTGTCCAT GCACGTGATTGCGCGCCGGCGGCGGGCGTAA (SEQ ID NO:86) t ιr

Estll4 (Amino Acid) MGRSRVLKAVFLVACLVGRLAAHAEASPIWYGDSLSDNGNLFALTGGVAPPSP PYFNGRFSNGPNAVEYLAAALGSPLEDFAVGGATTGLGVNGDPGGSPTSLGAAGL 5 PGLQTTFAATQGTLGPYVGGLFWWAGPNDFLSPSPLDTNAFQIANRAVSNILGV VASLQALGVERILVPGMPDLGLTPALQPIAGAATAFTDLFNSMLRAGLPNDVLYL DTATffRSIVADPGAYGLTNVTTPCLIGATVCANPDQYLFWDGIHPTTAGHAILGN ALVAQAVPEPATMVLVLTGLSMHVIARRRRA (SEQ ED NO:87)

10 Sinorhizobium meliloti Smel (SMal993) (DNA) ATGACAATCAACAGCCATTCATGGAGGACGTTAATGGTGGAAAAGCGCTCAG TACTGTGCTTTGGGGATTCGCTGACATGGGGCTGGATTCCGGTGAAGGGATC CTCACCGACCTTGCGCTATCCCTATGAACAACGGTGGACCGGCGCAATGGCC

15 GCGAGGCTTGGCGACGGTTACC AC ATC ATCGAAGAGGGGCTGAGCGCCCGC A CCACCAGCCTCGACGACCCCAACGACGCGCGGCTCAACGGCAGCACCTACCT GCCCATGGCACTCGCCAGCCACCTCCCACTCGACCTCGTCATCATCATGCTGG GCACGAACGACACGAAATCCTATTTCCACCGCACGCCTTACGAGATCGCCAA CGGCATGGGCAAGCTAGTCGGCCAGGTGCTGACCTGCGCCGGTGGCGTCGGC

20 ACGCCATATCCCGCGCCGAAGGTGCTTGTCGTCGCTCCGCCGCCGCTCGCGCC GATGCCCGACCCGTGGTTCGAAGGCATGTTCGGCGGCGGCTACGAGAAGTCG AAGGAACTCTCCGGCCTCTACAAGGCGCTTGCCGATTTCATGAAGGTCGAGT TTTTCGCCGCCGGTGATTGCATTTCCACCGATGGGATCGACGGCATTCACCTC TCGGCGGAAACCAACATCAGACTCGGGCACGCGATCGCGGACAAAGTTGCG

25 GCGTTGTTC (SEQ ED NO:88)

Sinorhizobium meliloti Smel (SMal993) (Amino Acid) MTINSHSWRTLMVEKRSVLCFGDSLTWGWB?VKGSSPTLRYPYEQRWTGAMAA 30 RLGDGYHIIEEGLSARTTSLDDPNDARLNGSTYLPMALASHLPLDLVEMLGTNDT KSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLWAPPPLAPMPDPWF EGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLG HAIADKVAALF (SEQ ED NO:89)

35 Sinorhizobium meliloti Smell (Q92XZ1) (DNA) ATGGAGGAGACAGTGGCACGGACCGTTCTATGCTTCGGAGATTCCAACACTC ACGGCCAGGTACCTGGCCGCGGACCGCTTGATCGCTACCGACGCGAACAGCG CTGGGGCGGTGTTCTGCAAGGCCTGCTCGGCCCGAACTGGCAGGTTATCGAA

40 GAAGGCCTGAGCGGACGCACGACCGTGCATGACGATCCGATCGAAGGTTCGC TCAAGAACGGCCGGACCTATCTGCGCCCCTGTCTGCAGAGCCATGCACCACT GC821-2 ^>-~\

CGACCTTATCATCATTATGCTCGGCACCAATGACCTGAAGCGGCGCTTCAACA TGCCACCGTCCGAGGTCGCAATGGGCATCGGCTGTCTCGTGCACGATATCCG AGAACTCTCGCCCGGCCGGACCGGCAACGATCCCGAAATCATGATCGTCGCC CCGCCGCCGATGCTGGAAGATCTCAAGGAATGGGAGTCGATTTTCTCAGGCG CACAGGAAAAATCTCGCAAGCTGGCGCTGGAGTTCGAGATAATGGCGGATTC TCTGGAGGCGCATTTCTTCGACGCCGGTACGGTCTGCCAGTGTTCGCCGGCCG ATGGCTTCCACATCGACGAGGATGCCCACCGCCTGCTCGGCGAGGCTCTCGC CCAGGAAGTGCTGGCGATCGGGTGGCCCGATGCGTAA (SEQ ED NO:90)

10 Sinorhizobium meliloti Smell (Q92XZ1) (Amino Acid) MEETVARTVLCFGDSNTHGQVPGRGPLDRYRREQRWGGVLQGLLGPNWQVIEE GLSGRTTVHDDPIEGSLKNGRTYLRPCLQSHAPLDLIEMLGTNDLKRRFNMPPSE VAMGIGCLVHDIRELSPGRTGNDPEIMIVAPPPMLEDLKEWESIFSGAQEKSRKLA LEFEIMADSLEAHFFDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA

15 (SEQ ED NO:91)

Sinorhizobium meliloti SmeEI (Q9EV56) (DNA)

20 ATGAAGACAGTCCTTTGCTACGGTGACAGTCTGACCTGGGGATACGATGCAA CCGGTTCCGGCCGGCATGCGCTGGAGGACCGTTGGCCGAGCGTGCTGCAGAA GGCGCTCGGTTCGGACGCGCATGTCATCGCCGAAGGGCTGAACGGGCGGACG ACCGCCTATGACGACCATCTCGCCGATTGCGACCGGAACGGCGCGCGTGTCC TCCCGACGGTCCTGCACACCCACGCGCCACTCGATCTCATCGTGTTCATGCTC

25 GGCTCGAACGACATGAAGCCGATCATTCACGGCACCGCTTTCGGCGCGGTGA AGGGCATCGAGCGCCTCGTCAATCTGGTGCGCAGGCACGACTGGCCGACGGA AACGGAGGAGGGGCCCGAGATTCTCATCGTCTCGCCGCCGCCGCTCTGCGAG ACGGCCAACAGCGCCTTTGCCGCCATGTTCGCGGGCGGGGTCGAGCAATCCG CAATGCTGGCGCCGCTTTATCGCGATCTCGCCGACGAGCTCGACTGCGGCTTC

30 TTCGACGGCGGATCGGTGGCCAGGACGACGCCGATCGACGGTGTCCACCTCG ACGCGGAGAACACCCGGGCGGTCGGCAGAGGGTTGGAGCCTGTCGTGCGGA TGATGCTCGGGCTTTAA (SEQ ED NO:92)

35 Sinorhizobium meliloti Smelll (Q9EV56) (Amino Acid) MKTVLCYGDSLTWGYDATGSGRHALEDRWPSVLQKALGSDAHVIAEGLNGRTT AYDDHLADCDRNGARVLPTVLHTHAPLDLIVFMLGSNDMKPIIHGTAFGAVKGIE RLVNLVRRHDWPTETEEGPEILIVSPPPLCETANSAFAAMFAGGVEQSAMLAPLY RDLADELDCGFFDGGSVARTTPIDGVHLDAENTRAVGRGLEPWRMMLGL

40 (SEQ ED NO:93) i ''' ^l -^{1 ,} " " ' ' "' « GC821-2

Agrobacterium tumefaciens Atu III (AAD02335) (DNA) ATGGTGAAGTCGGTCCTCTGCTTTGGCGATTCCCTCACCTGGGGATCAAATGC 5 GGAAACGGGTGGCCGGCACAGCCATGACGATCTTTGGCCGAGCGTCTTGCAG AAGGCGCTCGGTCCTGACGTGCATGTGATTCACGAAGGTCTGGGTGGTCGCA CCACCGCCTATGACGACAACACCGCCGATTGCGACCGCAACGGCGCGCGGGT TCTTCCGACGTTGTTGCACAGCCATGCGCCGCTGGATCTGGTGATTGTCATGC TCGGGACCAACGACCTGAAGCCGTCAATCCATGGATCGGCGATCGTTGCCAT 0 GAAGGGTGTCGAAAGGCTGGTGAAGCTCACGCGCAACCACATCTGGCAGGTG CCGGACTGGGAGGCGCCTGACGTGCTGATCGTCGCACCGCCGCAGCTGTGTG AAACGGCCAATCCGTTCATGGGCGCGATCTTTCGTGATGCGATCGATGAATC GGCGATGCTGGCGTCCGTTTACCGGGACCTTGCCGACGAGCTTGATTGCGGCT TTTTCGATGCGGGTTCCGTCGCCCGAACGACGCCGGTGGATGGCGTTCATCTC 5 GATGCTGAAAATACGCGGGCCATCGGGCGGGGGCTGGAGCCCGTCGTTCGCA TGATGCTCGGACTTTAA (SEQ ED NO:94)

Agrobacterium tumefaciens Atu El (AAD02335) (Amino Acid)0 MVKSVLCFGDSLTWGSNAETGGRHSHDDLWPSVLQKALGPDVHVIHEGLGGRT TAYDDNTADCDRNGARVLPTLLHSHAPLDLVIVMLGTNDLKPSIHGSAIVAMKG VERLVKLTRNHIWQVPDWEAPDVLIVAPPQLCETANPFMGAIFRDABDESAMLAS VYRDLADELDCGFFDAGSVARTTPVDGVHLDAENTRAIGRGLEPWRMMLGL (SEQ ED NO:95)5

Mesorhizobium loti Mlo I (Q98MY5) (DNA) ATGAAGACGGTGCTTTGCTACGGCGACTCGCTGACCTGGGGCTACAATGCCG0 AAGGCGGCCGCCATGCGCTGGAAGACCGCTGGCCGAGCGTGCTGCAAGCAG CGTTAGGCGCCGGCGTGCAAGTGATTGCCGATGGCCTCAACGGCCGCACCAC GGCCTTCGACGATCATCTGGCCGGTGCTGATCGCAACGGCGCCAGGCTGCTG CCGACGGTCCTGACGACGCACGCGCCGATCGACCTGATCATCTTCATGCTCG GCGCCAACGACATGAAGCCTTGGATCCACGGCAATCCGGTCGCAGCCAAGCA5 AGGCATCCAGCGGTTGATCGACATCGTGCGTGGTCACGACTACCCGTTCGAC TGGCCGGCGCCGCAGATCCTGATCGTCGCGCCGCCTGTAGTCAGCCGCACCG AAAATGCCGACTTCAAGGAAATGTTCGCCGGTGGCGATGACGCCTCGAAGTT TTTGGCACCGCAATATGCCGCGCTCGCCGACGAAGCCGGCTGTGGCTTCTTCG ACGCCGGCAGCGTGGCCCAAACCACACCGCTCGATGGCGTTCACCTCGATGC0 CGAAAACACGCGAGAAATCGGCAAGGCGCTGACGCCGATCGTGCGCGTCAT GCTGGAATTGTAA (SEQ ID NO:96) I * " - 1 >• v -. H 'W "^|l".-' , GC821-2 ^ '"

Mesorhizobium loti Mlo I (Q98MY5) (Amino Acid) MKTVLCYGDSLTWGYNAEGGRHALEDRWPSVLQAALGAGVQVIADGLNGRTT 5 AFDDHLAGADRNGARLLPTVLTTHAPEDLΠFMLGANDMK WIHGNPVAAKQGIQ RLIDIVRGHDYPFDWPAPQILIVAPPWSRTENADFKEMFAGGDDASKFLAPQYA ALADEAGCGFFDAGSVAQTTPLDGVHLDAENTREIGKALTPIVRVMLEL (SEQ ED NO:97) 10 Moraxella bovis Mbo (AAK53448) (DNA) ATGAAAAAATCCGCCTTTGCCAAATACTCAGCACTTGCCCTAATGGTTGGGAT GTGCCTGCACACCGCTTACGCCAAGGAGTTTAGCCAAGTCATCATTTTTGGGG ACAGCTTGTCCGATACAGGTCGCCTAAAAGATATGGTCGCCCGAAAAGATGG 15 CACCCTTGGCAACACCTTACAGCCATCTTTTACCACCAACCCCGACCCTGTAT GGTCAAGCTTATTTGCCCAAAGTTATGGCAAAACCGCCAGTCCCAACACGCC TGACAATCCCACTGGCACTAACTATGCCGTGGGCGGAGCTCGCTCTGGCTCG GAGGTCAATTGGAATGGTTTTGTGAATGTACCCTCCACCAAAACGCAAATCA CCGACCATTTGACCGCCACAGGTGGCAAAGCCGACCCTAATACCCTGTATGC 20 CATTTGGATTGGCTCTAATGACTTAATTTCAGCTTCTCAAGCCACCACAACAG CCGAAGCCCAAAACGCCATTAAAGGTGCGGTAACTCGCACCGTGATAGACAT CGAAACACTCAATCAAGCAGGGGCGACAACCATTTTGGTGCCAAATGTGCCT GATTTGAGCCTCACGCCCCGAGCCATCTATGGCGAAAGCCTCATGGCAGGCG TGCAAGACAAAGCCAAACTCGCCTCAAGTCTGTATAATAGCGGTCTGTTTGA 25 AGCATTAAATCAATCCACCGCCAACATCATCCCTGCCAACACCTTTGCCCTAC TCCAAGAAGCGACCACAAATAAAGAAGCCTTTGGTTTTAAAAACACGCAAGG CGTGGCGTGTCAAATGCCCGCTCGTACCACAGGGGCGGATGATGTGGCTTCT ACTTCCTTGGCATGTACCAAAGCCAATCTTATAGAAAACGGGGCAAATGACA CCTACGCCTTTGCCGATGACATTCACCCATCGGGACGCACGCACCGCATTTTG 30 GCACAGTATTACCGTTCTATCATGGACGCCCCTACTCACATGGGTAAACTCTC AGGCGAGCTTGTCAAAACAGGTTCAGCCCACGACCGTCATGTTTACCGTCAG CTTGACAGGCTTAGTGGCTCACAGCACAGCATTTGGGCAAACGTCTATGCCA GCGACCGTACCGACCCCACCACCCAAATCGGCTTGGACGTGGCAGGTTCATC AAGCCATACAGGGGCGTATCTGAGCCACCAAAACCAAGATTATGTGCTGGAT 35 GACACCCTATCATCAGATGTCAAAACCATTGGCATGGGGCTGTATCATCGCC ATGACATCGGCAATGTCCGTCTAAAAGGCGTGGCAGGTATCGACCGACTTAG CGTGGATACGCACCGCCATATCGACTGGGAGGGGACAAGCCGTTCGCACACC GCAGATACCACCGCCAGACGTTTTCATGCAGGGCTACAAGCCAGCTATGGCA TAGACATGGGCAAAGCCACCGTGCGTCCGCTTATCGGCGTACATGCCCAAAA 40 AGTCAAAGTAAATGACATGACCGAGAGCGAATCAACTTTATCCACCGCCATG

CGTTTTGGCGAGCAAGAACAAAAGTCCCTACAAGGCGAGATTGGCGTCGATG TGGCTTATCCGATTAGCCCTGCTTTGACTCTGACGGGCGGTATCGCTCACGCT CATGAGTTTAACGATGATGAACGCACCATTAATGCCACTTTAACCTCCATTCG TGAATACACGAAGGGCTTTAATACAAGCGTTAGCACCGACAAATCTCACGCC ACCACCGCTCATCTGGGCGTACAAGGGCAACTTGGCAAGGCAAATATTCATG

CAGGCGTTCACGCCACCCACCAAGACAGCGATACAGACGTGGGTGGTTCGCT TGGGGTTCGCTTGATGTTTTAA (SEQ ED NO:98)

Moraxella bovis Mbo (AAK53448) (Amino Acid) MKKS AFAKYS ALALMVGMCLHTAYAKEFSQVEFGDSLSDTGRLKDMVARKDG TLGNTLQPSFTTNPDPVWSSLFAQSYGKTASPNTPDNPTGTNYAVGGARSGSEVN WNGFVNVPSTKTQITDHLTATGGKADPNTLYAIWIGSNDLISASQATTTAEAQNA E GAVTRTVEDIETLNQAGATTILVPNVPDLSLTPRAJYGESLMAGVQDKAKLASS LYNSGLFEALNQSTANEPANTFALLQEATTNKEAFGFKNTQGVACQMPARTTGA DDVASTSLACTKANLIENGANDTYAFADDFFLPSGRTHRILAQYYRSIMDAPTHMG KLSGELVKTGSAHDRΪTVYRQLDRLSGSQHSIWANVYASDRTDPTTQIGLDVAGS SSHTGAYLSHQNQDYVLDDTLSSDVKTIGMGLYHRHDIGNVRLKGVAGΠDRLSV DTHRHROWEGTSRSHTADTTARRFHAGLQASYGROMGKATVRPLIGVHAQKVKV NDMTESESTLSTAMRFGEQEQKSLQGEIGVDVAYPISPALTLTGGIAHAHEFNDD ERTINATLTSIREYTKGFNTSVSTDKSHATTAHLGVQGQLGKANIHAGVHATHQD SDTDVGGSLGVRLMF (SEQ ED NO:99)

Chromobacterium violaceum Cvi (Q7NRP5) (DNA) ATGCGCTCTATCGTCTGCAAAATGCTGTTCCCTTTGTTGCTGCTGTGGCAGCT GCCCGCCCTGGCCGCCACCGTGCTGGTGTTCGGCGACAGCCTGTCCGCCGGC TACGGCCTGGCCCCGGGCCAGGGATGGGCGGCGCTGCTGGCGCGCGACCTCT CGCCCCGGCACAAGGTGGTCAACGCCAGCGTGTCCGGCGAAACCAGCGCCGG CGGCCTGTCCAGGCTGCCCGACGCGCTCGCCCGCCACCAGCCCGACGTGCTG GTGCTGGAACTCGGCGCCAACGATGGCCTGCGCGGCCTGCCGATGGCTGACA TGAGGCGCAACCTGCAGCGGATGATAGACCTGGCCCAGGCGCGCAAGGCCA AGGTGCTGCTGGTGGGCATGGCGCTGCCACCCAACTATGGCCCCCGCTACGG CGCCGAGTTCCGCGCCGTTTATGACGATTTGGCCCGCCGCAACCGCCTGGCCT ACGTGCCGCTGCTGGTCGAGGGCTTCGCCGGCGACCTCGGCGCCTTCCAGCC CGACGGCCTGCATCCCCGCGCGGAGAAGCAGGCCACCATGATGCGCACGGTC

AAGGCAAAACTGCCAGTGAAATAA (SEQ ED NO: 100)

Chromobacterium violaceum Cvi (Q7NRP5) (Amino Acid) MRSEVCKMLFPLLLLWQLPALAATVLVFGDSLSAGYGLAPGQGWAALLARDLSP

RHKWNASVSGETSAGGLSRLPDALARHQPDVLVLELGANDGLRGLPMADMRR ^■ » * ^• „^■* - - GC821-2

NLQRMEDLAQARKAKVLLVGMALPPNYGPRYGAEFRAVYDDLARRNRLAYVPL LVEGFAGDLGAFQPDGLHPRAEKQATMMRTVKAKLPVK (SEQ ED NO: 101)

br/o vulnificus Vvu (AA007232) (DNA) ATGTTTTTCCTTTCTAGCGTCGCACACGCAACCGAGAAAGTGTTAATTCTTGG CGACAGCCTAAGTGCAGGATACAACATGTCTGCAGAGCAGGCTTGGCCTAAT TTGTTACCAGAAGCATTGAATACATACGGAAAAAACGTAGAAGTGATCAACG

10 CC AGTATCTCTGGAGAC AC AACCGGCAATGGACTATCTCGTCTGCCTGAGTTG TTAAAAACGCACTCACCAGACTGGGTGCTTATTGAGTTGGGTGCCAATGATG GCTTGCGAGGTTTCCCGCATAAAGTGATCTCTTCAAACCTTTCGCGAATGATT CAACTCAGTAAAGCCTCAGACGCTAAAGTCGCATTGATGCAAATTCGTGTAC CGCCTAACTATGGCAAGCGCTACACCGATGCATTTGTCGAACTCTACCCTACG 15 CTTGCTGAACATC ACC AAGTCCCGTTGCTCCCCTTTTTCTTAGAGGAAGTGAT CGTGAAACCGGAATGGATGATGCCTGATGGCTTACACCCAATGCCCGAAGCT CAGCCTTGGATCGCTCAATTTGTTGCAAAAACGTTTTACAAACATCTCTAA (SEQ E NO:102)

20 Vibrio vulnificus Vvu (AA007232) (Amino Acid) MFFLSSVAHATEKVLILGDSLSAGYNMSAEQAWPNLLPEALNTYGKNVEVENASI SGDTTGNGLSRLPELLKTHSPDWVLIELGANDGLRGFPHKVISSNLSRMIQLSKAS DAKVALMQIRVPPNYGKRYTDAFVELYPTLAEHHQVPLLPFFLEEVIVKPEWMM

25 PDGLHPMPEAQPWIAQFVAKTFYKHL (SEQ ED NO: 103)

Ralstonia eutropha Reu (ZP00166901) (DNA) ATGCCATTGACCGCGCCGTCTGAAGTCGATCCGCTGCAAATCCTGGTCTATGC

30 CGATTCGCTTTCGTGGGGCATCGTGCCCGGCACCCGCCGGCGGCTTCCCTTCC CGGTTCGCTGGCCAGGCCGGCTCGAACTCGGCCTGAACGCCGACGGCGGCGC CCCGGTCCGCATCATCGAGGACTGCCTGAACGGCCGGCGCACCGTCTGGGAC GACCCATTCAAACCGGGCCGCAACGGCTTGCAAGGGCTGGCGCAGCGCATCG AGATCCATTCCCCGGTGGCGCTCGTGGTTTTGATGCTGGGCAACAACGATTTC

35 CAGTCCATGCATCCGCACAACGCCTGGCATGCGGCACAGGGCGTCGGCGCGC TGGTCCACGCCATCCGGACGGCGCCGATCGAACCGGGAATGCCGGTGCCGCC GATCCTGGTGGTGGTGCCGCCGCCGATCCGCACGCCCTGCGGGCCGCTCGCG CCCAAGTTCGCCGGCGGCGAACACAAGTGGGCAGGCCTGCCCGAGGCGCTGC GCGAACTGTGCGCCACTGTCGACTGCTCGCTGTTCGATGCGGGTACCGTGATC

40 CAGAGCAGTGCCGTCGACGGCGTACACCTTGACGCCGATGCCCATGTCGCCC --^■- s , " ^» .'^■ " " " - i GC821-2

TGGGCGATGCCCTGCAACCGGTCGTTCGTGCGCTGCTCGCCGAATCCTCGGG ACATCCCTCCTAA (SEQ ED NO: 104)

5 Ralstonia eutropha Reu (ZP00166901) (Amino Acid) MPLTAPSEVDPLQILVYADSLSWGIVPGTRRRLPFPVRWPGRLELGLNADGGAPV RIIEDCLNGRRTVWDDPFKPGRNGLQGLAQRIEIHSPVALWLMLGNNDFQSMHP HNAWHAAQGVGALVHAIRTAPIEPGMPVPPILWVPPPEITPCGPLAPKFAGGEH KWAGLPEALRELCATVDCSLFDAGTVIQSSAVDGVHLDADAHVALGDALQPW 10 RALLAESSGHPS (SEQ ED NO: 105)

Salmonella typhimurium Stm (AAC38796) (DNA)

15 ATGACCCAAAAGCGTACCCTGCTAAAATACGGCATACTCTCGCTGGCGCTGG CCGCGCCATTATCTGCCTGTGCGTTTGACTCTCTTACGGTGATTGGCGATAGC CTTAGCGATACCGGTAATAACGGTCGCTGGACCTGGGATAGTGGTCAAAATA AGCTCTACGACGAACAGTTGGCCGAACGATATGGGCTGGAATTAAGCCCTTC CAGCAATGGCGGCTCTAATTATGCCGCCGGCGGCGCGACGGCGACCCCGGAA

20 TTAAACCCGCAGGATAATACCGCGGATCAGGTACGGCAGTGGCTTGCCAAAA CGGGGGGAAAAGCCGACCACAACGGTTTGTATATTCACTGGGTCGGCGGAAA CGATCTGGCGGCGGCCATCGCGCAACCAACCATGGCACAGCAAATAGCCGGT AATAGCGCCACTAGCGCGGCGGCGCAGGTAGGGCTGTTACTGGATGCCGGCG CCGGGCTGGTCGTGGTGCCAAACGTACCGGATATTAGTGCGACGCCAATGCT

25 TCTGGAGGCGGTAATCACCGCTGGGCTGGGCGCAGCGGCGCCCCCGGCGCTA AAAGCGGCGTTAGATGCGCTGGCGGAGGGCGCTACGCCCGATTTCGCCAGTC GGCAACAGGCGATCCGCAAGGCGCTGCTGGCGGCGGCTGCAACGGTAAGCA GCAATCCATTTATTCAGCAACTGCTCGTTGAACAACTGCTGGCGGGCTATGAA GCGGCGGCAGGGCAGGCGTCAGCTCTGACCGATTATTATAATCAGATGGAAG

30 AGAAGGGGCTGGAGCAACACGGCGGCAATATAGCCCGTGCCGATATCAACG GCCTCTTTAAGGAAATTCTTGCCAACCCGCAGGCGTTTGGTCTGACAAATACC GTAGGTATGGCCTGCCCGCCTGGCGTATCCGCTTCGGCGTGCTCCTCGGCAAT GCCTGGATTTAATGCGTCGCAGGACTATGTGTTTGCCGATCATTTACATCCCG GTCCGCAGGTCCATACCATTATTGCGCAATATATTCAGTCGATCATTGCCGCG

35 CCGGTACAGGCGACATACCTGAACCAAAGCGTTCAGTCGATGGCGCAAGGCA GTCGTACCACGCTTGACAGCCGTTATCAGCAGCTTCGCCAGGGGGAAAATCC TGTTGGTTCGCTGGGCATGTTCGGCGGATACAGCGGGGGATATCAACGTTAT GATAATAATGAGGCCGACGGGAACGGTAATCATAATAATCTGACGGTTGGCG TCGATTATCAGCTTAACGAGCAGGTTCTGCTGGGAGGGCTGATAGCCGGTTCT

40 CTGGATAAGCAACATCCTGACGATAATTATCGTTATGATGCCCGCGGTTTTCA if .-^■ < ι

GGCCGCCGTATTCAGCCATTTACGCGCCGGTCAGGCGTGGCTGGATAGCGAT TTACACTTTCTGTCCGCTAAATTCAGTAACATTCAGCGCAGTATAACGCTCGG TGCGCTAAGACGGGTGGAAGAGGGCGAAACCAACGGTCGGCTGTCGGGCGC GAGCTTAACCAGCGGTTATGATTTTGTCATGGTGCCGTGGTTAACGACCGGAC CGATGCTGCAATATGCATGGGATTACAGCCACGTTAATGGTTATAGCGAGAA GCTCAATACCAGTACATCAATGCGTTTTGGTGACCAAAACGCCCATTCGCAG GTGGGTAGCGCGGGTTGGCGTCTGGATCTTCGCCACAGCATCATTCACTCCTG GGCGCAGATTAATTATCGCCGTCAGTTTGGCGATGATACGTATGTGGCGAAC GGCGGCCTTAAATCGACCGCGCTGACGTTTAGCCGCGACGGAAAAACGCAGG ATAAAAACTGGGTTGATATCGCGATTGGCGCAGATTTTCCGCTGTCGGCAAC GGTGTCCGCTTTCGCCGGGCTGTCGCAAACGGCAGGGTTAAGCGATGGCAAT CAAACCCGTTATAACGTTGGGTTTAGCGCCCGATTTTAA (SEQ ED NO: 106) Salmonella typhimurium Sim (AAC38796) (Amino Acid) MTQKRTLLKYGILSLALAAPLSACAFDSLTVIGDSLSDTGNNGRWTWDSGQNKL YDEQLAERYGLELSPSSNGGSNYAAGGATATPELNPQDNTADQVRQWLAKTGG KADHNGLYIHWVGGNDLAAAIAQPTMAQQIAGNSATSAAAQVGLLLDAGAGLV WPNVPDISATPMLLEAVITAGLGAAAPPALKAALDALAEGATPDFASRQQAIRK ALLAAAATVSSNPFIQQLLVEQLLAGYEAAAGQASALTDYYNQMEEKGLEQHG GNIARADINGLFKEILANPQAFGLTNTVGMACPPGVSASACSSAMPGFNASQDYV FADHLHPGPQVHTEAQYIQSEAAPVQATYLNQSVQSMAQGSRTTLDSRYQQLRQ GENPVGSLGMFGGYSGGYQRYDNNEADGNGNHNNLTVGVDYQLNEQVLLGGLI AGSLDKQHPDDNYRYDARGFQAAVFSHLRAGQAWLDSDLHFLSAKFSNIQRSrr LGALRRVEEGETNGRLSGASLTSGYDFVMVPWLTTGPMLQYAWDYSHVNGYSE KLNTSTSMRFGDQNAHSQVGSAGWRLDLRHSIIHSWAQINYRRQFGDDTYVAN GGLKSTALTFSRDGKTQDKNWVDIAIGADFPLSATVSAFAGLSQTAGLSDGNQTR YNVGFSARF (SEQ ED NO: 107)

In total, nine of the new "GDSL"-type esterases were identified in 6 metagenomic libraries and BRAIN's esterase/lipase library. Eight of these genes were heterologously expressed in E. coli and the resulting enzymes analyzed for activity in the assays described herein. The characterization of these enzymes for perhydrolase activity revealed that one displayed the desired activity. A second one was predicted to show this activity due to the presence of amino acids conserved among this group of enzymes. ii ii„t-i t ιuι ^»"ii'.'' - - ^s GC821-2 <3 ^

Comparison of the sequences of enzymes for which the presence or absence of the desired perhydrolase activity was determined led to the identification of 19 amino acid positions which were conserved among the enzymes which displayed the desired perhydrolase activity. Thus, it is contemplated that these conserved amino acids are essential for the perhydrolase reaction and/or is a structural feature of perhydrolase enzymes. One of the identified structural motifs ("G/ARTT") conserved among esterases with the desired perhydrolase activity was used to design degenerate primers which provided the means to focus the screening on true perhydrolases among "GDSL"-type esterases. Indeed, the use of these "G/ARTT" primers led to the identification of enzymes with the desired perhydrolase activity from the metagenome. However, it is not intended that the use of the metagenome be limited to any particular assay method. Indeed, it is contemplated that the metagenome be searched by assaying for a particular enzyme activity or activities desired (e.g., perhydrolysis and/or acyltransferase (cofactor dependent or independent) activity). In addition, screening using poly and/or monoclonal anti-sera directed against a protein of interest finds use in the present invention. In additional embodiments, the metagenome is searched using degenerate primer sets based on the sequence of the protein of interest. In addition, the knowledge of the structure/function relationship of perhydrolases allowed searching for these enzymes in genome sequences of cultivable microorganisms. Of 16 "GDSL"-type esterases identified in different bacterial isolates, the corresponding genes of 10 enzymes were amplified and heterologously expressed in E. coli. The resulting enzyme samples of seven clones were analyzed using the assays described herein. Of five samples characterized to date, 4 enzymes indeed showed the desired activity and all results confirmed the proposed relationship between primary structural determinants and the function of perhydrolases. Thus, an enzyme library of 19 "GDSL"- type esterases comprising at least 6 perhydrolases with the desired perhydrolase activity : it i if '"ii" ■.•^{■ ιr}"i" " , u GC821-2

was set up. The identified correlation between the stracture and function of perhydrolases provides a definition of the sequence space used by enzymes with the desired perhydrolase activity. Comparisons were made of protein sequences of enzymes for which the absence or presence of the desired perhydrolase activity. This revealed a correlation between the presence of certain amino acids and the capability to perform perhydrolase reactions. This knowledge was used to identify enzymes containing these conserved amino acids in sequenced genomes from cultivable microorganisms. The following enzymes were identified and experiments to amplify the genes from the genomic DNA of the corresponding strains using specific primers were performed.

Table 1. "GDSL' ype Esterases with a "GRTT"-Motif From Bacterial Isolates

Isolate Protein Acronym Amplicon Expression Identifier Vector Sinorhizobium Smal993 Sme I yes pLO_SmeI meliloti Sinorhizobium Q92XZ1 Sme ll yes pET26_SmeII meliloti Sinorhizobium Q9EV56 Sme III yes pET26_SmeEI meliloti Agrobacterium Q9KWB1 Arh l no rhizogenes Agrobacterium Q9KWA6 Arh II no rhizogenes s ■ ..^{■ ■} » GC821-2

Agrobacterium AAD02335 Atu m yes pET26_Atuiπ tumefaciens Mesorhizobium loti Q98MY5 Mlo l yes pET26_Mlo Mesorhizobium loti ZP_00197751 Mlo π no - Ralstonia Q8XQI0 Rso no - solanacearum Ralstonia eutropha ZP_00166901 Reu yes n.d. Moraxella bovis AAK53448 Mbo yes ρET26_Mbo Burkholderia ZP_00216984 Bee no - cepacia Chromobacterium Q7NRP5 Cvi yes pET26_Cvi violaceum Pirellula sp. NP_865746 Psp n.d. n.d. Vibrio vulnificus AA007232 Vvu yes pET26_Vvu Salmonella AAC38796 Sty yes pET26_Sty typhimurium

In the cases of A. rhizogenes, M. loti (enzyme II), R. solanacearum and B. cepacia no amplicon could be generated. It was thought that this was probably due to genetic differences between the strains used in this investigation and those used for the sequencing of the genes deposited in the public domain databases. One reason might be that the corresponding genes are located on plasmids which are not present in the strains used in this investigation. However, it is not intended that the present invention be limited to any particular mechanism or theory.

The amplicons from all other strains were sequenced. In many cases there were differences between the sequence from the databases and the sequence determined during the development of the present invention. By sequencing two clones from independent amplifications, mutations introduced by the polymerase could be nearly excluded. The sequences of the genes and the deduced amino acid sequences of "GDSL"-type esterases with a "GRTT"-motif or variations from bacterial isolates are provided below:

SMal993_Sinorhizobium meliloti (Sme I) (SEQ ED NOS:88 and 89) 10 Q92XZ1 Sinorhizobium meliloti (Sme E) (SEQ ED NOS:90 and 91) Q9EY56_Sinorhizobium meliloti (Sme El) (SEQ ED NOS:92 and 93) AAD02335_Agrobacterium tumefaciens (Atu III) (SEQ ED NOS: 94 and 95) Q98MY5_Mesorhizobium loti (Mlo I) (SEQ ED NOS:96 and 97) ZP_00166901_Rα/sto 'α eutropha (Reu) (SEQ ED NOS:104 and 105) 15 AAK53448_ or xe// bovis (Mbo) (SEQ ED NOS: 98 and 99) Q KP5_Chromobacterium violaceum (Cvi) (SEQ ED NOS:100 and 101) AA007232_Vibrio vulnificus (Vvu) (SEQ ED NOS:102 and 103) AAC38796_Salmonella typhimurium (Stm) (SEQ ID NOS: 106 and 107)

20 Q9KWBl_Agrobacterium rhizogenes (Arh I) MICHKGGEEMRSVLCYGDSNTHGQIPGGSPLDRYGPNERWPGVLRRELGSQWY VIEEGLSGRTTVRDDPIEGTMKNGRTYLRPCLMSHAILDLVIIMLGTNDLKARFGQ 25 PPSEVAMGIGCLVYDIRELAPGPGGKPPEIMWAPPPMLDDIKEWEPIFSGAQEKS RRLALEFEEADSLEVHFFDAATVASCDPCDGFHINREAHEALGTALAREVEAIGW R (SEQ ED NO: 108) ATGATTTGCCATAAAGGTGGGGAGGAAATGCGGTCAGTCTTATGCTACGGCG 30 ACTCGAATACGCACGGCCAGATTCCGGGGGGCTCACCGCTCGACCGATACGG GCCGAACGAGCGCTGGCCTGGCGTTTTGAGACGGGAGCTTGGAAGCCAGTGG TATGTGATCGAGGAGGGCCTGAGTGGCCGCACGACGGTTCGCGACGATCCGA TCGAGGGCACGATGAAAAACGGCCGGACCTACCTGCGTCCGTGCCTCATGAG CCACGCGATCCTCGATCTCGTGATTATCATGCTCGGGACGAACGACCTGAAA 35 GCGCGCTTCGGTCAACCGCCATCGGAAGTGGCGATGGGGATCGGCTGCCTCG TCTACGATATCAGGGAGCTGGCGCCCGGACCGGGCGGCAAGCCCCCCGAAAT CATGGTGGTTGCTCCGCCGCCGATGCTGGACGATATCAAGGAATGGGAACCC .. , t • ^• .- , ' „ GC821-2

ATATTTTCCGGCGCCCAGGAGAAATCCCGGCGTCTCGCGCTTGAGTTTGAAAT TATTGCTGATTCGCTTGAAGTACACTTCTTTGACGCCGCGACCGTCGCATCGT GTGATCCTTGCGATGGTTTTCACATCAACCGGGAAGCGCATGAAGCCTTGGG AACAGCGCTTGCCAGGGAAGTGGAGGCGATCGGTTGGAGATGATGA (SEQ ED NO: 109)

Q9KWA6_Agrobacterium rhizogenes (Arh II) MAESRSILCFGDSLTWGWIPVPESSPTLRYPFEQRWTGAMAAALGDGYSEEEGLS 10 ARTTS VEDPNDPRLNGS AYLPMALASHLPLDLVELLGTNDTKSYFRRTPYEIANG MGKLAGQVLTSAGGIGTPYPAPKLLIVSPPPLAPMPDPWFEGMFGGGYEKSLELA KQYKALANFLKVDFLDAGEFVKTDGCDGIHFSAETNITLGHAIAAKVEAE^JSQEA KNAAA (SEQ ED NO: 110)

15 ATGGCAGAGAGCCGCTCAATATTATGTTTTGGGGATTCACTCACATGGGGTTG GATTCCGGTACCGGAGTCGTCGCCGACGCTCAGATATCCCTTTGAGCAGCGCT GGACCGGTGCAATGGCTGCGGCACTCGGTGACGGCTATTCAATCATCGAGGA AGGCCTTTCCGCCCGCACGACCAGCGTCGAGGATCCGAACGATCCCAGGCTG AACGGCAGCGCCTACCTGCCGATGGCGCTCGCCAGCCATCTGCCGCTCGATC

20 TCGTCATCATCCTTCTCGGCACCAACGACACCAAGTCCTATTTCCGCCGCACG CCCTATGAGATCGCCAACGGCATGGGCAAGCTTGCCGGACAGGTTCTGACCT CGGCCGGCGGGATCGGCACGCCCTACCCTGCCCCGAAGCTTCTGATCGTTTC GCCGCCGCCGCTCGCTCCCATGCCTGACCCGTGGTTCGAAGGCATGTTCGGTG GCGGTTACGAAAAGTCGCTCGAACTCGCAAAGCAGTACAAGGCGCTCGCCAA

25 CTTCCTGAAGGTCGACTTCCTCGACGCCGGCGAGTTTGTAAAGACCGACGGC TGCGATGGAATCCATTTCTCCGCCGAGACGAACATCACGCTCGGCCATGCGA TCGCGGCGAAGGTCGAAGCGATTTTCTCACAAGAGGCGAAGAACGCTGCGGC TTAG (SEQ ED NO: 111)

30 ZP_Q0197751_Mesorhizobium loti (Mlo II) MKTILCYGDSLTWGYDAVGPSRHAYEDRWPSVLQGRLGSSARVIAEGLCGRTTA FDDWVAGADRNGARILPTLLATHSPLDLVIVMLGTNDMKSFVCGRAIGAKQGME RIVQIEIGQPYSFNYKVPSILLVAPPPLCATENSDFAEIFEGGMAESQKLAPLYAAL 35 AQQTGCAFFDAGTVARTTPLDGIHLDAENTRAIGAGLEPWRQALGL (SEQ ED NO: 112) ATGAAGACCATCCTTTGTTACGGTGACTCCCTCACTTGGGGCTATGATGCCGT CGGACCCATGAAGACCATCCTTTGTTACGGTGACTCCCTCACTTGGGGCTATG 40 ATGCCGTCGGACCCTCACGGCATGCTTATGAGGATCGATGGCCCTCCGTACTG t ;r __. i -'" ij. > • r * IS, ,1' " 'If , ' ,, ⁱ GC821-2 ^

CAAGGCCGCCTCGGTAGCAGTGCGCGGGTGATCGCCGAGGGGCTTTGCGGCC GCACAACTGCGTTTGACGACTGGGTCGCTGGTGCGGACCGGAACGGTGCGCG CATCCTGCCGACGCTTCTTGCGACCCATTCACCGCTTGACCTCGTTATCGTCA TGCTCGGGACGAACGACATGAAATCGTTCGTTTGCGGGCGCGCTATCGGCGC 5 CAAGCAGGGGATGGAGCGGATCGTCCAGATCATCCGCGGGCAGCCTTATTCC TTCAATTATAAGGTACCGTCGATTCTTCTCGTGGCGCCGCCGCCGCTGTGCGC TACCGAAAACAGCGATTTCGCGGAAATTTTTGAAGGTGGCATGGCTGAATCG CAAAAGCTCGCGCCGCTTTATGCCGCGCTGGCCCAGCAAACCGGATGCGCCT TCTTCGATGCAGGCACTGTGGCCCGCACGACACCGCTCGACGGTATTCACCTC 10 GATGCTGAAAACACGCGCGCCATTGGTGCCGGCCTGGAGCCGGTGGTCCGCC AAGCGCTTGGATTGTGA (SEQ ED NO: 113)

Q8XQlO_Ralstonia solanacearum (Rso) 15 MQQILLYSDSLSWGBPGTRRRLPFAARWAGVMEHALQAQGHAVRIVEDCLNGR TTVLDDPARPGRNGLQGLAQRIEAHAPLALVILMLGTNDFQAffRHTAQDAAQG VAQLVRAIRQAPffiPGMPWPVLrVv PAITAPAGAMADKFADAQPKCAGLAQAY RATAQTLGCHVFDANSVTPASRVDGIHLDADQHAQLGRAMAQWGTLLAQ (SEQ ED NO:114) 20 ATGCAACAGATCCTGCTCTATTCCGACTCGCTCTCCTGGGGCATCATCCCCGG CACCCGCCGGCGCCTGCCGTTCGCCGCCCGCTGGGCCGGGGTCATGGAACAC GCGCTGCAGGCGCAAGGGCACGCCGTGCGCATCGTCGAAGACTGCCTCAATG GACGCACCACGGTGCTCGACGATCCCGCGCGGCCGGGGCGCAACGGACTGCA 25 GGGGCTCGCGCAGCGGATCGAAGCGCACGCCCCGCTTGCCCTGGTCATCCTG ATGCTCGGCACCAACGACTTCCAGGCGATCTTCCGGCACACCGCCCAGGACG CGGCGCAAGGCGTGGCGCAGCTGGTGCGGGCCATCCGCCAGGCGCCGATCGA ACCCGGCATGCCGGTGCCGCCCGTGCTGATCGTGGTGCCGCCGGCCATCACC GCGCCGGCCGGGGCGATGGCCGACAAGTTTGCCGACGCGCAGCCCAAGTGCG 30 CCGGCCTTGCGCAGGCCTATCGGGCAACGGCGCAAACGCTAGGCTGCCACGT CTTCGATGCGAACAGCGTCACGCCGGCCAGCCGCGTGGACGGCATCCACCTC GATGCCGACCAGCATGCGCAGCTGGGCCGGGCGATGGCGCAGGTCGTCGGG ACGCTGCTTGCGCAATAA (SEQ ID NO: 115) 35 ZP 00216984 Burkholderia cepacia (Bee) ATGACGATGACGCAGAAAACCGTGCTCTGCTACGGCGATTCGAACACGCATG GCACACGCCCGATGACGCATGCTGGCGGACTGGGGCGGTTTGCACGCGAAGA ACGCTGGACCGGCGTGCTGGCGCAAACGCTCGGTGCGAGCTGGCGGGTCATT 40 GAAGAAGGGTTGCCCGCGCGTACGACCGTGCATGACGATCCGATCGAAGGCC -„ ' - ' - •<« ■" .' . -rm GC821-2 1 ^

GGCACAAGAATGGTTTGTCGTATCTGCGCGCGTGCGTCGAAAGCCACTTGCC CGTCGATGTCGTCGTGCTGATGCTCGGGACCAACGATCTGAAGACACGCTTCT CGGTCACGCCCGCCGACATCGCGACATCGGTCGGCGTATTGCTTGCCAAGAT CGCTGCGTGCGGCGCCGGTCCGTCCGGTGCGTCACCGAAGCTCGTGCTGATG GCGCCTGCGCCGATCGTCGAGGTCGGATTCCTCGGCGAGATCTTTGCGGGCG GCGCAGCGAAGTCGCGGCAGCTCGCGAAGCGGTACGAACAGGTGGCAAGCG ATGCCGGTGCGCACTTTCTCGATGCCGGCGCGATCGTCGAGGTGAGCCCGGT GGATGGCGTTCACTTCGCGGCCGATCAGCATCGTGTGCTCGGGCAGCGGGTC GCTGCCCTTCTGCAGCAGATTGCGTAA (SEQ ED NO: 116) 10 MTMTQKTVLCYGDSNTHGTRPMTΗAGGLGRFAREERWTGVLAQTLGASWRVI EEGLPARTTVHDDPIEGRHKNGLSYLRACVESHLPVDVVVLMLGTNDLKTRFSV TPADIATSVGVLLAKIAACGAGPSGASPKLVLMAPAPIVEVGFLGEffAGGAAKSR QLAKRYEQVASDAGAHFLDAGAIVEVSPVDGVHFAADQHRVLGQRVAALLQQI 15 A (SEQ ED NO:117)

NP_865746 Pirellula sp (Psp) 20 MHSILEYGDSLSWGIEPGTRRRFAFHQRWPGVMEIELRQTGEDARVIEDCLNGRRT VLEDPE PGRNGLDGLQQRIEINSPLSLWLFLGTNDFQSVHEFHAEQSAQGLALL VDAIRRSPFEPGMPTPKILLVAPPTVHHPKLDMAAK-FQNAETKSTGLADAIRKVS TEHSCEFFDAATVTTTSWDGVHLDQEQHQALGTALASTIAEILADC (SEQ ED NO: 118) 25 ATGCATTCAATCCTCATCTATGGCGATTCTCTCAGTTGGGGAATCATTCCCGG CACGCGTCGTCGCTTCGCGTTCCATCAGCGTTGGCCGGGCGTCATGGAGATTG AACTGCGACAAACTGGAATCGATGCCCGCGTCATCGAAGACTGCCTCAATGG CCGACGAACCGTCTTGGAAGATCCAATCAAACCCGGACGCAATGGCCTGGAT 30 GGTTTGCAGCAACGGATCGAAATCAATTCACCTCTGTCACTGGTCGTGCTCTT TCTGGGGACCAACGATTTCCAGTCCGTCCACGAATTCCATGCCGAGCAATCG GCACAAGGACTCGCACTGCTTGTCGACGCCATTCGTCGCTCCCCTTTCGAACC AGGAATGCCGACACCGAAAATCCTGCTTGTCGCACCACCGACGGTTCACCAC CCGAAACTTGATATGGCGGCGAAGTTCCAAAACGCGGAAACGAAATCGACG 35 GGACTCGCAGATGCGATTCGCAAGGTCTCAACAGAACACTCCTGCGAATTCT TCGATGCGGCCACGGTCACCACAACAAGTGTCGTCGACGGAGTCCATCTCGA TCAAGAACAACATCAAGCACTCGGTACCGCACTGGCATCGACAATCGCTGAA ATACTAGCAGACTGTTGA (SEQ ID NO: 119) 40 •- ,-. if .^• it,? - nfrf -'it' .' -"it" ,tι '" , ι GC821-2 ^' ~j

As indicated above, the above sequences are the protein sequences and the coding sequences of "GDSL-type" esterases with a "GRTT'-motif or similar motifs from different bacterial isolates. The DNA sequences represent the target-DNA from which specific primers were deduced. All amplicons were ligated as Niel/ &oI-fragments to pET26 thereby eliminating the/?e/ -leader sequence of this vector. All of the "GDSL- type" esterases from these isolates were expressed in E. coli Rosetta (DE3) at 28°C. The expression was induced by addition of 100 μM IPTG at an O.D.sso = 1 and the cells were harvested 20 h after induction. Only the cells expressing the enzymes from M. bovis and S. typhimurium were collected 4 h after induction, since previous experiments had shown 10 that the highest activity could be obtained at this point of time. Table 2 summarizes the expression experiments.

Table 2: Expression and Characterization of "GDSL"-type Esterases From Bacterial Isolates for Perhydrolase Activity

Strain Enzyme Expression Solubility³ Activity Perhydrolase GRTT Level² 4 Activity -Motif S. meliloti Sme l +++ ++ 5770,0 yes ARTT S. meliloti Sme ll +++ +++ 85,0 yes GRTT S. meliloti Sme m +++ ++ 746,5 n.d. GRTT A. tumefaciens Atu m n.d⁵. n.d. n.d. n.d. GRTT M. loti Mlo l +++ ++ 1187,3 yes GRTT M. bovis Mbo + n.d. 25,2 yes ARTT C. violaceum Cvi + + 2422,7 n.d. GETS V vulnificus Vvu n.d. n.d. n.d. n.d. GDTT R. eutropha Reu n.d. n.d. n.d. n.d. GRRT S. typhimurium¹ Sty + n.d. 17,2 no SRTT outer membrane localized autotransporter protein - „ , .,

2 expression level: + moderate overexpression; ++ strong overexpression; very strong overexpression as judged from SDS-PAGE-analysis ³ as judged by SDS-PAGE-analysis towards _jp-nitrophenyl butyrate not determined

With the exception of the enzyme from S. typhimurium, all other enzymes tested 10 showed the desired perhydrolase activity, confirming the correlation between the presence of certain conserved amino acids an the capability to perform perhydrolase reactions. Although the enzyme from S. typhimurium contains the GRTT-motif, it is different from the other enzymes by the location of this motif downstream from block V. In all other enzymes, this motif is located between block I and IE, indicating that it might have a 15 different function in the enzyme from S. typhimurium. Thus, the absence of perhydrolase activity in the enzyme from S. typhimurium also supports the identified stracture/function-relationship of the perhydrolases provided by the present invention.

20 Screening of New "GDSL-type" Esterases in Metagenome Libraries i) Library S279 The full-length sequence of the gene from clone M75bA2 was completed, as provided below.

25 1 tgggcggttt cgcggagtcg agcagggaga gatgctcctg ggtcgtacga gttggtacgg g r f r g v e q g e m l l g r t s w y

30 61 aggcatcgtt gaagatctca cgcctgcttg aatgcgcgcg gatatggaac ggaccggccg g g i v e d l t p a - m r a d m e r t g 35 121 cgctggcgat cggtgtcggc gtggggctgg cgagcctgag cccggtcgcg ctggcgacgc GC821-2 r>> hhh.

r a g d r c r r g a g e p e p g r a g d 181 cgccgcgggg caccgtgccg gtgttcaccc gatcggggac agcctgacgg acgagtattt 5 a a a g h r a g v h p i σ d s i t d e y 241 tgagccgttc ttccagtggg ggttctgcgg gaagtcgtgg gccgagattt tggtggagac

10 f e p f f q g f c g k s w a e i l v e 301 ggggcgggcg agcatgggcc cgacggcgca gcaggcgggg atcagcgagc cggagggatg t g r a s m g p t a q q a g i s e p e g 15 361 gtcggatccg cggaacacgg ggtatcagca caactgggcg cggtactcgt ggagctcctc w s d p r n t g y q h n w a r y s s s

20 421 agacgcgctg accgaggagt cgccgggggc gacgctgagc gtgctgcttg gggcggagta s d a l t e e s p g a t l s v l l g a e 481 cgcggtggtg ttcattggga ccaacgactt caatccgtcg tggccggcgt atcagagcgt 25 y a v v f i q t n d f n p s w p a y q s 541 gtatctgagc cagtggagcg acgagcagat cgacacgtac gtgaacgggg tggtgcagaa

30 v y l s q s d e q i d t y v n g v v q 601 catcgcgcag atggtggact cgctgaagtc ggtcggggcg aaggtggtgc ttgcgccgcc n i a q m v d s l k s v g a k v v l a p

35 661 ggtggatttt cagttcgcgg ggttcctgcg gaactcatgc ccggatccga tgctgcgcga p v d f q f a g f l r n s c p d p m l r

40 721 gcaggcgggt attctgacac ggaagtgcca cgaccgggtg cggtcgatgg cgcggcagaa e q a g i l t r k c h d r v r s m a r q 781 gcacgtggtg ttcgtggaca tgtggcggct gaaccgcgat ttgttcggca acgggttcgc

45 k h v v f v d m w r l n r d l f g n g f _, ,.' 'I,„P > Iiⁱl>,ⁱ' ^I' •' T' lⁱ .. ι GC821-2 ^'"^""> ^"^)

841 gatcagctac ggccttcgga acacggtgcg cgtgggggac tcggagatcg ggctgcaact a i s y g l r n t v r v g d s e i g l q 901 ggccgggctg acgggatcgg cggggctggt tccggacggg atccatccgc agcgggtggt l a g l t g s a g l v p d q i h p q r v 961 gcaggggatc tgggcgaatg cgttcatcgt gggtctgaac gcgcatgggg cgaacatcgc v q g i w a n a f i v g l n a h g a n i 1021 gcccatcggc gaggcggaga tgtgcgcgat ggggggggtc gtgtacgggg gaacggacac a p i g e a e m c a m g g y g g t d 1081 gctggcgaac ttcctgccgc cggtcgcggg ctacgtggag gacttccgca acgcggggga t l a n f l p p v a g y v e d f r n a g 1141 cttcgtgtgc acggcggact tcaaccatga ccttggcgtg acgccgacgg acatcttcgc d f v c t a d f n h d l g v t p t d i f 1201 gttcatcaac gcgtggttca tgaatgatcc ctcggcgcgg atgagcaacc cggagcacac a f i n a w f m n d p s a r m s n p e h 1261 gcagatcgag gacatcttcg tgtttctgaa tctgtggctg gtggggtgct gaggcagagt t q i e d i f v f l n 1 1 v g c - g r 1321 gggaaggggg tcagcccact tcgcgcgtct ggaagaggat gacggcgacg gagaggaaga v g r g s a h f a r l e e d d g d g e e

In the sequence of S279_M75bA2 provided above (DNA, SEQ ID NO:80; and amino acid sequence, SEQ ID NO:81), the coding sequence running from position 104 through 1312 is shown on a grey background. Conserved structural motifs are shown underlined and in bold. The derived amino acid sequence showed the highest homology to a hypothetical protein (Y17D7A.2) from Caenorhabditis elegans (BlastP2; swisspir), although with a f .* f-, ., , -- '' - , , GC821-2 .

very high E-value of 2.5 (i.e., indicating a non-reliable hit). The fact that no esterase is among the homologous proteins identified by the BlastP2-analysis indicates that this enzyme is a rather unusual "GDSL-type" esterase. Furthermore, the enzyme is characterized by unusually long peptides between the N-terminus and the "GDSL'Vmotif and the "DXXH"-motif of block V (containing the active site aspartic acid and histidine) and the C-terminus. The very C-terminal sequence shows similarity to a membrane lipoprotein lipid attachment site. A corresponding signal sequence of lipoproteins was not identified. The gene encoding M75bA5 was amplified but no further efforts were taken for this enzyme since it did not have the conserved amino acids typical of the perhydrolase of the present invention. ii) Library S248 The clone carrying the sequence-tag SP7_3j5h which could have been part of a gene encoding a "GDSL"-type esterase was identified (M31bAl 1), and the sequence was elongated. This facilitated the determination that this sequence did not encode a "GDSL- type" esterase, because block V could not be identified. The generation of this amplicon can be explained by an "unspecific" hybridization of primer 5h with the first mismatches at nucleotides 10, 14 and 15 from the 3 '-terminus of the primer. The sequence showed the highest homology to a hypothetical protein (KO3E5.5) from Caenorhabditis elegans with an E-value of 1.6, indicating a non-reliable hit. The sequence-tag from clone S248_M3 IbAl 1 is provided below.

1 cggaattatc atgctgggtt ttaatgacca gcgcgagagg atcaacgaca acctcgatta r n y h a g f - - p a r e d q r q p r l g i i m l q £ n d q r e r i n d n l d e l s c w v l m t s a r g s t t t s i 61 ctgggacgcc taccactccg tcctgggcga gagacagttt tattccggca attccaagat l g r l p l r p g r e t v l f r q f q d . GC821-2 ^ ^

y w d a y h s v l g e r q f y s g n s k t g t p t t p s w a r d s f i p a i p r 121 gttcgtcccc atcaccaaga tcgcggtgaa ggcgcgcaag acccggttca ccaatcagat 5 v r p h h q d r g e g a q d p v h q s d m f v p i t k i a v k a r k t r f t n q e s s p s p r s r - r r a r p g s p i r ^■4 o — oo-o-o 181 ttttcctcag tccggccgca acgtcgatgt caccaccacg gacggcacac tcccccacgc 10 f s s v r p q r r c h h h g r h t p p r i f p q s g r n v d v t t t d g t 1 p h f f l s p a a t s m s p p r t a h s p t oco_co_ooo 241 caccatgtcc ctggtcgagc actacatccg ggcctgccgc ctgcgcaccc agatcgttcc 15 h h v p g r a l h p g l p p a h p d r s a t m s l v e h y i r a c r l r t q i v p p c p w s s t t s g p a a c a p r s f 301 ggccctgatc gttaacggcg attgcgaagg catgtacagc atctatgtcg gctggtcgaa 20 g p d r - r r l r r v q h l c r l v e p a l i v n g d e e g m y s i y v g w s r p - s l t a i a k a c t a s m s a g r 361 aaccaccaag catgttgttt cacgtgaaac aaagccggtc gaaagcgacg gcatggaatt 25 n h q a c e f t - n k a g r k r r h g i k t t k h v v s r e t k p v e s d g m e k p p s m l f h v k q s r s k a t a w n 421 tcccgaactg ggcgaagccg acgacatcac cgaagaaacg . cttgagtgtg gccttcccga 30 s r t g r s r r h h r r n a - v w p s r f p e l g e a d d i t e e t l e e g l p f p n w a k p t t s p k k r l s v a f p 481 catcgaattg atctcggacg ccgatcttct cgtccttcca ccagcgccga caacattcca 35 h r i d i g r r s s r p s t s a d n i p d i e l i s d a d l l v l p p a p t t f t s n - s r t p i f s s f h q r r q h s 541 aggcgcttga gatgggcggg ttcggtcacg atcttgcgcc gtggacaagg gcaaggtccg 40 r r l r w a g s v t i l r r g q g q g p q g a - d g r v r s r s c a v d k g k v k a l e g g f g h d l a p w t r a r s 601 cagatgatcg acgaggcgcg atcaccgaga tgccgcgacg atctgtcgac gctatgtcac 45 q m i d e a r s p r c r d d i s t i c h r r - s t r r d h r d a a t i c r r y v a d d r r g a i t e m p r r s v d a m s GC821-2 C^"^

661 cagcgcatgt ccgacggtgg aatgcaagac aggtnggntn gatcgggg{SEQ ID NO: 120) q r m s d g g m q d r ? ? ? s g(SEQ ID N0:121) t s a c p t v e c k t g ? ? d r (SEQ ID NO: 122) p a h v r r w n a r q ? ? ? i g(SEQ ID NO:123)

In the above sequence-tag of the clone S248_M31bAl 1, the primers 3j and 5h are indicated. Hybridization between primer and template is indicated by arrows, mismatches by open circles. Putative conserved structural motifs are indicated in bold and underlined. Several further sequence-tags were generated using different primer pairs of the primers 2 and 5 but none turned out to encode a "GDSL"-type esterases. The screening of this library was completed. iii) Library M091 The elongation of the amplicon SP3_lj5h, which was identified in the insert-DNA of clone M24dG12 proved that the corresponding sequence does not encode a "GDSL"- type esterase. Whereas the sequence encoding a putative block V (DGTHP; SEQ ED NO: 124) was found, the corresponding sequence encoding block I was missing. The amplicon was generated due to an "unspecific" hybridization of primer lj with the first mismatches at positions 5, 10, 11 and 12 from the 3'-terminus of the primer. The sequence-tag of clone M091_ M24dG12 s shown below:

1 gcctgatggc ttcgagttcg tcgaattcac ctcgccccag cccggcgtgc tggaggcggt a - w l r v r r i h l a p a r r a g g g p d g f e f v e f t s p q p g v l e a l m a s s s s n s p r p s p a c w r r 61 gtttgaaaag ctgggtttca ccctggtcgc caagcaccgg tccaaggatg tggtgctgta v - k a g f h p g r q a p v q g c g a v v f e k l g f t l v a k h r s k d v v l GC821-2 ^

e l k s w v s p w s p s t g p r m w e e 121 ccgccagaac ggcatcaact tcatcctgaa ccgcgagccc cacagccagg ccgcctactt 5 p p e r h q l h p e p r a p q p g r l l y r q n g i n f i l n r e p h s q a a y t a r t a s t s s - t a s p t a r p p t

10 181 tggtgccgag catggcccct ccgcctgtgg cctggccttc cgtgtgaagg atgcgcataa w c r a w p l r l w p g l p e e g c a - f g a e h g p s a c g l a f r v k d a h 15 l v p s m a p p p v a w p s v - r rα r i 241 ggcttataac cgcgcgctgg aactgggcgc ccagcccatc gagatcccca ccggccccat g l - p r a g t g r p a h r d p h r p h

20 k a y n r a l e l g a q p i e i p t g p r l i t a r w n w a p s p s r s p p a p cccooo — 0000— O-O COD- — o 301 ggaactgcgc ctgcccgcca tcaagggcat tggcggcgcc gcctctgtat ttgatcgacc

25 g t a p a r h q g h w r r r l c i - s t m e l r l p a i k g i g g a a s v f d r w n c a c p p s r a l a a p p l y l i d

30 361 gctttgaaga cggcaagtcc atctacgaca tcgacttcga gttcatcgaa ggcgtggacc a l k t a s p s t t s t s s s s k a w t p l - r r q v h l r h r l r v h r r r g 35 r f e d g k s i y d i d f e f i e g v d 421 gccgccccgc ggggcatggc ctgaacgaga tcgatcacct cacgcacaac gtgtaccggg a a p r g m a - t r s i t s r t t c t g 40 P P P ^r g a w p e r -d r s p h a q r v p r r p a g h q 1 n e i d h l t h n v y r 481 gccgcatggg cttctgggcc aacttctacg aaaagctgtt caacttccgc gaaatccgct

45 a a w a s g p t s t k s c s t s a k s a g p h g 1 1 g q l l r k a v q l p r n p g r m g f w a n f y e k l f n f r e i r 541 acttcgacat ccagggcgaa tacacgggcc tgacctccaa ggccatgacc gcgcccgacg

50 t s t s r a n t r a - P P ^r P ~ P ^r P t l l r h p g r i h g p d l q g h d r a r y f d i q g e y t g I t s k a m t a p d

55 601 gcaagattcg catcccgctg aacgaagagt ccaagcaggg cggcggccag atcgaagaat a r f a s r - t k s p s r a a a r s k n i .. n_|ⁱ.„μi l ir,.' "l" ,- "l"., l i GC821-2 ^f^) ^)

r q d s h p a e r r v q a g r r p d r r g k i r i p l n e e s k q 9 g g q i e e 661 ttttgatgca attcaacggc gagggcattc agcacatcgc gctgatctgc gacaacctgc f - c n s t a r a f s t s r - s a t t c i f d a i q r r g h s a h r a d l r q p f l m q f n g e g i q h i a l i c d n l 721 tggacgtggt ggacaagctg ggcatggccg gcgtgcagct ggccaccgcg cccaacgagg w t w w t s w a w p a c s w p p r p t r h g 1 d m 781 tctattacga aatgctggac acccgcctgc ccggccacgg ccagccggtg cccgagctgc s i t k c w t p a c p a t a s r c p s c g l l r n a g h p p a r p r p a g a r a v y y e m l d t r l p g h g q p v p e l - o-oo— cooo-o 841 agtcgcgcgg catcttgctg gacggcacca cggccgacgg cacgcacccg cctgctagct s r a a s c w t a p r p t a r t r l l a a v a r h l a g r h h g r r h a p a c - q s r g i l l d g t t a d σ t h p p a s ooo-o 901 tcagatcttc tccacgccca tgctgggccc ggtgttcttc gaattcatcc agcgcgaggg s d l l h a h a g p g v l r i h p a r g l q i f s t p m l g p v f f e f i q r e f r s s p r p c w a r e s s n s s s a r 961 cgactaccgc gacggctttg gcgaaggcaa cttcaaggcg ctgttcgagt cgctggaacg r i p r r l w r r q l q g a v r v a g t g d y r d g f g e g n f k a l f e s l e a t t a t a l a k a t s r r c s s r w n 1021 cgaccagatc cgccgtggtg tgctgaacac ataagacatc agacatccag ggttaaccct r p d p p w c a e h i r h q t s r v n p r d q i r r g v l n t - d i r h p g i t a t r s a v v c - t h k t s d i q g ^_ P 1081 gcacaggtgc ctatactgcg cgctccccgg aactcaaaag gatcccgatg tcgctccgta GC821-2

a q v p i l r a p r n s k g s r c r s v l h r c l y e a l p g t q k d p d v a p c t g a y t a r s p e l k r i p m s i r

1141 gcaccctgtt cagcaccctt ttggccggcg cagccactgt cgcgctggcg cagaacccgt a p e s a p f w p a q p l s r w r r t r - h p q h p f g r r s h c r a g a e p s t l f s t l l a g a a t v a l a q n p

1201 ctgcccgctc acatcg (SEQ ID NO: 125) 1 p a h i (SEQ ID NO:126) v c p 1 t s (SEQ ID NO: 127) s a r s h (SEQ ID NO: 128)

Sequence-tag of the clone M091_M24dG12. The primers lj and 5h are indicated in the above sequence-tag of the clone M09 l_M24dGl 2. Hybridization between primer and template is indicated by arrows, mismatches by open circles. Putative conserved structural motifs are depicted in bold and underlined. A further sequence-tag (SPl_2b5h) was generated using the primer pair 2b/5h. A BlastX-analysis of the sequence from this tag yielded the highest homology to an arylesterase from Agrobacterium tumefaciens, with 70% identity. The single clone carrying the corresponding gene was identified (M4aEl 1) and the full length sequence determined to be as shown below:

1 atgaagacca ttctcgccta tggcgacagc ctgacctatg gggccaaccc gatcccgggc m k t i l a y q d s 1 t y g a n p i p g 61 gggccgcggc atgcctatga ggatcgctgg cccacggcgc tggagcaggg gctgggcggc g p r h a y e d r w p t a l e q g l g g 121 aaggcgcggg tgattgccga ggggctgggt ggtcgcacca cggtgcatga cgact^gttt k a r v i a e g l g q r t t v h d d w f

181 gcgaatgcgg acaggaacgg tgcgcgggtg ctgccgacgc tgctcgagag ccattcgccg a n a d r n g a r v l p t l i e s h s p

241 ctcgacctga tcgtcatcat gctcggcacc aacgacatca agccgcatca cgggcggacg - GC821-2 ^

I d l i v i m l a n d i k p h h g r t 301 gccggcgagg ccgggcgggg catggcgcgg ctggtgcaga tcatccgcgg gcactatgcc a g e a g r g m a r l v q i i r g h y a 5 361 ggccgcatgc aggacgagcc gcagatcatc ctcgtgtcgc cgccgccgat catcctcggc g r m q d e p q i i l v s P P P i i l g 421 gactgggcgg acatgatgga ccatttcggc ccgcacgaag cgatcgccac ctcggtggat 10 d w a d m m d h f g p h e a i a t s v d 481 ttcgctcgcg agtacaagaa gcgggccgac gagcagaagg tgcatttctt cgacgccggc f a r e y k k r a d e q k v h f f d a g 15 541 acggtggcga cgaccagcaa ggccgatggc atccacctcg acccggccaa tacgcgcgcc t v a t t s k a d σ i h 1 d p a n t r a 601 atcggggcag ggctggtgcc gctggtgaag caggtgctcg gcctgtaa(SEQ ID NO: 129) i g a g l v p l v k q v l g l -{SEQ ID NO: 130) 20 In the above sequence, the conserved structural motifs are shown in bold and underlined. The BlastP-analysis with the deduced full length amino acid sequence identified the same hit with a identity of 48%. The primary stracture of this enzyme 25 showed the "GRTT'-motif proving the usefulness of the primers directed towards block 2 for the identification of "GRTT'-esterases. The gene was amplified to introduce unique restriction enzyme recognition sites and the absence of second site mutations was confirmed by sequencing. The gene was ligated to pET26 and was expressed in E. coli Rosetta (DE3). The vector map is provided in Figure 5. Expression and control strains 30 were cultivated in LB in the presence of kanamycin (25 μg/ml), chloramphenicol (12.5 μg/ml), and 1% glucose. At an ODsso of 1, expression was induced by addition of 100 μM IPTG. Samples were taken at 2, 4, and 20 hours after induction. Cells were separated from the culture supernatant by centrifugation and after resuspending in sample buffer, they wee incubated for 10 minutes at 90°C. An amount of cells representing an 35 ODsso of 0.1 was applied to a 4-12% acryl amide gradient gel, which was stained with Coomassie Brilliant Blue R250. GC821-2

Strong overexpression of the gene was detected aheady 2 h after induction with 100 μM IPTG, as determined by SDS-PAGE analysis of crude cell extracts from E. coli Rosetta (DE3) pET26_M4aEl 1. The amount of protein representing M4aEl 1 (calculated size 23.2 kDa) increased further over time. Esterase activity of crude cell extracts from strains expressing the "GDSL"-type esterase M4aEl 1 was determined. An amount of cells corresponding to an O.D.sso = 2 were resuspended in 200 μl of 5mM Tris/HCl pH 8.0, and lysed by ultrasonication. Then, 20 μl of each sample were used to determine the esterase activity towards p- nitrophenyl butyrate in a total volume of 200 μl. The activity was corrected for the background activity of the control strain. The activity towards 7-nitrophenylbutyrate reached about 125 nmol/ml x min 20 h after induction. In addition, SDS-PAGE analysis of the soluble and insoluble fraction of crude cell extracts from E. coli Rosetta (DE3) pET26_M4aEl 1 was conducted. Cells from a culture induced with 100 μM EPTG and harvested 4 h and 20h after induction were lysed by ultrasonication and separated into soluble and insoluble fraction by centrifugation.

Sample buffer was added and directly comparable amounts of soluble and insoluble fractions were applied to a 4-12% acryl amide gradient gel, which was stained with Coomassie Brilliant Blue R250. The results of this analysis of the solubility revealed that M4aEl 1 is partially (estimated 80%) soluble. The screening of the library M091 was completed. Thus, in total nine different "GDSL' ype esterases were identified in 6 different large insert metagenomic libraries and the esterases/lipases BRAIN's library comprising more than 4.3 Gbp. Eight of these genes were heterologously expressed in E. coli. The resulting enzyme samples of seven clones were characterized for the desired perhydrolase activity. Two of the enzymes displayed this activity. Table 3 summarizes the screening, expression and characterization of the metagenomic "GDSL"-type esterases. , , GC821-2 "

Table 3: Expression and Characterization of Metagenomic "GDSL"-Type Esterases

GDSL -type Homology Expression Solubility Activity Perhydrolase Esterase Level Activity S248 M2bBl l 12.9% 136 - S248 M40cD4 14.8% 50 -/+⁶ S248 M44aA5 12.4% 75 -/+ S261 M2aA12 36.9% 72 +' S279 M70aE8 11.9% + 167 - S279 M75bA2 5.7% n.d n.d. n.d. n.d. M091 M4aEll 33.9% ++ 125 n.d. Estl05 4.3% . n.d. Estl l4 7.8% n.d. n.d. 13 identity to the prototype enzyme from M. smegmatis calculated with the dialign algorithm (Morgenstern etal, 1996) expression level: + moderate overexpression; ++ strong overexpression; very strong overexpression as judged from SDS-PAGE-analysis as judged by SDS-PAGE-analysis towards ^-nitrophenyl butyrate; given as nmol/(ml x min) not determined

10 perhydrolysis activity 2x background perhydrolase activity more than 2x background

15 Engineering of the Perhydrolase Based on the structure of the perhydrolase, residues which may alter substrate specificity (e.g., Km, kcat, Vmax, chain length, etc.) and or the multimeric nature of the protein were identified. However, it is not intended that the present invention be limited to any particular residues. Nonetheless, site saturation libraries of residues D10, L12, 20 T13, W14, W16, S54, A55, N94, K97, Y99, P146, W149, F150, 1194, F196, are constracted, as well as combinatorial libraries of residues: E51A, Y73A, H81D, T127Q and single mutations of the active site residues D192A, HI 95 A and a site saturation _ i "' t- i ,■' "--» •« .^■"!•> P it .' ' " -ι „„,ι ⁱi, ^t GC821-2 "~^

library of the conserved D95. Methods for production of such libraries are known to those skilled in the art and include commercially available kits as the Stratagene Quikchange Site-directed mutagenesis kit and/or Quikchange Multi-Site-directed mutagenesis kit. 5 Perhydrolase Activity The use of enzymes obtained from microorganisms is long-standing. Indeed there are numerous biocatalysts known in the art. For example, U.S. Patent No. 5,240,835 (herein incorporated by reference) provides a description of the transacylase activity of 10 obtained from C. oxydans and its production. In addition, U.S. Patent No. 3,823,070 (herein incorporated by reference) provides a description of a Corynebacterium that produces certain fatty acids from an n-paraffin. U.S. Patent No.4,594,324 (herein incorporated by reference) provides a description of a Methylcoccus capsulatus that oxidizes alkenes. Additional biocatalysts are known in the art (See e.g., U.S. Patent Nos. 15 4,008,125 and 4,415,657; both of which are herein incorporated by reference). EP 0280 232 describes the use of a C. oxydans enzyme in a reaction between a diol and an ester of acetic acid to produce monoacetate. Additional references describe the use of a C. oxydans enzyme to make chiral hydroxycarboxyhc acid from a prochiral diol. Additional details regarding the activity of the C. oxydans transacylase as well as the culture of C. 20 oxydans, preparation and purification of the enzyme are provided by U.S. Patent No. 5,240,835 (incorporated by reference, as indicated above). Thus, the transesterification capabilities of this enzyme, using mostly acetic acid esters were known. However, the determination that this enzyme could carry out perhydrolysis reaction was quite unexpected. It was even more surprising that these enzymes exhibit very high 25 efficiencies in perhydrolysis reactions. For example, in the presence of tributyrin and water, the enzyme acts to produce butyric acid, while in the presence of tributyrin, water and hydrogen peroxide, the enzyme acts to produce mostly peracetic acid and very little GC821-2 ^ -, ^

butyric acid. This high perhydrolysis to hydrolysis ratio is a unique property exhibited by the perhydrolase class of enzymes of the present invention and is a unique characteristic that is not exhibited by previously described lipases, cutinases, nor esterases. The perhydrolase of the present invention is active over a wide pH and temperature range and accepts a wide range of substrates for acyl transfer. Acceptors include water (hydrolysis), hydrogen peroxide (perhydrolysis) and alcohols (classical acyl transfer). For perhydrolysis measurements, enzyme is incubated in a buffer of choice at a specified temperature with a substrate ester in the presence of hydrogen peroxide. Typical substrates used to measure perhydrolysis include esters such as ethyl acetate, triacetin, tributyrin, ethoxylated neodol acetate esters, and others. In addition, the wild type enzyme hydrolyzes nitrophenylesters of short chain acids. The latter are convenient substrates to measure enzyme concentration. Peracid and acetic acid can be measured by the assays described herein. Nitrophenylester hydrolysis is also described. Although the primary example used during the development of the present invention is the M. smegmatis perhydrolase, any perhydrolase obtained from any source which converts the ester into mostly peracids in the presence of hydrogen peroxide finds use in the present invention.

Substrates In some preferred embodiments of the present invention, esters comprising aliphatic and/or aromatic carboxylic acids and alcohols are utilized with the perhydrolase enzymes of the present invention. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. In additional embodiments, triacetin, tributyrin, neodol esters, and/or ethoxylated neodol esters serve as acyl donors for peracid formation. GC821-2 ' ,

Cleaning and Detergent Formulations The detergent compositions of the present invention are provided in any suitable form, including for example, as a liquid diluent, in granules, in emulsions, in gels, and 5 pastes. When a solid detergent composition is employed, the detergent is preferably formulated as granules. Preferably, the granules are formulated to additionally contain a protecting agent (See e.g., U.S. Appln. Ser. No. 07/642,669 filed January 17, 1991, incorporated herein by reference). Likewise, in some embodiments, the granules are formulated so as to contain materials to reduce the rate of dissolution of the granule into

10 the wash medium (See e.g., U.S. Patent No. 5,254,283, incorporated herein by reference in its entirety). In addition, the perhydrolase enzymes of the present invention find use in formulations in which substrate and enzyme are present in the same granule. Thus, in some embodiments, the efficacy of the enzyme is increased by the provision of high local concentrations of enzyme and substrate (See e.g., U.S. Patent Application Publication

15 US2003/0191033, herein incorporated by reference). Many of the protein variants of the present invention are useful in formulating various detergent compositions. A number of known compounds are suitable surfactants useful in compositions comprising the protein mutants of the invention. These include nonionic, anionic, cationic, anionic or zwitterionic detergents (See e.g., U.S. Patent Nos

20 4,404, 128 and 4,261 ,868). A suitable detergent formulation is that described in U.S. Patent No. 5,204,015 (previously incorporated by reference). Those in the art are familiar with the different formulations which find use as cleaning compositions. As indicated above, in some preferred embodiments, the detergent compositions of the present invention employ a surface active agent (i.e., surfactant) including anionic, non-ionic and

25 ampholytic surfactants well known for their use in detergent compositions. Some surfactants suitable for use in the present invention are described in British Patent Application No. 2 094 826 A, incorporated herein by reference. In some embodiments, t ._' ( ._m,ι ι I .. GC821-2 ^ ^)

mixtures surfactants are used in the present invention. Suitable anionic surfactants for use in the detergent composition of the present invention include linear or branched alkylbenzene sulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefin sulfonates; alkane sulfonates and the like. Suitable counter ions for anionic surfactants include alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alkanolamines having 1 to 3 alkanol groups of carbon number 2 or 3. Ampholytic surfactants that find use in the present invention include quaternary ammonium salt sulfonates, betaine-type ampholytic surfactants, and the like. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule. Nonionic surfactants that find use in the present invention generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like. In some preferred embodiments, the surfactant or surfactant mixture included in the detergent compositions of the present invention is provided in an amount from about 1 weight percent to about 95 weight percent of the total detergent composition and preferably from about 5 weight percent to about 45 weight percent of the total detergent composition. In various embodiments, numerous other components are included in the compositions of the present invention. Many of these are described below. It is not intended that the present invention be limited to these specific examples. Indeed, it is contemplated that additional compounds will find use in the present invention. The descriptions below merely illustrate some optional components. Proteins, particularly the perhydrolase of the present invention can be formulated into known powdered and liquid detergents having pH between 3 and 12.0, at levels of about .001 to about 5% (preferably 0.1% to 0.5%) by weight. In some embodiments, _„ GC821-2 ^' ^)

these detergent cleaning compositions further include other enzymes such as proteases, amylases, mannanases, peroxidases, oxido reductases, cellulases, lipases, cutinases, pectinases, pectin lyases, xylanases, and/or endoglycosidases, as well as builders and stabilizers. 5 In addition to typical cleaning compositions, it is readily understood that perhydrolase variants of the present invention find use in any purpose that the native or wild-type enzyme is used. Thus, such variants can be used, for example, in bar and liquid soap applications, dishcare formulations, surface cleaning applications, contact lens cleaning solutions or products, , waste treatment, textile applications, pulp-bleaching,

10 disinfectants, skin care, oral care, hair care, etc. Indeed, it is not intended that any variants of the perhydrolase of the present invention be limited to any particular use. For example, the variant perhydrolases of the present invention may comprise, in addition to decreased allergenicity, enhanced performance in a detergent composition (as compared to the wild-type or unmodified perhydrolase).

15 The addition of proteins to conventional cleaning compositions does not create any special use limitations. In other words, any temperature and pH suitable for the detergent are also suitable for the present compositions, as long as the pH is within the range in which the enzyme(s) is/are active, and the temperature is below the described protein's denaturing temperature. In addition, proteins of the invention find use in

20 cleaning, bleaching, and disinfecting compositions without detergents, again either alone or in combination with a source of hydrogen peroxide, an ester substrate (e.g., either added or inherent in the system utilized, such as with stains that contain esters, pulp that contains esters etc), other enzymes, surfactants, builders, stabilizers, etc. Indeed it is not intended that the present invention be limited to any particular formulation or application.

25

Substrates „ -

GC821-2 β>

In some preferred embodiments of the present invention, esters comprising aliphatic and/or aromatic carboxylic acids and alcohols are utilized with the perhydrolase enzymes in the detergent formulations of the present invention. In some preferred embodiments, the substrates are selected from one or more of the following: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleic acid. Thus, in some preferred embodiments, detergents comprising at least one perhydrolase, at least one hydrogen peroxide source, and at least one ester acid are provided.

Hydmlases In addition to the perhydrolase described herein, various hydrolases find use in the present invention, including but not limited to carboxylate ester hydrolase, thioester hydrolase, phosphate monoester hydrolase, and phosphate diester hydrolase which act on ester bonds; a thioether hydrolase which acts on ether bonds; and α-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase, acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl-peptide hydrolase which act on peptide bonds, all these enzymes having high perhydrolysis to hydrolysis ratios (e.g., >1). Preferable among them are carboxylate ester hydrolase, and peptidyl-peptide hydrolase. Suitable hydrolases include: (1) proteases belonging to the peptidyl-peptide hydrolase class (e.g., pepsin, pepsin B, rennin, trypsin, chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin, aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein, gastrisin, cathepsin D, bromelin, keratinase, chymotrypsin C, pepsin C, aspergillopeptidase B, urokinase, carboxypeptidase A and B, and aminopeptidase); (2) carboxylate ester hydrolase including carboxyl esterase, lipase, pectin esterase, and chlorophyllase; and (3) enzymes having high perhydrolysis to hydrolysis ratios. Especially effective among them are lipases, as well as esterases that - C T " GC821-2

exhibit high perhydrolysis to hydrolysis ratios, as well as protein engineered esterases, cutinases, and lipases, using the primary, secondary, tertiary, and/or quaternary stractural features of the perhydrolases of the present invention. The hydrolase is incorporated into the detergent composition as much as required 5 according to the purpose. It should preferably be incorporated in an amount of 0.0001 to 5 weight percent, and more preferably 0.02 to 3 weight percent,. This enzyme should be used in the form of granules made of crade enzyme alone or in combination with other enzymes and/or components in the detergent composition. Granules of crade enzyme are used in such an amount that the purified enzyme is 0.001 to 50 weight percent in the 10 granules. The granules are used in an amount of 0.002 to 20 and preferably 0.1 to 10 weight percent. In some embodiments, the granules are formulated so as to contain an enzyme protecting agent and a dissolution retardant material (i.e., material that regulates the dissolution of granules during use).

15 Cationic. Surfactants and T^ng-Chain Fatty Acid Salts Such cationic surfactants and long-chain fatty acid salts include saturated or fatty acid salts, alkyl or alkenyl ether carboxylic acid salts, a-sulfofatty acid salts or esters, amino acid-type surfactants, phosphate ester surfactants, quaternary ammonium salts including those having 3 to 4 alkyl substituents and up to 1 phenyl substituted alkyl 20 substituents. Suitable cationic surfactants and long-chain fatty acid salts include those disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference. The composition may contain from about 1 to about 20 weight percent of such cationic surfactants and long-chain fatty acid salts.

25 Buil rs In some embodiments of the present invention, the composition contains from about 0 to about 50 weight percent of one or more builder components selected from the w

GC821-2 ^

group consisting of alkali metal salts and alkanolamine salts of the following compounds: phosphates, phosphonates, phosphonocarboxylates, salts of amino acids, * aminopolyacetates high molecular electrolytes, non-dissociating polymers, salts of dicarboxylic acids, and aluminosilicate salts. Examples of suitable divalent sequestering agents are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference. In additional embodiments, compositions of the present invention contain from about 1 to about 50 weight percent, preferably from about 5 to about 30 weight percent, based on the composition of one or more alkali metal salts of the following compounds as the alkalis or inorganic electrolytes: silicates, carbonates and sulfates as well as organic alkalis such as triethanolamine, diethanolamine, monoethanolamine and triisopropanolamine.

Anti-Rftrifiposition Agents In yet additional embodiments of the present invention, the compositions contain from about 0.1 to about 5 weight percent of one or more of the following compounds as antiredeposition agents: polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose. In some preferred embodiments, a combination of carboxymethyl-cellulose and/or polyethylene glycol are utilized with the composition of the present invention as useful dirt removing compositions.

Bleaching Agents The use of the perhydrolases of the present invention in combination with additional bleaching agent(s) such as sodium percarbonate, sodium perborate, sodium sulfate/hydrogen peroxide adduct and sodium chloride/hydrogen peroxide adduct and/or a photo-sensitive bleaching dye such as zinc or aluminum salt of sulfonated phthalocyanine further improves the detergent effects. In additional embodiments, the perhydrolases of I ' , ill »i IUt "* -'' ^,r,f ' i E "' " .- ' GC821-2 n ~\

the present invention are used in combination with bleach boosters (e.g., TAED and/or NOBS).

Bluing Agents and Flnnrespent Dyes 5 In some embodiments of the present invention, bluing agents and fluorescent dyes are incorporated in the composition. Examples of suitable bluing agents and fluorescent dyes are disclosed in British Patent Application No. 2 094 826 A, the disclosure of which is incorporated herein by reference.

10 Caking Inhibitors In some embodiments of the present invention in which the composition is powdered or solid, caking inhibitors are incorporated in the composition. Examples of suitable caking inhibitors include p-toluenesulfonic acid salts, xylenesulfonic acid salts, acetic acid salts, sulfosuccinic acid salts, talc, finely pulverized silica, clay, calcium 15 silicate (e.g., Micro-Cell by Johns Manville Co.), calcium carbonate and magnesium oxide.

Antioxidants The antioxidants include, for example, tert-butyl-hydroxytoluene, 4,4'- 20 butylidenebis(6-tert-butyl-3-methylphenol), 2,2'-butylidenebis(6-tert-butyl-4- methylphenol), monostyrenated cresol, distyrenated cresol, monostyrenated phenol, distyrenated phenol and l,l-bis(4-hydroxy-phenyl)cyclohexane.

Snlπhi liters 25 In some embodiments, the compositions of the present invention also include solubilizers, including but not limited to lower alcohols (e.g., ethanol, benzenesulfonate salts, and lower alkylbenzenesulfonate salts such as p-toluenesulfonate salts), glycols GC821-2 O

such as propylene glycol, acetylbenzene-sulfonate salts, acetamides, pyridinedicarboxylic acid amides, benzoate salts and urea. In some embodiments, the detergent composition of the present invention are used in a broad pH range of from acidic to alkaline pH. In a preferred embodiment, the detergent composition of the present invention is used in mildly acidic, neutral or alkaline detergent wash media having a pH of from above 4 to no more than about 12. In addition to the ingredients described above, perfumes, buffers, preservatives, dyes and the like also find use with the present invention. These components are provided in concentrations and forms known to those in the art. In some embodiments, the powdered detergent bases of the present invention are prepared by any known preparation methods including a spray-drying method and a granulation method. The detergent base obtained particularly by the spray-drying method and/or spray-drying granulation method are preferred. The detergent base obtained by the spray-drying method is not restricted with respect to preparation conditions. The detergent base obtained by the spray-drying method is hollow granules which are obtained by spraying an aqueous slurry of heat-resistant ingredients, such as surface active agents and builders, into a hot space. After the spray-drying, perfumes, enzymes, bleaching agents, inorganic alkaline builders may be added. With a highly dense, granular detergent base obtained such as by the spray-drying-granulation method, various ingredients may also be added after the preparation of the base. When the detergent base is a liquid, it may be either a homogeneous solution or an inhomogeneous dispersion. The detergent compositions of this invention may be incubated with fabric, for example soiled fabrics, in industrial and household uses at temperatures, reaction times and liquor ratios conventionally employed in these environments. The incubation conditions (t.e., the conditions effective for treating materials with detergent compositions according to the present invention), are readily ascertainable by those of GC821-2

skill in the art. Accordingly, the appropriate conditions effective for treatment with the present detergents correspond to those using similar detergent compositions which include wild-type perhydrolase. As indicated above, detergents according to the present invention may 5 additionally be formulated as a pre-wash in the appropriate solution at an intermediate pH where sufficient activity exists to provide desired improvements softening, depilling, pilling prevention, surface fiber removal or cleaning. When the detergent composition is a pre-soak (e.g., pre-wash or pre-treatment) composition, either as a liquid, spray, gel or paste composition, the perhydrolase enzyme is generally employed from about 0.00001%

10 to about 5% weight percent based on the total weight of the pre-soak or pre-treatment composition. In such compositions, a surfactant may optionally be employed and when employed, is generally present at a concentration of from about 0.0005 to about 1 weight percent based on the total weight of the pre-soak. The remainder of the composition comprises conventional components used in the pre-soak (e.g., diluent, buffers, other

15 enzymes (proteases), etc.) at their conventional concentrations.

Cleaning Compositions Comprising Perhydrolase The cleaning compositions of the present invention may be advantageously employed for example, in laundry applications, hard surface cleaning, automatic

20 dishwashing applications, as well as cosmetic applications such as dentures, teeth, hair and skin. However, due to the unique advantages of increased effectiveness in lower temperature solutions and the superior color-safety profile, the enzymes of the present invention are ideally suited for laundry applications such as the bleaching of fabrics. Furthermore, the enzymes of the present invention find use in both granular and liquid

25 compositions. The enzymes of the present invention also find use in cleaning additive products. Cleaning additive products including the enzymes of the present invention are ideally GC821-2 ^

suited for inclusion in wash processes where additional bleaching effectiveness is desired. Such instances include, but are not limited to low temperature solution cleaning applications. The additive product may be, in its simplest form, one or more of the enzymes of the present invention. Such additive may be packaged in dosage form for 5 addition to a cleaning process where a source of peroxygen is employed and increased bleaching effectiveness is desired. Such single dosage form may comprise a pill, tablet, gelcap or other single dosage unit such as pre-measured powders or liquids. A filler or carrier material may be included to increase the volume of such composition. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate

10 and silicate as well as talc, clay and the like. Filler or carrier materials for liquid compositions may be water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to; methanol, ethanol, propanol and isopropanol. The compositions may contain from about 5% to about 90% of such materials. Acidic fillers can be used to reduce pH.

15 Alternatively, the cleaning additive may include activated peroxygen source defined below or the adjunct ingredients as defined below. The cleaning compositions and cleaning additives of the present invention require an effective amount of the enzymes provided by the present invention. The required level of enzyme may be achieved by the addition of one or more species of the M. smegmatis

20 perhydrolase, variants, homologues, and/or other enzymes or enzyme fragments having the activity of the enzymes of the present invention. Typically, the cleaning compositions of the present invention comprise at least 0.0001 weight percent, from about 0.0001 to about 1, from about 0.001 to about 0.5, or even from about 0.01 to about 0.1 weight percent of at least one enzyme of the present invention.

25 In some embodiments, the cleaning compositions of the present invention comprise a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, said peroxygen source being selected from the group GC821-2

consisting of: (i) from about 0.01 to about 50, from about 0.1 to about 20, or even from about 1 to 10 weight percent of a per-salt, an organic peroxyacid, urea hydrogen peroxide and mixtures thereof; 5 (ii) from about 0.01 to about 50, from about 0.1 to about 20, or even from about 1 to 10 weight percent of a carbohydrate and from about 0.0001 to about 1, from about 0.001 to about 0.5, from about 0.01 to about 0.1 weight percent carbohydrate oxidase; and (iii) mixtures thereof. 10 Suitable per-salts include those selected from the group consisting of alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphates, alkalimetal persulphates and mixtures thereof. The carbohydrate may be selected from the group consisting of mono- carbohydrates, di-carbohydrates, tri-carbohydrates, oligo-carbohydrates and mixtures 15 thereof. Suitable carbohydrates include carbohydrates selected from the group consisting of D-arabinose, L-arabinose, D-Cellobiose, 2-Deoxy-D-galactose, 2-Deoxy-D-ribose, D- Fractose, L-Fucose, D-Galactose, D-glucose, D-glycero-D-gulo-heptose, D-lactose, D- Lyxose, L-Lyxose, D-Maltose, D-Mannose, Melezitose, L-Melibiose, Palatinose, D- Raffinose, L-Rhamnose, D-Ribose, L-Sorbose, Stachyose, Sucrose, D-Trehalose, D- 20 Xylose, L-Xylose and mixtures thereof. Suitable carbohydrate oxidases include carbohydrate oxidases selected from the group consisting of aldose oxidase (IUPAC classification EC 1.1.3.9), galactose oxidase (IUPAC classification ECl.1.3.9), cellobiose oxidase (IUPAC classification ECl.1.3.25), pyranose oxidase (IUPAC classification EC 1.1.3.10), sorbose oxidase (IUPAC 25 classification EC 1.1.3.11) and/or hexose oxidase (IUPAC classification EC 1.1.3.5), Glucose oxidase (IUPAC classification ECl.1.3.4) and mixtures thereof. In some preferred embodiments, the cleaning compositions of the present GC821-2 P5

invention also include from about 0.01 to about 99.9, from about 0.01 to about 50, from about 0.1 to 20, or even from about 1 to about 15 weight percent a molecule comprising an ester moiety. Suitable molecules comprising an ester moiety may have the formula:

wherein R is a moiety selected from the group consisting of H or a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; in one aspect of the present invention, R may comprise from 1 to 50,0000 carbon atoms, from 1 to 10,000 carbon atoms, or even from 2 to 100 carbon atoms; 2 2 each R is an alkoxylate moiety, in one aspect of the present invention, each R is independently an ethoxylate, propoxylate or butoxylate moiety; R is an ester-forming moiety having the formula: R⁴CO- wherein R⁴ may be H, substituted or unsubstituted alkyl, alkenyl,5 alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl, in one aspect of the present invention, R⁴ may be substituted or unsubstituted alkyl, alkenyl, alkynyl, moiety comprising from 1 to 22 carbon atoms, an aryl, alkylaryl, alkylheteroaryl, or heteroaryl moiety comprising from 4 to 22 carbon atoms or R may be a substituted or unsubstituted C1-C22 alkyl moiety or0 R⁴ may be a substituted or unsubstituted C1-C12 alkyl moiety; x is 1 when R is H; when R¹ is not H, x is an integer that is equal to or less than the number of carbons in R p is an integer that is equal to or less than x m is an integer from 0 to 50, an integer from 0 to 18, or an integer from 05 to 12, and n is at least 1. In one aspect of the present invention, the molecule comprising an ester moiety is 1 9 1 an alkyl ethoxylate or propoxylate having the formula R O_x[(R )_m(R )_n]_P wherein: GC821-2 -^)

R is an C₂-C₃₂ substituted or unsubstituted alkyl or heteroalkyl moiety; each R is independently an ethoxylate or propoxylate moiety; R is an ester-forming moiety having the formula: R CO- wherein R⁴ may be H, substituted or unsubstituted alkyl, alkenyl, 5 alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl, in one aspect of the present invention, R may be a substituted or unsubstituted alkyl, alkenyl, or alkynyl moiety comprising from 1 to 22 carbon atoms, a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl, or heteroaryl moiety comprising from 4 to 22 carbon atoms or R⁴ may be a substituted or 10 unsubstituted Ci-G.2 alkyl moiety or R may be a substituted or unsubstituted C1-C12 alkyl moiety; x is an integer that is equal to or less than the number of carbons in R p is an integer that is equal to or less than x m is an integer from 1 to 12, and 15 n is at least 1. In one aspect of the present invention, the molecule comprising the ester moiety has the formula: R¹O_x[(R²)_m(R³)„]_P

20 wherein R is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, said R¹ moiety that comprises an amine moiety being selected from the group consisting of a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; in one aspect of Applicants' invention R may comprise from 1 to 50,000 carbon atoms, from 1 to 10,000 carbon

25 atoms, or even from 2 to 100 carbon atoms;

is independently an ethoxylate, propoxylate or butoxylate moiety; '- is ,« κ„ it «,,.it ""it" ..^• '" »" iUf '"'!'" ' .. GC821-2 '"^"" ^

R is an ester-forming moiety having the formula: R CO- wherein R maybe H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl, in one aspect of the present invention, R⁴ may be a substituted or unsubstituted alkyl, alkenyl, or alkynyl moiety comprising from 1 to 22 carbon atoms, a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl, or heteroaryl moiety comprising from 4 to 22 carbon atoms or R may be a substituted or unsubstituted d-C22 alkyl moiety or R may be a substituted or unsubstituted C1-C12 alkyl moiety; x is 1 when R is H; when R¹ is not H, x is an integer that is equal to or less than the number of carbons in R¹ p is an integer that is equal to or less than x m is an integer from 0 to 12 or even 1 to 12, and n is at least 1. In any of the aforementioned aspects of the present invention, the molecule comprising an ester moiety may have a weight average molecular weight of less than 600,000 Daltons, less than 300,000 Daltons, less than 100,000 Daltons or even less than 60,000 Daltons. Suitable molecules that comprise an ester moiety include polycarbohydrates that comprise an ester moiety. The cleaning compositions provided herein will typically be formulated such that, during use in aqueous cleaning operations, the wash water will have a pH of from about 5.0 to about 11.5, or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from about 3.0 and about 9.0. Granular laundry products are typically formulated to have a pH from about 9 to about 11. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, _ i GC821-2

etc., and are well known to those skilled in the art. When the enzyme(s) of the present invention is/are employed in a granular composition or liquid, it may be desirable for the enzyme(s) to be in the form of an encapsulated particle to protect such enzyme from other components of the granular 5 composition during storage. In addition, encapsulation is also a means of controlling the availability of the enzyme(s) during the cleaning process and may enhance performance of the enzyme(s). In this regard, the enzyme(s) may be encapsulated with any encapsulating material known in the art. The encapsulating material typically encapsulates at least part of the enzyme(s).

10 Typically, the encapsulating material is water-soluble and/or water-dispersible. The encapsulating material may have a glass transition temperature (Tg) of 0°C or higher. Glass transition temperature is described in more detail in WO 97/11151, especially from page 6, line 25 to page 7, line 2. The encapsulating material may be selected from the group consisting of

15 carbohydrates, natural or synthetic gums, chitin and chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes and combinations thereof. When the encapsulating material is a carbohydrate, it maybe typically selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. Typically, the

20 encapsulating material is a starch. Suitable starches are described in EP 0 922 499; US 4,977,252; US 5,354,559 and US 5,935,826. The encapsulating material may be a microsphere made from plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof; commercially available microspheres that can be used are those

25 supplied by Expancel of Stockviksverken, Sweden under the trademark EXPANCEL®, and those supplied by PQ Corp. of Valley Forge, Pennsylvania U.S.A. under the _ti , , , if"¹ it,-, it^" ,,'- !&-!> •„„!^! >W ^II "^{' l! •}"* " *""' '"'¹ GC821-2 O (?

tradename PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL® and SPHERICEL®.

Processes of Making and Using the Cleaning Compositions nf 5 the Present Invention The cleaning compositions of the present invention can be formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. 5,879,584; U.S. 5,691,297; U.S. 5,574,005; U.S. 5,569,645; U.S. 5,565,422 Del Greco et al.; U.S. 5,516,448; U.S. 5,489,392; and U.S. 10 5,486,303; all of which are incorporated herein by reference.

Adjunct Materials in Addition to the Fnzymes nf the Present Invention. Hydrogen Permride, and/or Hydrogen Permride Source and Material Comprising an F.ster Moiety 15 While not essential for the purposes of the present invention, the non-limiting list of adjuncts illustrated hereinafter are suitable for use in the instant cleaning compositions and may be desirably incorporated in certain embodiments of the invention, for example to assist or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the cleaning composition as is the case with perfumes, 20 colorants, dyes or the like. It is understood that such adjuncts are in addition to the enzymes of the present invention, hydrogen peroxide and/or hydrogen peroxide source and material comprising an ester moiety. The precise nature of these additional components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the cleaning operation for which it is to be used. Suitable 25 adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed GC821-2

peracids, polymeric dispersing agents, clay soil removal anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids and/or pigments. In addition to the disclosure below, suitable examples of such other adjuncts and levels of use are found in U.S. Patent Nos. 5,576,282, 6,306,812, and 6,326,348, herein incorporated by reference. The aforementioned adjunct ingredients may constitute the balance of the cleaning compositions of the present invention. Surfactants - The cleaning compositions according to the present invention may comprise a surfactant or surfactant system wherein the surfactant can be selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof. The surfactant is typically present at a level of from about 0.1% to about 60%, from about 1% to about 50% or even from about 5% to about 40% by weight of the subject cleaning composition. Builders - The cleaning compositions of the present invention may comprise one or more detergent builders or builder systems. When a builder is used, the subject cleaning composition will typically comprise at least about 1%, from about 3% to about 60% or even from about 5% to about 40% builder by weight of the subject cleaning composition. Builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicate builders polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-trihydroxy benzene-2, 4, 6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, , - r

GC821-2

polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof. Chelating Agmts - The cleaning compositions herein may contain a chelating agent, Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof. When a chelating agent is used, the cleaning composition may comprise from about 0.1% to about 15% or even from about 3.0% to about 10% chelating agent by weight of the subject cleaning composition. Deposition Aid - The cleaning compositions herein may contain a deposition aid. Suitable deposition aids include, polyethylene glycol, polypropylene glycol, polycarboxylate, soil release polymers such as polytelephthalic acid, clays such as Kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite, and mixtures thereof. Dye Transfer inhibiting Agents - The cleaning compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N- vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinyhmidazoles or mixtures thereof. When present in a subject cleaning composition, the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the cleaning composition. Dispersants - The cleaning compositions of the present invention can also contain dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. F ^yymes - The cleaning compositions can comprise one or more detergent enzymes which provide cleaning performance and/or fabric care benefits. Examples of GC821-2 O

suitable enzymes include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. A typical combination is cocktail of conventional applicable enzymes like protease, lipase, cutinase and/or cellulase in conjunction with amylase. F.n7 me Stabilizers - Enzymes for use in detergents can be stabilized by various techniques. The enzymes employed herein can be stabilized by the presence of water- soluble sources of calcium and/or magnesium ions in the finished compositions that provide such ions to the enzymes. Catalytic Metal Complexes - The cleaning compositions of the present invention may include catalytic metal complexes. One type of metal-containing bleach catalyst is a catalyst system comprising a transition metal cation of defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, an auxiliary metal cation having little or no bleach catalytic activity, such as zinc or aluminum cations, and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and water-soluble salts thereof. Such catalysts are disclosed in U.S. 4,430,243. If desired, the compositions herein can be catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art and include, for example, the manganese-based catalysts disclosed in U.S. 5,576,282. Cobalt bleach catalysts useful herein are known, and are described, for example, in U.S. 5,597,936; and U.S. 5,595,967. Such cobalt catalysts are readily prepared by known procedures, such as taught for example in U.S. 5,597,936, and U.S. 5,595,967. Compositions herein may also suitably include a transition metal complex of a ._„

P . v*- U .Si ."'^" ""!'" ^t- ^„: GC821-2 ^•"^">

macropolycyclic rigid ligand - abreviated as "MRL". As a practical matter, and not by way of limitation, the compositions and cleaning processes herein can be adjusted to provide on the order of at least one part per hundred million of the active MRL species in the aqueous washing medium, and will preferably provide from about 0.005 ppm to about 5 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of the MRL in the wash liquor. Preferred transition-metals in the instant transition-metal bleach catalyst include manganese, iron and chromium. Preferred MRL's herein are a special type of ultra-rigid ligand that is cross-bridged such as 5,12-diethyl-l,5,8,12-tetraazabicyclo[6.6.2] 10 hexadecane. Suitable transition metal MRLs are readily prepared by known procedures, such as taught for example in WO 00/332601, and U.S. 6,225,464.

Method o T Tse 15 The cleaning compositions disclosed herein of can be used to clean a situs inter alia a surface or fabric. Typically at least a portion of the situs is contacted with an embodiment of Applicants' cleaning composition, in neat form or diluted in a wash liquor, and then the situs is optionally washed and/or rinsed. For purposes of the present invention, washing includes but is not limited to, scrubbing, and mechanical agitation. 20 The fabric may comprise most any fabric capable of being laundered in normal consumer use conditions. The disclosed cleaning compositions are typically employed at concentrations of from about 500 ppm to about 15,000 ppm in solution. When the wash solvent is water, the water temperature typically ranges from about 5 °C to about 90 °C and, when the situs comprises a fabric, the water to fabric mass ratio is typically from 25 about 1 : 1 to about 30:1.

EXPERIMENTAL The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be . , -

GC821-2 ^ ^

construed as limiting the scope thereof. In the experimental disclosure which follows, the following abbreviations apply: °C (degrees Centigrade); rpm (revolutions per minute); H₂O (water); HC1 (hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons); gm (grams); μg and ug (micrograms); mg (milligrams); ng (nanograms); μl and ul (microliters); ml (milliliters); mm (millimeters); nm (nanometers); μm and urn (micrometer); M (molar); mM (millimolar); μM and uM (micromolar); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); MgCfe (magnesium chloride); NaCl (sodium chloride); OD280 (optical density at 280 nm); OD₆oo (optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); TAED (N,N,N'N'-tetraacetylethylenediamine); w/v (weight to volume); v/v (volume to volume); Per (perhydrolase); per (perhydrolase gene); Ms (M. smegmatis); MS (mass spectroscopy); BRAIN (BRAIN Biotechnology Research and Information Network, AG,

Zwingenberg, Germany); TIGR (The Institute for Genomic Research, Rockville, MD); AATCC (American Association of Textile and Coloring Chemists); WFK (wfk Testgewebe GmbH, Braggen-Bracht, Germany); Amersham (Amersham Life Science, Inc. Arlington Heights, IL); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, CA); Pierce (Pierce Biotechnology, Rockford, IL); Amicon (Amicon, Inc., Beverly, MA); ATCC

(American Type Culture Collection, Manassas, VA); Amersham (Amersham Biosciences, Inc., Piscataway, NJ); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, NJ); BioRad (BioRad, Richmond, CA); Clontech (CLONTECH Laboratories, Palo Alto, CA); Difco (Difco Laboratories, Detroit, MI); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, MD); Novagen (Novagen, Inc., Madison, WI); Qiagen (Qiagen, Inc.,

Valencia, CA); Invitrogen (Invitrogen Corp., Carlsbad, CA); Genaissance (Genaissance Pharmaceuticals, Inc., New Haven, CT); DNA 2.0 (DNA 2.0, Menlo Park, CA); MIDI , _, GC821-2 ^~ ^"~^

(MIDI Labs, Newark, DE) ivivoGen (InvivoGen, San Diego, CA); Sigma (Sigma Chemical Co., St. Louis, MO); Sorvall (Sorvall Instruments, a subsidiary of DuPont Co., Biotechnology Systems, Wilmington, DE); Stratagene (Stratagene Cloning Systems, LΛ Jolla, CA); Roche (Hoffmann La Roche, Inc., Nutley, NJ); Agilent (Agilent Technologies, Palo Alto, CA); Minolta (Konica Minolta, Ramsey, NJ); and Zeiss (Carl Zeiss, Inc., Thornwood, NY). In the following Examples, various media were used. 'TS" medium (per liter) was prepared using Tryptone (16 g) (Difco), Soytone (4 g) (Difco), Casein hydrolysate (20 g) (Sigma), K₂HPO₄ (10 g), and d H₂O (to 1 L). The medium was sterilized by autoclaving. Then, sterile glucose was added to 1.5% final concentration. Streptomyces Production Medium (per liter) was prepared using citric acid(H₂O) (2.4 g), Biospringer yeast extract (6 g), (NH₄)2SO4 (2.4 g), MgSO4-7 H₂O (2.4 g), Mazu DF204 (5 ml), trace elements (5 ml). The pH was adjusted to 6.9 with NaOH. The medium was then autoclaved to sterilize. After sterilization, CaCl2-2 H2O (2 mis of 100 mg/ml solution), KH2PO4 (200 ml of a 13% (w/v) solution at ρH6.9), and 20 mis of a 50% glucose solution were added to the medium. In these experiments, a spectrophotometer was used to measure the absorbance of the products formed after the completion of the reactions. A reflectometer was used to measure the reflectance of the swatches. Unless otherwise indicated, protein concentrations were estimated by Coomassie Plus (Pierce), using BSA as the standard.

EXAMPLE 1 Enzyme Analysis In this Example, methods to assess enzyme purity and activity used in the subsequent Examples and throughout the present Specification are described.

Enzyme Activity Assay (pNB Assay) This activity was measured by hydrolysis of /?-nitrophenylbutyrate. The reaction mixture was prepared by adding 10 ul of 100 mM/ nifrophenylbutyrate in dimethylsulfoxide to 990 ml of 100 mM Tris-HCl buffer, pH 8.0 containing 0.1 % triton X-100. The background rate of hydrolysis was measured before the addition of enzyme at 410 nm. The reaction was initiated by the addition of 10 ul of enzyme to 990 ml of the reaction and the change of absorbance at 410 nm was measured at room temperate (~23°C). The background corrected results are reported as δA4io/min ml or

10 δAtio/min/mg protein.

Transesterification Transesterification was measured by GC separation of products in buffered aqueous reactions. Reactions to measure ethyl acetate transesterification with propanol

15 contained in 1 ml of 50 mM KPO4, pH 7.0; 200 mM ethyl acetate, 200 mM 1 -propanol, and enzyme. Reactions to measure ethyl acetate transesterification with neopentyl glycol (NPG) contained in 1 ml of 50 mM KPO4, pH 7.0; 303 mM ethyl acetate, 100 mM NPG, and enzyme. The reactions were incubated at the indicated temperatures and for the indicated times. Separations were preformed using a 30M FFAP column (Phenomenex).

20 The inlet split ratio was approximately 1:25, the injector was 250°C, head pressure of 10 psi He, and detection was by FED at 250°C. The chromatography program was 40°C initial for 4 min, followed by a gradient of 15°C/min to 180°C. Components eluted in the following order and were not quantified; ethyl acetate, ethyl alcohol, propyl acetate, propyl alcohol, acetic acid, NPG diacetate, NPG monoacetate, and NPG.

25 Perhydrolase Used in Crystallography Studies This perhydrolase preparation was used for crystallography studies. In addition, P C ',•'^■"

unlabelled protein was grown and purified in similar manner. A 500 ml preculture of E. coli BL21(DΕ3)/ρLysS/ρMSATNcol-l was grown in a baffled 2.8 L Fembach flask on LB containing 100 ug/ml carbenicillin. After overnight culture at 37°C and 200 rpm on a rotary shaker, the cells were harvested by centrifugation and resuspended in M9 medium 5 containing: glucose, 2 g/L; Na₂HPO , 6 g/L; KH₂PO₄, 3 g/L; NH₄C1, 1 g/L; NaCl, 0.5 g/L; thiamine, 5 mg/L; MgSO , 2 mM; CaCl₂, 100 uM; Citric acid.H₂0, 40 mg/L; MnSO₄.H₂0, 30 mg/L; NaCl, 10 mg L; FeSO₄.7H₂0, 1 mg/L; CoCl₂.6H₂0, 1 mg/L; ZnSO «7H₂0, 1 mg/L; CuSO₄«5H₂0, 100 ug/L; H₃BO₃.5H₂O, 100 ug/L; and NaMoO₄«2H2θ, 100 ug/L; and supplemented with carbenicillin, 100 mg/L. The 10 resuspended cells were used to inoculate six Fembach flasks containing 500 ml each of M9 medium supplemented with carbenicillin (100 mg/L). The cultures were incubated at 20°C and 200 rpm on a rotary shaker until the OD₆oo reached about 0.7 at which time 100 mg/L of lysine, threonine, and phenylalanine and 50 mg/L of leucine, isoleucine, valine, and selenomethionine were added. After further incubation for 30 min, IPTG was 15 added to a final concentration of 50 uM. The cultures were then incubated overnight (~15hr) and harvested by centrifugation. The cell pellet was washed 2 times with 50 mM KPO₄ buffer, pH 6.8. The yield was 28.5 gm wet weight of cells to which was added 114 ml of 100 mM KPO₄ buffer, pH 8.2 and 5 mg of DNase. This mixture was frozen at - 80°C and thawed 2 times. 20 The thawed cell suspension was lysed by disruption in a French pressure cell at 20K psi. The unbroken cells and cell membrane material were sedimented by centrifugation at 100K times g for 1 hour. The supernatant crude extract, 128 ml (CE) was then placed in a 600 ml beaker and stirred for 10 minutes in a 55°C water bath to precipitate unstable proteins. After 10 min the beaker was stirred in ice water for 1 min 25 followed by centrifugation at 15K times g for 15 min. The supernatant from this procedure, HT, contained 118 ml. The HT extract was then made 20% saturating in (NH₄)2SO₄ by the slow addition of 12.7 g of (NH₄)₂SO . This was loaded on to a 10 cm GC821-2 ^ ^

X 11.6 cm Fast Flow Phenyl Sepharose (Pharmacia) column equilibrated inlOO mM KPO₄ buffer, pH 6.8, containing 20% saturation (109 g/L) (NH )₂SO₄. After loading the extract the column was washed with 1700 ml of starting buffer and eluted with a two step gradient. The first step was a linear 1900 ml gradient from start buffer to the same buffer without (NH₄)2SO , the second was a 500 ml elution with 100 mM KPO , pH 6.8 containing 5% EtOH. Active fractions, 241 ml, were pooled, diluted 100 % with water and loaded onto a 1.6 mm X 16 mm Poros HQ strong anion exchange column equilibrated in 100 mM Tris-HCl, pH 7.6. After loading the extract, the column was washed with 5 column volumes of starting buffer. The protein was eluted with a 15 column volume gradient from start buffer to start buffer containing 175 mM KCl. The active fractions were pooled and concentrated using a Centriprep 30 (Millipore) to 740 μl. Figure 6 provides a purification table showing the enzyme activity of the enzyme of the present invention through various steps in the purification process. The present application must be used to determine the respective values of the parameters of the present invention. Unless otherwise noted, all component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources. Enzyme components weights provided herein are based on total active protein.

All percentages and ratios were calculated by weight unless otherwise indicated. All percentages and ratios were calculated based on the total composition unless otherwise indicated.

EXAMPLE 2 j ^* S-„ B . t - GC821-2 "^* ^

Determination of Ratio Between Peracid and Acid Formation In this Example, methods for determining the ratio of perhydrolysis to hydrolysis are described. In particular, this Example provides methods for determining the ratio between peracid formation (i.e., perhydrolysis) and acid formation (i.e., hydrolysis) resulting from enzyme activity on an ester substrate in the presence of peroxide in an aqueous system.

A. Determination of Pfirhy rnlysis tn Hydrolysis Ratio 10 Preparation of Substrate The substrates were prepared as described herein. Ethyl acetate (EtOAc) and other water soluble esters were diluted in a desired buffer to a concentration of 10 mM of ester. Tributyrin and other water insoluble substrates were prepared by making substrate swatches. Polyester swatches were cut from non-dyed polyester fabric (Polycotton, PCW 15 22) using a 5/8 inch punch and placed in a 24- well microtiter plate (Costar, Cell Culture Plate). The insoluble ester was diluted to 1.03 M in hexane. Then, 10 μL of the insoluble ester solution were then adsorbed onto the polyester swatch.

Determination of Hydrolysis (GC Assay) 20 The hydrolytic assay described below was used to determine the amount of substrate hydrolysis. In this assay, the assay solution was comprised of 50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogen peroxide, and 20 mM potassium chloride in a total volume of 0.99ml and an amount of enzyme that would generate 20 nmoles of acetic acid per minute at 25°C. 25 For measuring water insoluble ester hydrolysis, the reaction mixture was added to the insoluble ester fabric swatch. The swatch was prepared as described above ("Preparation of Substrate"). All the other conditions for the assay were the same except GC821-2 ^

for exclusion of other ester substrates. Hydrolytic activity was measured by monitoring the increase of acids generated by the enzyme from acyl donor substrates using gas chromatography coupled with flame ionization detection. The assay was conducted by first pipetting 50 μL of assay solution 5 containing all the components except the enzyme into 200 mL of methanol (HPLC grade) to determine the amount of acid in the assay solution at time 0. Then, 10 μL of enzyme were added to the assay solution to a desired final concentration which produced approximately 20 nanomoles of acid per minute. A timer was started and 50 μL aliquots were taken from the assay solution and added to 200 μL of methanol at various times,0 typically 2, 5, 10, 15, 25, 40, and 60 minutes, after addition of the enzyme. These methanol-quenched samples were then injected into a gas chromatograph coupled with a flame ionization detector (Agilent 6890N) and analyzed for hydrolytic components, acetic, and butyric acids. Gas chromatography was conducted using a nitroterephthalic acid modified polyethylene glycol column (Zebron FFAP; with5 dimensions: 30 m long, 250 um diameter, 250 nm film thickness). A 3 μL aliquot of sample was applied to the column by a splitless injection under constant a helium flow of 1.0 mL/minute. The inlet was maintained at a temperature of 250°C and was purged of any remaining sample components after 2 minutes. When analyzing acetic acid, the temperature of the column was maintained at 75 °C for 1 minute after injection, increased0 25°C/minute to 100°C, then increased 15°C/minute to 200°C. When analyzing butyric acid, the temperature of the column was controlled as described above, except the temperature was additionally increased 25°C/minute to 225°C and held at 225°C for 1 minute. The flame ionization detector was maintained throughout the chromatography at 250°C and under constant hydrogen flow of 255 mL/minute, air flow of 200 mL/minute, and a combined column and makeup helium flow of 30 mL/minute. The amount of hydrolyzed acid in the sample was then determined by integrating the acid peak in the chromatogram for total ion counts and calculating the acid iKi ϋ • . GC821-2 f~

from the ion count using a standard curve generated under the above conditions for acetic and butyric acids at varying concentrations in the assay solution (without enzyme).

Determination of Perhydrolysis (OPD Assay) 5 The perhydrolytic activity assay described below was used to determine the amount of peracid formed in the reaction. In these assays, the solution comprised 50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogen peroxide, 20 mM potassium chloride, and 10 mM O-phenylenediamine. When using water insoluble ester as the acyl donor, an ester adsorbed fabric 10 swatch was used as the substrate, prepared as described above ("Preparation of Substrate"). Perhydrolytic activity was measured by monitoring the absorbance increase at 458 nm of oxidized O-phenylenediamine (OPD) by peracid generated with the enzyme. The perhydrolytic activity assay solution was prepared in the same manner as the hydrolytic 15 activity assay solution, except that OPD was added to the assay solution to a final concentration of lOmM. The OPD solution was prepared immediately before conducting the assay by dissolving 72mg OPD (Sigma- Aldrich, dihydrochloride) in 19.94 mL of the same buffer and the pH was adjusted by slowly adding 60 μL of 13.5 M potassium hydroxide. The pH was measured and if needed, small quantities of potassium hydroxide 20 were added to return the pH to the original pH of the buffer. Then, 495 μL of this OPD solution were added with the other assay components to a final assay volume of 0.990 mL. An assay quenching solution was also prepared by dissolving 36mg OPD in 20 mL 100 mM citric acid and 70% ethanol. The assay was typically conducted at 25°C. The assay was started by pipetting 25 100 μL of assay solution before the addition of the enzyme into 200 μL of quenching solution to determine the amount of perhydrolytic components and background absorbance in the assay solution at time 0. Then, 10 μL of enzyme were added to the „p„ j -,* - _ _ [ i - i,,

assay solution to a desired final concentration which produced approximately 10 nanomoles of peracid per minute. A timer was started and 100 μL aliquots were taken from the assay solution and added to 200 μL of quenching solution at various times, typically 2, 5, 10, 15, 25, 40, and 60 minutes, after adding the enzyme. The quenched 5 assay solutions were incubated for 30 minutes to allow any remaining peracid to oxidize the OPD. Then, 100 μL of each quenched assay solution was transferred to a 96- well microtiter plate (Costar) and the absorbance of the solution was measured at 458 nm by a spectrophotometric plate reader (Molecular Devices, SpectraMAX 250). The amount of peracid in each quenched sample was calculated using a standard curve generated under 10 the above conditions with peracetic acid at varying concentrations in the assay solution (without enzyme).

Perhydrolysis /Hydrolysis ratio: Perhydrolysis/ Hydrolysis ratio= Perhydrolysis measured in the Perhydrolysis 15 assay/(Total acid detected in the hydrolysis assay-Perhydrolysis measured in the perhydrolysis assay)

The results of these experiments are provided in Figures 7, 10 and Figure 11. Figure 7 provides a graph which shows the ratio of perbutyric acid to butyric acid 20 generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 40 minutes. Figure 10 shows the ratio of perbutyric acid to butyric acid generated by various enzymes from 10 mM tributyrin and 29 mM hydrogen peroxide in 4, 10, and 30 minutes. Figure 11 shows the ratio of peracetic acid to acetic acid generated by various enzymes from 10 mM triacetin and 29 mM hydrogen peroxide in 4 and 10 minutes. The 25 results obtained in these experiments indicated that M. smegmatis perhydrolase homologues exhibited a ratio above 1 in the OPD/GC assays described above, while other classes of enzymes exhibited ratios significantly below 1. GC821-2 -^•

Table 2-lprovides data showing the perhydrolysis activity of various homologues described herein on triacetin, as compared to the wild-type M. smegmatis perhydrolase. The results provided in Table 2-2 indicate that the perhydrolase has activity over a broad range of substrates. In addition to the results provided in these Tables, Figures 8 and 9 provide data showing that the perhydrolase of the present invention has broad pH and temperature range activities.

Table 2-2. Peracid Production by 1 ppm Wild-Type Perhydrolase with 29 mM H2O2 and Various Esters nmol Peracetic Acid / min f .•^■' lt-i' ., 41 'Mi ' it ^■ - GC821-2 C% €)

Ester lOmM of lOmM of lOmM of Ester Ester with Ester on Polycott* on 0.5% Swatch Neodol Ethyl Acetate 5.00 Butyl Acetate 8.06 8.72 Hexyl Acetate 7.96 5.86 Octyl Acetate 8.03 0.48 Ethyl Propionate 0.90 1.43 Butyl Propionate 2.47 3.39 Hexyl Propionate 4.00 2.66 Isoamyl Acetate 7.83 17.69 Cifronellyl Acetate 7.25 4.27 Cifronellyl 2.85 3.21 Propionate Dodecyl Acetate 3.95 0.19 Neodol 23-3 2.25 8.77 Acetate Neodol 23-6.5 2.73 10.12 Acetate Neodol 23-9 2.97 10.20 Acetate Ethylene Glycol 13.30 Diacetate Propylene Glycol 13.17 Diacetate Triacetin 11.91 Tributyrin 0.66 2.70 Ethyl 0.49 Methoxyacetate Linalyl Acetate 0.30 Ethyl Butyrate 0.31 Ethyl Isobutyrate 0.10 Ethyl 2- 0.11 methylbutyrate Ethyl Isovalerate 0.37 Diethyl Maleate 0.75 Ethyl Glycolate 1.91 - i ->^v w ■.'-> 't» ^u "' fi" •^■' "" ^«■ ' "i" .,» GC821-2 ^{' ""}^ ^/^~ -

B. Typical Perhydrolase Peracid Generation Assay 5 Perhydrolase is active over a wide pH and temperature range and accepts a wide range of substrates for acyl transfer. Acceptors include water (hydrolysis), hydrogen peroxide (perhydrolysis) and alcohols (classical acyl transfer). For perhydrolysis measurements enzyme was incubated in the buffer of choice at a specified temperature with a substrate ester in the presence of hydrogen peroxide. Typical substrates used to 10 measure perhydrolysis include ethylacetate, triacetin, tributyrin, ethoxylated neodol acetate esters, and others. In addition, the wild type enzyme was found able to hydrolyze nitrophenylesters of short chain acids. The latter are convenient substrates to measure enzyme concentration. In some embodiments, peracid acid and acetic acid were measured by the ABTS or HPLC assays as described below. Nifrophenylester hydrolysis 15 is also described below.

£. ARTS Assay (one milliliterV This assay provides a determination of peracetic acid produced by perhydrolase. This protocol was adapted from Karst etal, Analyst, 122:567-571 [1997]). Briefly, a 20 100 μL aliquot of solution to be analyzed was added to 1 mL 125 mM K citrate pH 5, 1 mM ABTS, 50 μM KI. Absorbance was measured at 420 nm for highest sensitivity. However, multiple additional wavelengths were sometimes used over the broad absorption spectrum of ABTS. Calibration curves were constructed based on known peracid concentration series. 25 D. TTPT.C (Model - Agilent 1100) Determination of Perhydrolase Reaction Products- For determination of the ratio of perhydrolysis to hydrolysis of the perhydrolase GC821-2 ^ ^

reaction, perhydrolase reaction samples were quenched by acidification to a final concentration of 0.24% methanesulfonic acid, and the products were separated by reverse phase HPLC on a Dionex OA column (cat #062903; Dionex Corporation, Sunnyvale, CA). The mobile phase was 100 mM NaPO4, pH 3.9 (buffer was prepared by titrating 100 mM Na2PO4 with methanesulfonic acid to pH 3.9) run under isocratic conditions at

30 C. Detection was at 210 nm. Concentrations of products were calculated by comparison of the integrated peak areas against calibration standards.

E_ Nifrophenylester Hydrolysis Kinetic Assay Enzyme and substrate were incubated in 100 mM Tris/HCl pH 8.0 (or 50 mM

B(OH)₃ pH 9.5 or another buffer). Absorbance at 402 nm was monitored. In some experiments, the assay was carried out in standard 1 mL cuvettes, while in other experiments, microtiter plate wells were used. The latter method was used for the screening of mutant libraries. Enzyme concentration was determined by comparison to standard curves obtained under the same reaction conditions.

F. Para-nifrophenylcaproate Hydrolysis Assay The pNC6 subsfrate solution was prepared by mixing ImM pNC6 (100 mM stock solution), 1 ml DMSO, 19 mis 1 OOmM Phosphate (ρH8), and glycerol to a final concentration of 10%. To assay samples, 10 μl of the cell lysate were added to 190 μl of the subsfrate solution, and assayed at 405 nm for 15 minutes in a spectrophotometer. The results are presented as the average of two experiments.

C Para-nitrophenyl Acetate (pNA) Hydrolysis Assay Aliquots of the lysed cell supernatant were diluted 1-100 in 100 mM phosphate buffer (pH 8). To assay the samples, 5 μl of the 1-100 diluted cell supernatant were _ll if ,.' i| ,ιι ...ιt (f.„μ 11 >' ⁱ *sJ^l , ^J

placed into each well of a microtiter plate. Then, 195 μl of reaction buffer/subsfrate mix (1 mM pNA, 100 mM phosphate, pH 8, 10% glycerol ) were added, and the absorbance rate at 405 nm was measured over 3 minutes (kinetics program, microtiter plate reader). The results are presented as the average of two experiments.

EXAMPLE 3 Assays Including Detergent Compositions In this Example, assay systems used to screen for superior perhydrolase activity in detergents with particular substrates are provided. These assays include those that measure peracid degradation of perhydrolase, as well as the peracid synthesis activity of the enzyme. Materials and Methods for Peracetic Acid Formation (PAF) and Peracetic Acid Degradation (PAD) Assays This section provides the materials and methods used to screen for a superior perhydrolases in Ariel with C9E2OAC ester subsfrate

Materials: Ariel Futur without bleach, perfume, or enzymes (P&G, Ariel "C") C9E2OAc (P&G) 30% Hydrogen Peroxide (Sigma) 32% Peroxyacetic acid ("peracid", PAA)(Sigma cat#) MW = 76.05; 4.208M Citric Acid, anhydrous MW=192.12 Potassium Hydroxide MW=56.11 ABTS (Sigma cat# A1888) MW=548.68 Potassium Iodide MW=166.0 Potassium Phosphate , mono and di-basic Stock solutions- .1, S . Ji li .i. 11 ■■ ,T l, ,'' "

GC821-2 T

Ariel detergent stock: Ariel Futur without bleach, perfume, or enzymes ("Ariel C") was dissolved in water to 6.72 g/L. It was stirred at room temp for 30 minutes, then allowed to settle. Then, it was divided into convenient aliquots and stored at 4°C, until used. When made and used fresh, the solution was filtered, instead of settled

100 mM C9E2OAc in Ariel detergent stock: First, 30 μl C9E2OAc was added to 970 μl Ariel detergent stock, using a positive displacement pipet. It was sonicated in a bath sonicator until a milky dispersion was formed (15-60 seconds). The dispersion was stable for about two hours. When used, 10 μl of dispersion per ml of reaction mix were used.

42 mM Peroxyacetic acid stock: Right before use, the Sigma 32% PAA solution was diluted 1:100 in water. Then 5.7 μl of the 42 mM stock per ml of reaction mix was added.

2 M hydrogen peroxide: One ml of 30% Sigma hydrogen peroxide was added to 3.41 ml water. This solution was prepared fresh, right before use. It was used at 10 μl per ml of reaction mix. 125 mM Citrate buffer pH 5.0: This was prepared to 24.0 grams per liter. It was made up in 800 ml, and titrated to pH 5.0 with 50% KOH. The volume was adjusted to 1 liter and stored at room temperature.

100 mM ABTS stock: This was prepared using 549 mg of ABTS in 10 ml of water. It was frozen at -80°C, in convenient aliquots in opaque Eppendorf tubes. The stock was stable indefinitely when kept frozen in the dark. ABTS will precipitate when thawed from -80°C but goes back into solution upon mixing. In use, 10 μl of ABTS stock was used per ml of ABTS reagent. 250 mM KI: This was prepared as 415 mg in 10 ml water. It was kept at 4°C. It was diluted to 25 mM working stock, and 2 ul of working stock was used per ml of ABTS reagent.

25 mM Potassium Phosphate buffer, pH 8.0:

Method: The night prior to performance of the assays, the plates containing lysed cells that contain perhydrolase were checked to be sure that they were frozen twice. On the day of £_ £ ,.' if„i( .^,j! i!„ιι ^• 'ir,.' ^,r"l^[. ii. ,[| HI" , ;;;|ι ipi GC821-2 ^

the assay, 30 to 45 minutes were allowed for the plates to thaw. The ABTS reagent was prepared and the Multidrop (Multidrop 384 instrument, ThermoElecfron) to fill the detection plates with 200 μl per well. Store the filled plates covered at room temperature in the dark until needed. Dilutions of the standards were prepared so that when 20 μl of

5 the diluted standard were added to the 180 μl of the reaction mix, the concenfration in the well was 1 ppm. Four 4 two-fold serial dilutions were prepared to a set of six standards: 1, 0.5, 0.25, 0.125, and 0.0625 ppm final concentration in the wells. To test, 20 μl of the standards were added to the thawed 1:10 dilution plate. The reaction mixtures were prepared and the Multidrop used to fill one reaction plate for each0 plate to be assayed (180μl/well). Note that the reaction mixtures are different for the PAF and PAD assays.

Peracid Hydrolysis (Peracid Degradation, PAD) Assay:5 This assay measures the amount of peracetic acid remaining after a 100 minute incubation with enzyme in an Ariel detergent background. The amount of peracid remaining is detected by reacting an aliquot of the reaction mixture with the ABTS detection reagent. In this assay, 20 μl enzyme samples from the thawed 1:10 dilution plate were0 transferred, one column at a time with an 8 channel pipetter, into the corresponding column of the pre-filled PAD reaction plate. A timer was started as soon as transfer occurred from the first column; subsequent columns were transferred at 15 second intervals (i.e., the last column was finished 2 min. 45 sec. after starting the first one). The plate was mixed for 30 seconds on the thermomixer (750 rpm, to avoid splashing). The5 plate was then transferred to a humidified chamber at 25°C. The plate was incubated for a total of 100 minutes from the time the first column of enzyme was added. At 100 minutes incubation, the reaction plate was removed from the incubator. Then, 20 ul , if -■•^" .'i-tt ii 'u' "^{r ,}i' "^t ,"iι GC821-2 ^

aliquots of the reaction mixture were transferred to an ABTS reagent plate, in the same order and with the same 15 second time interval that the enzyme samples were originally added to the reaction plate. The ABTS plate was allowed to sit at room temperature for three minutes after the last column of reaction mixture was added. The plate was then read on the specfrophotometric plate reader at 420 and 740 nm.

Perhydrolysis (Peracid Formation, PAF) Assay Multidrop Optimized Protocol: Screening for a Superior Perhydrolysis in Ariel with C9E2OAC Ester Substrate The same materials and stock solutions described above for PAD were used in these experiments, as indicated below. Method: The methods were designed to assay 20 μl aliquots from a 1 :100 dilution plate. The 20 μl 1 : 100 dilution assay plates were produced during the process of obtaining the protein concentrations and were stored at -80°C. The plates were thawed for about 30 to 45 minutes before use. Dilutions of the S54V standards were prepared, so that when 2 μl of the diluted standard are added to the 20 μl of the 1:100 diluted cell lysate, the concentration in the well was 0.1 ppm. Four two-fold serial dilutions were prepared to produced a set of six standards: 0.1, 0.05, 0.025, 0.0125, and 0.00625 ppm final concenfration in the wells. Then, 2 ul of the standards were added to the thawed 20 ul 1:100 dilution assay plates in the wells indicated.5

Perhydrolysis (Peracid formation, PAF) Assay: This assay measures the amount of peroxyacetic acid that is produced in 10 - - ^•* - ' '^t GC821-2

minutes from the C9E2OAc substrate in an Ariel detergent background. The amount of peracid formed is detected after 10 minutes by reacting an aliquot of the reaction mixture with the ABTS detection reagent.

The Multidrop was used to deliver 180 μl/well of the PAF reaction mix to the prepared 1 : 100 dilution plate. The timer was started and the reaction plate was placed on the thermomixer, with the temperature set at 25°C. The plate was covered and the solutions mixed for 30 seconds at 750 φm. The plate was then allowed to rest on the thermomixer without mixing, for a total of 10 minutes from the time the reaction mix was added. At 10 minutes, the Multidrop was used to add 20μl/well of the lOx ABTS reagent. The lOx reagent was a milky suspension. The thermomixer was used to briefly shake the plate. The ABTS reagent quickly went into solution. The plate was allowed to sit at room temperature for three minutes after the ABTS reagent was added. The plate was then read on the spectrophotometric plate reader at 420 nm.

EXAMPLE 4 Cloning of Mycobacterium smegmatis Perhydrolase In this Example, the cloning of M. smegmatis perhydrolase is described. An enzyme with acyltransferase activity was purified from Corynebacterium oxydans (now Mycobacterium parafortuitum ATCC19686). Two peptide sequences were obtained from the purified protein. One peptide was determined by Edman degradation from cyanogen bromide cleavage of the purified enzyme using methods known in the art. The sequence of this peptide was determined to be KVPFFD AGS VISTDGVDGI (SEQ ID

NO:3). The second peptide was analyzed using N-terminal sequencing and was found to have the GTEUULSFGDSLTWGWEPV (SEQ ID NO:4). A BLAST search against the „ GC821-2 "")

TIGR unfinished genome database identified a sequence of potential interest in Mycobacterium smegmatis, which is shown below:

MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLS ARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALG MSVLVTQVLTSAGGVGTTYPAPKVLWSPPPLAPMPHPWFQLIFEGGEQKTTELA RVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL (SEQ ID NO:2). 0 The corresponding DNA sequence of the gene is: 5'- ATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCTGGGGCTGGGTCCC CGTCGAAGACGGGGCACCCACCGAGCGGTTCGCCCCCGACGTGCGCTGGACC GGTGTGCTGGCCCAGCAGCTCGGAGCGGACTTCGAGGTGATCGAGGAGGGAC5 TGAGCGCGCGCACCACCAACATCGACGACCCCACCGATCCGCGGCTCAACGG CGCGAGCTACCTGCCGTCGTGCCTCGCGACGCACCTGCCGCTCGACCTGGTG ATCATCATGCTGGGCACCAACGACACCAAGGCCTACTTCCGGCGCACCCCGC TCGACATCGCGCTGGGCATGTCGGTGCTCGTCACGCAGGTGCTCACCAGCGC GGGCGGCGTCGGCACCACGTACCCGGCACCCAAGGTGCTGGTGGTCTCGCCG0 CCACCGCTGGCGCCCATGCCGCACCCCTGGTTCCAGTTGATCTTCGAGGGCG GCGAGCAGAAGACCACTGAGCTCGCCCGCGTGTACAGCGCGCTCGCGTCGTT CATGAAGGTGCCGTTCTTCGACGCGGGTTCGGTGATCAGCACCGACGGCGTC GACGGAATCCACTTCACCGAGGCCAACAATCGCGATCTCGGGGTGGCCCTCG CGGAACAGGTGCGGAGCCTGCTGTAA-3' (SEQ ID NO:l)5 Primers were designed based on the gene sequence to amplify and clone the gene. The primers used for amplification were:

MsRBSF: 5'-0 CTAACAGGAGGAATTAACCATGGCCAAGCGAATTCTGTGTTTCGGTGATTCC CTGACCT-3' (SEQ ID NO:5) ,.<- : ; « i .- ---. , - GC821-2 ^

MspetBamR: 5'- GCGCGCGGATCCGCGCGCTTACAGCAGGCTCCGCACCTGTTCCGCGAGGGCC ACCCCGA-3' (SEQ ED NO:6) The amplification of the gene was done by PCR using Tag DNA polymerase (Roche) per the manufacturer's instructions, with approximately 500 ng of chromosomal DNA from Mycobacterium smegmatis as the template DNA and the addition of 1% DMSO to the PCR reaction mix. Thirty picomoles of each of the primers MsRBSF and MspetBamR were added to the mix. The amplification cycle was: 30 cycles of (95°C for 1 min, 55°C for 1 min, 72°C for 1 min). The fragments obtained from the PCR reaction were separated on a 1.2% agarose gel and a single band of the expected size of 651 bp (coding sequence and stop codon) was identified. This band was cloned directly into the pCR2.1 TOPO cloning vector (Invitrogen) and transformed into E. coli Top 10 cells (Invitrogen) with selection on L agar (10 g/1 tryptone, 5 g/1 yeast extract, 5 g/1 NaCl, 20 g/1 agar) containing 100 micrograms/ml carbenicillin and X-gal (20 micrograms/ml, Sigma- Aldrich) for blue/white selection and incubated overnight at 37°C. Five white colonies were analyzed for the presence of the PCR fragment. Each colony was used to inoculate 5 mis of L broth (L agar without the addition of agar) containing 100 micrograms/ml carbenicillin and the cultures were grown overnight at 37°C with shaking at 200 φm. Plasmid DNA was purified from the cultures using the Quikspin kit (Qiagen). The presence of the correct fragment was determined by restriction enzyme digest with EcoRI to release the fragment, and sequencing using primers supplied by the pCR2.1 manufacturer (Invitrogen). The correct plasmid was designated pMS ATNcoI (See, Figure 12, for the map of this plasmid)). The sequence of this plasmid is provided below agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaag cgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgtt gtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctatttaggtgacactatagaat ie"* ϊ . t Λ t if _' „ GC821-2 ^ ^

actcaagctatgcatcaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttctaacagga ggaattaaccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccacc gagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggac tgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgac 5 gcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgc gctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctg gtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactga gctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtc gacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcagagcctgctgtaaaaggg 10 cgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaattcgccctatagtgagtcgtattaca attcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgc cagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctatacgtacggcagtttaaggtttac acctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggcgacggatggtgatccc cctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatga 15 tgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaa cgccattaacctgatgttctggggaatataaatgtcaggcatgagattatcaaaaaggatcttcacctagatccttttcacgtagaaa gccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaaga gaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccag ctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttctcgccgccaaggatctgatggcgcaggg 20 gatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgg gtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcagggg cgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggcca cgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggc aggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccgg 25 ctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatg atctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatct cgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctggg tgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctc gtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatt 30 tcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacaggtggcacttttcggggaaatgtgcgcggaacc cctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacgtgagga gggccaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgac cggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtc caggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggagg 35 tcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccct gcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctaaaacttcatttttaatttaaaagg atctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaa gatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgttt gccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagcc 40 gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgg i ; i

cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtg cacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccg aagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaac gcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatg 5 gaaaaacgccagcaacgcggcctttttacggttcctgggcttttgctggccttttgctcacatgttctttcctgcgttatcccctgattct gtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcga ggaagcggaag (SEQ ID NO: 13)

Construction of Perhydrolase T7 Expression Plasmid 10 The primer pair used to create pMS ATNco 1 was also used to create an Ncol site (CCATGG) in which the ATG is the start codon of the acyltransferase gene and a BamHI (GGATCC) just after the TAA stop codon. The plasmid pMSATΝcol was digested with NcoVBamHl as recommended by the manufacturer (Roche) and the 658 bp fragment containing the perhydrolase gene was purified using standard procedures known in the art 15 (e.g., Sambrook et al). The fragment was ligated using standard procedures known in the art (e.g., Sambrook et al) into the T7 promoter expression plasmid, pET16b (Novagen), also digested withNcoI/Rα Hl. The ligation reaction was transformed by standard procedures into E. coli Top 10 cells (Invitrogen) and selected on L agar containing 100 micrograms/ml carbenicillin overnight at 37°C. Ten colonies were picked from the 20 several transformants and used to inoculate 5 ml of LB containing 100 micrograms/ml carbenicillin. Cultures were grown overnight at 37°C with shaking at 200 φm. Plasmid DΝA was purified from the cultures using the Qiagen Quikspin kit (Qiagen). The presence of the correct fragment was determined by restriction enzyme digest with NcoVBamϋl as directed by the manufacturer. The correct plasmid was designated 25 pMSATΝcol- 1 (See, Figure 13, for the map of this plasmid). In this Figure, the following elements are indicated—LacI: gene encoding the Lad repressor protein, located at bp 1455-2534, ori: plasmid origin of replication at bp 4471, bla: The β-lactamase gene located at bp 6089-5232; T7 promoter: located at bpl068-1052; T7 terminator: located at bp 259-213, per: the M. smegmatis perhydrolase gene located at 981-334. The sequence «_„ S. .'" 'W .,„,n ⁱd,," Iⁱ >^■' l " - i ' GC821-2 -^ ^

of this plasmid is provided below: ttctcatgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaa atctaacaatgcgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcct cttgcgggatatccggatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttatt 5 gctcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatccgcgcgcttacagcaggctccgcacct gttccgcgagggccaccccgagatcgcgattgttggcctcggtgaagtggattccgtcgacgccgtcggtgctgatcaccgaac ccgcgtcgaagaacggcaccttcatgaacgacgcgagcgcgctgtacacgcgggcgagctcagtggtcttctgctcgccgccc tcgaagatcaactggaaccaggggtgcggcatgggcgccagcggtggcggcgagaccaccagcaccttgggtgccgggtac gtggtgccgacgccgcccgcgctggtgagcacctgcgtgacgagcaccgacatgcccagcgcgatgtcgagcggggtgcgc0 cggaagtaggccttggtgtcgttggtgcccagcatgatgatcaccaggtcgagcggcaggtgcgtcgcgaggcacgacggcag gtagctcgcgccgttgagccgcggatcggtggggtcgtcgatgttggtggtgcgcgcgctcagtccctcctcgatcacctcgaag tccgctccgagctgctgggccagcacaccggtccagcgcacgtcgggggcgaaccgctcggtgggtgccccgtcttcgacgg ggacccagccccaggtcagggaatcaccgaaacacagaattcgcttggccatggtatatctccttcttøaagttaaacaaaattattt ctagaggggaattgttatccgctcacaattcccctatagtgagtcgtattaatttcgcgggatcgagatctcgatcctctacgccgga5 cgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggc tcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctcc ttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataaggg agagcgtcgagatcccggacaccatcgaatggcgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattca gggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaacc0 aggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggca caacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcgg cgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcgg cggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagct gcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcg5 actgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgc gtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccgg ttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaa tgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgtta tatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggcc0 aggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctc cccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaat gtaagttagctcactcattaggcaccgggatctcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcg gggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttc ggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagcctt5 cgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccgacgcgctgggctacgtctt gctggcgttcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcagg ccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagcctaacttcgatcac tggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgccctatac cttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgctaacg0 gattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatc . - .„„ ,•^• GC821-2

gcgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgcgcatgatcgtgctcct gtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcga ctgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtc agcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcg 5 ctggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggca tgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacgga ggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactc aacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagctgcctcgcg cgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagc

10 agacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcgga gtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgt aaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggta tcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagca aaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg

15 ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgtt ccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctca gttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcc

20 agttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctftgatcttttctacggggtctgacgctcagtggaacgaaaactcacgtta agggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatat gagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctga ctccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcac

25 cggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcca gtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtgg tgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaa agcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccg

30 agttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcg gggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttta ctttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga atactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaata aacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaa

35 ataggcgtatcacgaggccctttcgtcttcaagaa (SEQ ID NO: 131)

This plasmid was transformed into the E. coli strain BL21(λDE3)pLysS (Novagen), which contains the gene encoding the T7 RNA polymerase, with selection on

LA containing 100 micrograms/ml carbenicillin. Cells were grown overnight at 37°C. One transformant was selected and the strain was designated MSATNcol.

Production of Perhydrolase in MSATNcol-1 Production of perhydrolase was done in cell culture. For example, 5 ml of LB with carbenicillin at a concenfration of 100 micrograms/ml was inoculated with a single colony of MSATNcol and grown overnight at 37°C with shaking at 200 φm. This culture was used to inoculate 100 ml of LB with carbenicillin at a concentration of 100 micrograms/ml (in a 250 ml baffled flask) to an ODβoo of 0.1. The cultures were grown at 30°C with shaking at 200 φm until they reached an ODβoo of 0.4. The expression of the perhydrolase gene was then induced by the addition of 100 micromolar IPTG and the incubation continued overnight. Cultures were harvested by centrifugation (10 min at 7000 φm, Sorvall SS34 rotor), the supernatant was removed and the pellets washed in 50 mM KPO , pH 6.8. The cells were centrifuged again, the supernatants removed and the wet weight of the cells was determined. The cells were resuspended in 100 mM KPO in a volume that was 4x the wet weight. The resuspended cells were frozen at -70°C. The cells were thawed and lysed in a French Pressure cell using standard procedures known in the art. The purification steps and assessment methods are provided in Example 1. Figure 6 provides a purification table showing the enzyme activity of the perhydrolase of the present invention through various steps in the purification process.

M. smegmatis Perhydrolase is in an Operon In additional experiments, it was determined that the M. smegmatis perhydrolase is part of an operon. The gene (phd) is the first gene in an operon that contains at least 2 genes, including phd, that are separated by 10 bp (GGCTGGGGGC [SEQ ED NO:7]) not including the TAA stop codon of phd. It is also possible that there are three genes in the operon, with the third being either 48 bp or 61 bp to the next ORF (open reading frame). ^,! .'^•' .» '" ' "' .. GC821-2

The latter two candidate genes have no significant homology to proteins in the database. A putative promoter was identified for M. smegmatis phd operon, TTGGGC (-35) SP (18) CCAGAT by sequence analysis and comparison with known M. smegmatis promoters (See e.g., Salazar et al, Microbiol., 149:773-784 [2003]). It is not intended that the present invention be limited to any particular promoter and/or constract design, as it is contemplated that other promoters and construct designs will find use in the present invention. The second gene in the phd operon encodes a protein (putative PBP-3) with the sequence:

10 rnUφaltwllvvglfisvvgcssspdpadrfsafaealgrkdaaaaaaqtsdpaaaeaaitamlagmgdaanvsvaaepee gddagatlkytwtwgegrdfgydttataaksgddwlitwsptvl^^ verahpesaaplaallapfdpttttesvtaqlnsttddrvtvmkheddlgqwdqlaqipgvtvreqgelltø elwhdritanagwsvylvdadgapaqqltstopkdtgpvrttldhτnqllaqqavaketφa\^^aisgstggilaaaqnpaa dpqgaiafsglyppgstfktittaaaldaglatodtovacpgeltienrtipnddnfdlgtvplssafshscntsmaalsdelppn

15 altdmakdfgigvdfinvpgltlΛlgnφnadn^ lppmtdalrarmmgtΛrtegtatalsdipdlgglrtgtaefgdn ^ g (SEQ ID NO:9)

The corresponding DNA sequence of the gene encoding the putative PBP-3:

20 atgcacttacgtcccgctctgacgtggctcctggttgtcggtctgttcatatcggtcgtcggatgttcgtcgtccccggatccggccg accggttctcggcgttcgccgaggcgctgggccgcaaggatgcggccgcggcggccgcccagaccagcgatccggcggcc gcggaggcggccatcaccgcgatgctggccgggatgggcgacgccgcgaacgtctcggtggccgccgaacccgaggaagg cgacgacgcgggcgcgacgctgaagtacacgtggacctggggtgagggccgcgacttcggctacgacaccaccgcgacggc ggccaaatccggtgacgactggctgatcacctggtcccccaccgtgttgcaccgcgacctcaccccggatctgcgcttccagtac

25 agcgaggacagcgaattgcagaccccggtgctcgaccgcaccggccagccgttgatgacatggcagaccgtcggtgtcatcac tgtcgaacgcgcacatccggagtcggccgcaccgctcgccgccctgctggcgcccttcgatccgaccaccaccaccgaatcgg tcaccgcacaactcaattcgacgaccgatgaccgcgtgacggtgatgaagctgcgcgaggacgatctgggtcaggtgcgcgat cagctcgcgcagatccccggcgtgaccgtgcgtgagcagggtgagctgctcaccgccgaccggcagctgtcctcgcccgccat cagcggcctggacgagctgtggcacgaccggatcaccgccaacgcgggctggtcggtgtacctggtcgacgccgacggtgca

30 cccgcacaacagctcacgtccacgccgcccaaggacaccgggcccgtgcgcaccacgctggacctgcgcatgcaactgctcg cgcagcaggccgtggccaaggagacccgcccggccgtggtggtcgcgatctccggatcgaccgggggcatcctggccgccg cacagaacccggccgccgatccgcaaggtgcgatcgcgttttcgggcctgtacccgccggggtcgacgttcaagaccatcacc acggcggcagccctcgacgcgggcctggccaccccggacacaccggtggcctgcccgggtgagctcaccatcgagaaccgc acgatccccaacgacgacaacttcgacctgggcaccgtgccgttgtcgtcggcgttctcgcactcctgcaacaccagcatggcc

35 gccctgtccgacgagctgccgcccaacgcactgaccgacatggcaaaggacttcgggatcggcgtcgacttcatggtgcccgg i!-"^1, £_« i£ j'- i i' "» .-. " •^•' ^{« ■} ' - GC821-2 ' --

cctgaccaccgtgaccggccgtgtccccaacgccgacaacgccgcccagcgtgtcgagaacggcatcggccagggcaccgt gaccgtcagcccgttcggcctcgccgtcgccgaggccagcctggcgcacggttcgacgatcctgccgacgctggtcgacggc gagaagaccacggccgacaccccgtcggtgccgttgccgcccaacatcaccgacgcgctgcgcgcgatgatgcgcggaacg gtcaccgagggcacggccaccgcgttgagcgacatccccgacctgggcggcaagaccggcacggcggaattcggcgacaac 5 acgcactcgcacggctggttcgcgggcatcgcgggcgacatcgcgttcgcgacgctggtggtcggcggcgactcgtcggcac cggccgtcgcgatctcaggagacttcctgcgccccgcgctcgccggctag (SEQ ID NO:8).

A standard BLAST search against the protein database identified homology with several penicillin binding proteins, class 3 (PBP-3). By sequence alignment and 10 comparison to literature (e.g., Goffin and Ghysen, Microbiol. Mol. Biol. Rev., 66:702-38 [2002]) the PBP was found to contain the required bar codes (conserved protein sequences that define a class of proteins) to place it in the SxxK superfamily of acyl transferases, with a C-terminal domain acyl transferase and an N-terminal domain of unknown function, but with homology to the Pen^r (i.e., penicillin resistant) protein 15 fusions of class B-like II and HT. This penicillin binding protein acyl transferase domain does not share significant homology with the perhydrolase of the present invention, although it does share homology with Co-A dependent acyl transferases known in the art. The amino acid sequence is provided below.

20 MHLRPALTWLLWGLFISWGCSSSPDPADRFSAFAEALGRKDAAAAAAQTSDP AAAEAAITAMLAGMGDAANVSVAAEPEEGDDAGATLKYTWTWGEGRDFGYDT TATAAKSGDDWLΓΓWSPTVLHRDLTPDLRFQYSEDSELQTPVLDRTGQPLMTWQ TVGVITVERAHPESAAPLAALLAPFDPTTTTESVTAQLNSTTDDRVTVMKLREDD LGQVRDQLAQEPGVTVREQGELLTADRQLSSPAISGLDELWHDRITANAGWSVYL 25 VDADGAPAQQLTSTPPKDTGPVRTTLDLRMQLLAQQAVAKETRPAVWAISGS TGGILAAAQNPAADPQGAIAFSGLYPPGSTFKTITTAAALDAGLATPDTPVACPG ELTIENRTEPNDDNFDLGTVPLSSAFSHSCNTSMAALSDELPPNALTDMAKDFGIG VDFMVPGLTTVTGRVPNADNAAQRVENGIGQGTVTVSPFGLAVAEASLAHGSTI LPTLVDGEKTTADTPSVPLPPNITDALRAMMRGTVTEGTATALSDIPDLGGKTGT 30 AEFGDNTHSHGWFAGIAGDIAFATLWGGDSSAPAVAISGDFLRPALAG (SEQ ID NO: 10)

The family-identifying bar codes provided in the above review were: (19) V (20) „ ■■' ^■

GC821-2 ^•^

G/A (140) PVxDRTG (142) TxDx3Q (22) TGGxLAx4PaxDP (13) SxxK (51) SCN (131) KTG (50) marked in bold letters in the above sequence. The letters represent the amino acid sequence defining the bar code; the numbers in brackets are the intervening number of amino acids between the particular bar codes; "x" represents any amino acid, (i.e., the amino acids are not conserved within the bar code but the number of amino acids (e.g., x3 corresponding to 3 intervening amino acids) is conserved). Based on these results and other data, as described herein, it is clear that the perhydrolase of the present invention represents a unique enzyme class.

EXAMPLE 5 Expression of the Perhydrolase in P. citrea In this Example, methods used to express the perhydrolase in P. citrea are described. The plasmid pMSATNcol was transformed into P. citrea by electroporation using the method essentially as known in the art (See e.g., Sambrook et al, supra) except that all cultures and recovery were done at 30°C. The transformants were plated on L agar + carbenicillin (200 μg/ml) and incubated overnight at 30^*C. Three transformants were picked for analysis. Each colony was used to inoculate a 30 ml culture of LB + carbenicillin (200 μg/ml) and grown overnight at 30°C with shaking at 200 φm. The cells were pelleted by centrifugation, washed one time in 50 mM phosphate buffer pH 7.2, and finally resuspended in 4x the wet cell weight of 100 mM phosphate buffer pH 8.0. The cells were lysed by treatment with lysozyme (2 μl of a 10 mg/ml solution per one ml of P. citrea culture) at 37°C for one hour. The cell debris was pelleted at 13,000 φm in a microfuge for 5 min. The resulting supernatant was used for further analysis in SDS-PAGE and Western blots, as well as assays for enzyme activity. SDS-PAGE analysis was carried out as known in the art (See e.g., Sambrook et al, supra) on the supernatants. Detection of the perhydrolase protein by Western blot "^l- |i_,_» £ i*' 'ir-f -.ii if. I¹ ii ,- lⁱ „„^|H -^it GC821-2 ^ ^

was done using an anti-perhydrolase polyclonal anti-sera (prepared from purified perhydrolase protein by Covance). The blot was developed as per manufacturer's suggestions using the ECL plus kit (Amersham). The enzymatic activity of the expressed perhydrolase was detected by the pNB (para-nitrophenylbutyrate) assay as described in Example 1, herein. The results are provided in the

Table 5-1. Enzymatic Activity of Perhydrolase Expressed by P. citrea Concentration Clone OD405 Rate (mgliter) P. citrea/ pMSATNcol 3.1129 0.47948 7.1922 Control (P. citrea) 2.6187 -9.8312 0

The SDS-PAGE and Western blot results, as well as the assay results indicated 10 that the perhydrolase is expressed by P. citrea and is active.

EXAMPLE 6 Expression of the Perhydrolase in Bacillus subtitis 15 The perhydrolase was expressed intracellularly in B. subtilis. A variety of promoters find use in this embodiment, including but not limited to pSPAC, pAprE, pAmyE, pVeg, pHpall. In some embodiments, the constract is present on a replicating plasmid (e.g., pBHl), while in other embodiments, it is integrated into the chromosome in one or more copies. Examples of sites for integration include, but are not limited to the 20 aprE, the amyE, the veg or the pps regions. Indeed, it is contemplated that other sites known to those skilled in the art will find use in the present invention.

A. Intracellular Expression of the Perhydrolase in Bacillus subtilis From ■ . i .•' £*. ' -' i ^, GC821-2 j

a Replicating Plasmid B. subtilis expresses a lipase/esterase encoded by the gene pnbA that hydrolyzes the pNB substrate used to detect activity of the perhydrolase. To identify B. subtilis strains expressing the perhydrolase after transformation with replicating or integrating 5 plasmids Has pnbA gene (the entire coding sequence) was first deleted from the desired host using the loxP cassette deletion method described in WO 03/083125, herein incoφorated by reference. It is also noted that other strains of Bacillus may contain one or more lipases/esterases capable of hydrolyzing the pNB or other subsfrate used as an indicator for perhydrolase activity. In some embodiments, for optimal expression and/or

10 activity detection it is necessary to delete one or more of the lipases/esterases from the hosts. The Bacillus subtilis strain used in this Example has the genotype Bacillus subtilis comKpnhA

aprE, nprE, degUHy32, oppA, spoIIE3501 and will be referred to as "B. subtilis pnbA" (See e.g., WO 03/083125, supra). In these experiments, a consensus Bacillus ribosome binding site (RBS) was used.

15 It is not intended that the consensus RBS be the only sequence used for expression, as a non-consensus RBS also finds use in the present invention. The RBS of pMSATNcol (See, Example 4) was changed to a Bacillus consensus RBS from the 16S rRNA (5'- ATAAGGAGGTGATC -3' [SEQ ID NO: 132]) of B. subtilis and a H dm site was added to the 5' end of the RBS by PCR using a primer (502rbsforward primer) containing the

20 desired changes. The reaction was carried out using an MJ Research PCR machine with 30 cycles of (1 min at 95°C, 1 min at 55°C, and 1 min at 72°C). Template DNA (pMSATrbs) was added to a 50 μl reaction (10 ng) and 10 picomoles of each primer were used. The ^'PCR-generated phd cassette was cloned into the PCR cloning vector, pCR-

25 Script CM (Stratagene) and transformed into E. coli Top 10 cells (Invitrogen) to make pAΗ502R. The complete sequence of this plasmid is provided below. - ifι-- - .H ft -•"^■ i J , tit if-ji 'Mi- .-'. H U ^,f" ifi" ~ M '^,"li GC821-2 ^

ctaaattgtøagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccg aaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtcca ctattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcacc ctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgac 5 ggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagc ggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcg caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgat taagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcacta tagggcgaattgggtaccgggccccccctcgaggtcgacggtatcgataagcttgatatcgaattcctgcagcccggggg

10 atccgcccaagcttaaggaggtgatctagaattccatggccaagcgaattctgtgtttcggtgattccctgacctggggc tgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagct cggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctca acggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgac accaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgc

15 gggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccct ggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatg aaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcg cgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaaaaggatccccgggaagcttgcatgggctagagcg gccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagc

20 tgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggt gcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagct gcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgc tgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggat aacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca

25 taggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagat accaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgccttt ctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagct gggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaa gacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttc

30 ttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagatta cgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgt taagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttcgaccgaataaatacctgtgacggaag atcacttcgcagaataaataaatcctggtgtccctgttgataccgggaagccctgggccaacttttggcgaaaatgagac

35 gttgatcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagttgtcgag attttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgta aagaacatxttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggccttttta aagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccgga attacgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaa

40 ctgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcg GC821-2 °

tgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgag tttcaccagttttgatttaaacgtggccaatatggacaacttcttcgccccgttttcaccatgggcaaatattatacgca aggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgctta atgaattacaacagtactgcgatgagtggcagggcggggcgtaatttttttaaggcagttattggtgcccttaaacgcct ggttgctacgcctgaataagtgataataagcggatgaatggcagaaattcgaaagcaaattcgacccggtcgtcggttca gggcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccggaagcagtgtgaccgtgtg cttctcaaatgcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatatgatcctttttttctgat caaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatg taacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca aaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagca tttatcaagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcac atttccccgaaaagtgccac (SEQ ID NO: 133)

Transformants were selected on L agar containing 100 μg/ml carbenicillin. The construct was confirmed by sequencing and biochemical assays (e.g., pNB activity assay)

Primer set for pAH502R construction:

502rbsForward primer:

5'- ccaagcttaaggaggtgatctagaattccatggccaagcgaattctgtgtttcg-3' (SEQ ID NO:134)

502Reverse Primer:

5'- ggggatccttttacagcaggctccgcacct-3' (SEQ ID NO: 135) The Hwcffll-RBS-phd-R mH I DNA fragment from pAΗ502R was cloned into the pSPAC containing vector, pMUTIN4 (See, Vagner et al, Microbiol., 144, 3097-3104 [1998]) creating the construct pAH503. The complete sequence of pAH503 is provided below:

ataattctacacagcccagtccagactattcggcactgaaattatgggtgaagtggtcaagacctcactaggcaccttaa aaatagcgcaccctgaagaagatttatttgaggtagcccttgcctacctagcttccaagaaagatatcctaacagcacaa gagcggaaagatgttttgttctacatccagaacaacctctgctaaaattcctgaaaaattttgcaaaaagttgttgactt tatctacaaggtgtggcataatgtgtggaattgtgagcgctcacaattaagcttaaggaggtgatctagaattccatggc caagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcg cccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcg cgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacct P C: US O **• ^{" !}M!_"0 WΑ ft GC821-2 ^'^ -^

gccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgc tgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctg gtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccac tgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccg 5 acggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctg ctgtaaaaggatccccagcttgttgatacactaatgcttttatatagggaaaaggtggtgaactactgtggaagttactg acgtaagattacgggtcgaccgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagc tggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctg gtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaa 10 actggcagatgcacggttacgatgcgcccatctacaccaacgtaacctatcccattacggtcaatccgccgtttgttccc acggagaatccgacgggttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattat ttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgt ctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgttggagtgacggcagt tatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaat 15 cagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcg agttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggc ggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggag cgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcg atgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaac 20 cgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaa caactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatg tggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccggcg atgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatc aggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcg 25 gcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtg ccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgat gggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctggg actgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacg ccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaaca 30 ccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgata acgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaa ggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgca accgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtga cgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgt 35 tggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcga tcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaac gctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctg attacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtgg tcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctgg 40 cgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgtttt

'"ii" ii„ _f|E "" jr „;_κ}ι GC821-2

gaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcg cgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatgg aaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggc gacgactcctggagcccgtcagtatcggcggaattacagctgagcgccggtcgctaccattaccagttggtctggtgtca aaaataataataaccgggcaggccatgtctgcccgtatttcgcgtaaggaaatccattatgtactatttcaagctaattc cggtggaaacgaggteatcatttccttccgaaaaaacggttgcatttaaatct^ atttgtaagatttgatgtttgagtcggctgaaagatcgtacgtaccaattattgtttcgtgattgttcaagccataacac tgtagggatagtggaaagagtgcttcatctggttacgatcaatcaaatattcaaacggagggagacgattttgatgaaac cagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccac gtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaact ggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcga ttaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcg gcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgt ggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctccc atgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccatta agttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacg ggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgc tggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcg gtagtgggatacgacgataccgaagacagctcatgtiatatcccgccgtca ccaccatcaaacaggattttcgcctgct ggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctg gcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttaggcatcgcatcctgtctcgc gtcgtcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgg gagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgata gcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgc acagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcgg ctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacat gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctg . acgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgt ggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcca ctggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaa ctacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagga tctcaagaagatcctfrgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcat gagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagt aaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagtt gcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgaga cccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactt . _{" '}

GC821-2 ^'^)

tatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgtt gttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaag gcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttgg ccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtg actggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacggga taataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatct taccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtt cttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaa ataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacatta acctataaaaataggcgtatcacgaggccctttcgtcttcaagaattgatcctctagcacaaaagaaaaacgaaatgata caccaatcagtgcaaaaaaagatataatgggagataagacggttcgtgttcgtgctgacttgcaccatatcataaaaatc gaaacagcaaagaatggcggaaacgtaaaagaagttatggaaataagacttagaagcaaacttaagagtgtgttgatagt gcagtatcttaaaatxttgtataataggaattgaagttaaattagatgctaaaaatttgtaattaagaaggagtgattac atgaacaaaaatataaaatattctcaaaacttlttaacgagtgaaaaagtactcaaccaaataata^aacaattgaattt aaaagaaaccgataccgtttacgaaattggaacaggtaaagggcatttaacgacgaaactggctaaaataagtaaacagg taacgtctattgaattagacagtcatctattcaacttatcgtcagaaaaattaaaactgaatactcgtgtcactttaatt caccaagatattctacagtttcaattccctaaca^acagaggtataaaattgttgggagtattccttaccatttaagcac acaaattattaaaaaagtggtxtttgaaagccatgcgtctgacatctatctgattgttgaagaaggattctacaagcgta ccttggatattcaccgaacactagggttgctcttgcacactcaagtctcgattcagcaattgcttaagctgccagcggaa tgctttcatcctaaaccaaaagtaaacagtgtcttaataaaacttacccgccataccacagatgttccagataaatattg gaagctatatacgtactxtgtttcaaaatgggtcaatcgagaatatcgtcaactgtttactaaaaatcagtttcatcaag caatgaaacacgccaaagtaaacaatttaagtaccgttacttatgagcaagtattgtctatttttaatagttatctatta tftaacgggaggaaataattctatgagtcgcttttgtaaatttggaaagttacacgttactaaagggaatgtagataaat tattaggtatactactgacagcttccaaggagctaaagaggtccctagactctagacccggggatctctgcagtcggatc tggtaatgactctctagcttgaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttg tttgtcggtgaacgctctcctgagtaggacaaatccgccgctctagctaagcagaaggccatcctgacggatggcctttt tgcgtttctacaaactcttgttaactctagagctgcctgccgcgtttcggtgatgaagatcttcccgatgattaattaat tcagaacgctcggttgccgccgggcgttttttatgcagcaatggcaagaacgttgctctaga (SEQ ID NO: 136)

The construction of pAH503 was confirmed by RFLP and pNB activity assays. The pSPAC-RBS-phd DNA cassette was isolated as a BglWSmal digest and then subcloned into the replicating plasmid pBHl, digested with RαmHl/EcoRV (See e.g., ΕP 0275509) to create pAH505 (See, Figure 14). The complete sequence of the plasmid is provided below. _r ,-,„ . GC821-2 ^

gatcttccaagatatcctaacagcacaagagcggaaagatgttttgttctacatccagaacaacctctgctaaaattcctgaaaaattt tgcaaaaagttgttgactttatctacaaggtgtggcataatgtgtggaattgtgagcgctcacaattaagcttaaggaggtgatctag aattccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagc ggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgag 5 cgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcac ctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctg ggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctggtggt ctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcg cccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacg 10 gaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaaaaggatccc atcgcatgcggtacctctagaagaagcttggagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaac ctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataa aaaaagcacctgaaaaggtgtctttttftgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttatttt gctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgtt 15 aaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaa tcaagggttftagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaact tctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaa agaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtca caaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaa 20 gaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattøataaaaatcttgttggtttt cgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaa gaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagt tcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacgga ttttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtg 25 gtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccg atgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgt tgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctc caaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaag aaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttg 30 gtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggaca accggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgctt acacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcc caaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaagga ccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgac 35 gagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaac tttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtg gatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagta attatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatg agcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggca 40 ttgccagtcggggatattaaaaagagtataggtttttattgcgataaactaggtttcactttggttcaccatgaagatggattcgcagtt -

GC821-2

ctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgctctcgtagtaatgattcaccggtttgtacag gtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggc atxttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagctt ttftcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctctt giatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaa ctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgag^ tgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggttUUtgUtctxttttctcgtaa^aaa aagaaaggtcttaaaggttttatggttttggtcggcactgccgacagcctcgcaggacacacactttatgaatataaagtatagtgtg ttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttat gcagtttgtagaatgcaaaaagtgaaatcagggg (SEQ ID NO: 137)

The ligation mixture for pAH505 was transformed into Bacillus subtilis pnbA. Correct transformants were verified by RFLP and sequencing of isolated plasmid DNA. One transformant was selected for analysis (B. subtilis pnbA/pAH505). Expression of the perhydrolase in Bacillus was assayed using the pNB Activity Assay described herein, after growth of the desired strain in shake flask. The data showed that the perhydrolase was expressed in B. subtilis pnbA.

B. Intracellular Expression of the Perhydrolase in B. subtilis pnbA by Integration into the Chromosome An additional constract useful to determine expression of the perhydrolase (act) gene integrated into the chromosome of 5. subtilis pnbA involved use of the spoVG promoter, which was found to drive expression of the perhydrolase gene in a non- replicating (i.e., integrating plasmid). In some embodiments, one site of integration is the aprE region of B. subtilis, although it is intended that integration occur at any suitable site. Indeed, it is not intended that the present invention be limited to this specific site nor this specific promoter, as various other suitable sites and promoters find use in the present invention. i

GC821-2 ^ '^"^

The configuration of the promoter/gene at the aprE locus in the chromosome of Bacillus subtilis was as follows: pAprE-aprE first 7 codons-translation stop-pSpoVG-ATG-perhydrolase gene from second codon

The clone was constracted as described below. The primers used were: Up5'F caggctgcgcaactgttgggaag (SEQ ED NO: 138)

FuaprEAct34R agtagttcaccaccttttccctatataaaagcattagtgtatcaatttcagatccacaattttttgcttctcactctttac (SEQ ED NO:139)

FuaprEAct4F

Aattgatacactaatgcttttatatagggaaaaggtggtgaactactatggccaagcgaattctgtgtttcggtg (SEQ ED NO:140)

-5,smI-DnAct504R gtgagaggcaitcggatccttttacagcaggctccg (SEQ ID NO : 141 )

PCR fusion is a technique well known in the art, in which two or more fragments of DNA are generated either by restriction digest or by PCR amplification. The fragments have overlapping segments, usually at least 18 bases long. In the instance that two fragments are used, the 3' end of fragment #1 has an overlapping sequence with the 5' end of fragment #2. The two fragments are used as template in a PCR reaction in which the primer set used hybridizes to the 5' end of fragment #1 (forward primer) and the 3' end of fragment #2 (reverse primer). During the amplification, the two regions of overlap hybridize forming a single template from which the two primers can amplify a full length fragment, a "fusion" of fragments #1 and #2. Multiple fragments of any length can be used in such a reaction, limited only by the ability of the chosen polymerase to l if, Jf i'S t ""«",.' ""II - ', :r GC821-2

amplify long DNA pieces. In the current example, the above construct was made by PCR fusion of two PCR products the above constract was made by PCR fusion of two PCR products. The first was a construct with the spoVG promoter added upstream of the phd gene. The second was the aprE promoter and first 7 codons of aprE, followed by a stop codon. Regions of 20 bp overlap were added on the 5' and 3' ends of the products respectively, to allow the PCR fusion reaction. The primer set FuaprEAct4F/BsmI-DnAct504R was used to amplify the perhydrolase gene from pAH505 as described above, which added the spoVG promoter sequence (contained within the primer) to the 5' end of the gene and changed the start codon from ATG to GTG. To create the second product (pAprE plus the first 7 codons of aprE) for the fusion, the primer set Up5'F/FuaprEAct34R was used to amplify a fragment from pBSFNASally. Figure 15 provides a map of this plasmid. The complete sequence of pBSFNASally is provided below. ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaa cccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactc caacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgagg tgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaa aggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacaccc gccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggc ctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgac gttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattggagctccaccgcggtggcggccgctcta gaactagtggatcccccgggctgcaggaattctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaa aaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaacagcggcgcaatggcggccgcatctg atgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatactt ttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacagg aatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttctgt atgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtcta ctaaaatattattccatctattacaataaattcacagaatagtcl ttaagtaagtctactctgaatttttttaaaaggagagggtaaaga gtgagaagcaaaaaattgtggatcagtttgctgtttgctttagcgttaatctttacgatggcgttcggcagcacatcctctgcccaggc ggcagggaaatcaaacggggaaaagaaatatattgtcgggtttaaacagacaatgagcacgatgagcgccgctaagaagaaag atgtcatttctgaaaaaggcgggaaagtgcaaaagcaattcaaatatgtagacgcagcttcagctacattaaacgaaaaagctgta aaagaattgaaaaaagacccgagcgtcgcttacgttgaagaagatcacgtagcacatgcgtacgcgcagtccgtgccttacggc . -

GC821-2 "~^ f^

gtatcacaaattaaagcccctgctctgcactctcaaggctacactggatcaaatgttaaagtagcggttatcgacagcggtatcgatt cttctcatcctgatttaaaggtagcaggcggagccagcatggttccttctgaaacaaatcctttccaagacaacaactctcacggaa ctcacgttgccggcacagttgcggctcttaataactcaatcggtgtattaggcgttgcgccaagcgcatcactttacgctgtaaaagt tctcggtgctgacggttccggccaatacagctggatcattaacggaatcgagtgggcgatcgcaaacaatatggacgttattaaca tgagcctcggcggaccttctggttctgctgctttaaaagcggcagttgataaagccgttgcatccggcgtcgtagtcgttgcggcag ccggtaacgaaggcacttccggcagctcaagcacagtgggctaccctggtaaatacccttctgtcattgcagtaggcgctgttgac agcagcaaccaaagagcatctttctcaagcgtaggacctgagcttgatgtcatggcacctggcgtatctatccaaagcacgcttcc tggaaacaaatacggcgcgttgaacggtacatcaatggcatctccgcacgttgccggagcggctgctttgattctttctaagcacc cgaactggacaaacactcaagtccgcagcagtttagaaaacaccactacaaaacttggtgattctttctactatggaaaagggctg atcaacgtacaggcggcagctcagtaaaacataaaaaaccggccttggccccgccggt^ atccgctccataatcgacggatggctccctctgaaaattttaacgagaaacggcgggttgacccggctcagtcccgtaacggcca agtcctgaaacgtctcaatcgccgcttcccggtttccggtcagctcaatgccgtaacggtcggcggcgttttcctgataccgggag acggcattcgtøatcggatcctctagagtcgatttttacaagaattagctttatataatttctgttn^ctaaagt^ acagaaatgtattgcaatcttcaactaaatccatttgattctctccaata^ cagtatacttttccatgttataacacataaaaacaacttagtttt^ gttttttactagtcatttaaaacgatacattaataggtacgaaaaagcaactttτtttgcgctta^aaccagtcataccωtaacttaagg gtaactagcctcgccggcaatagttacccttattatcaagataagaaagaaaaggatttttcgctacgctcaaatcctttaaaaaaac acaaaagaccacattttttaatgtggtctttattctt^ tttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagt actttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcatta tttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacatøaaacaagaaggatataaatttta ccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattg ggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagag ttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgcttttt ctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattca ttaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgtttatgaactctat tcaggaattgtcagataggcctaatgactggctxttataatatgagataatgccgactgtactttttacagtcggttttctaatgtcacta acctgccccgttagttgaagaaggtttttatattacagctccagatccatat∞^ ccaattgctttattgacgttgagcctcggaacccttaacaatcccaaaacttgtcgaatggtcggcttaatagctcacgctatgccga cattcgtctgcaagtttagttaagggttcttctcaacgcacaataaattttctcg^ gatagcaaaaaatgccattccaatacaaaaccacatacctataatcgaccggaattaattctccattttcttctgctatcaaaataaca gactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaa cagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcc cttttctgtaaagtttatttttcagaatact ttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgc aggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaa catttactcatgtctaltttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaataga gataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactct gaatttttttatcaagcttatcgataccgtcgacctcgagggggggcccggtacccagcttttgttccctttagtgagggttaattgcg cgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataa agtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgt j . ■>•■■ "it ti; u GC821-2 ' Λ

cgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgac tcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggata acgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccatagg ctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggc gtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagc gtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagc cactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactaga aggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccacc gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcaga^aaa^aggatctcaagaagatcctttgatctttt ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaat taaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctca gcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggcccca gtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgc agaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccggga^gctagagtaagtagttcgccagttaatagttt gcgcaacgttgttgccattgctacaggcatcg^ggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatca aggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca gtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaa ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcag aactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaa cccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgca aaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctc atgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac (SEQ ID NO: 142)

The two PCR products were subjected to fusion PCR as known in the art to create the 1.5 kb fusion. The resulting fusion product was then cloned into PCR2.1TOPO to produce pCP609 (See, Figure 16) and sequence below). caggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaa ggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcact atagggcgaattggagctccaccgcggtggcggccgctctagaactagtggatcccccgggctgcaggaattctccattttcttct gctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattc ccgatattggttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaata attttttcattctatcccttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcacgataatatccattgttctca cggaagcacacgcaggtcatttgaacgaatttxttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgaca -'' ii,« .;,"3t ii„ιι ""ii" .' ""ii" ^B' *,3 cr GC821-2 ^•^ ^

tttcagcataatgaacatttactcatgtctaltttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagc tatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtctttt aagtaagtctactctgaatitttttaaaaggagagggtaaagagtgagaagcaaaaaattgtggatctgaaattgatacactaatgctt ttatatagggaaaaggtggtgaactactatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcga 5 agacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcga ggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacct gccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcg caccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacc cggctcccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcg

10 agcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgat cagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcgg agcctgctgtaaaaggatccgaatgcctctcacaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcat ctagagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcg ttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa

15 cagttgcgcagcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtg accgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagc tctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaa<^gattagggtgatggttcacgt agtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaac aacactcaaccctatctcggtctattcttttgatttataagggatttt^

20 aatttaacgcgaattttaacaaaattcagggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacg gtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgca gtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggta aggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaaga gacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggct

25 atgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaag accgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgc agctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatccca ccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgacc accaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatc

30 aggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcga tgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatca ggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccg ctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttcc gtgtcgcccttattcccttttftgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatc

35 agttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaa tgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacact attctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctg ccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaac atgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg

40 cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatg GC821-2 Ω ^' -.ζ^

gaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcg tgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatata tactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtg 5 agttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttlxtttctgcgcgtaatctgctgcttgc - acaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagca gagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcg ctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggata aggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct

10 acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaac aggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattGtgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgca gccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgtt

15 ggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagct cactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaa acagctatgaccatgattacgccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgccctt (SEQ ID NO:143)

20 The plasmid PCP609 was digested with BamHI IXmal to release the fragment containing the pAprE-αprE-stop-pSpoVG-pAc? construct and ligated into pBSFNASally digested with-X αI 5cfl to give the plasmid pCP649. Figure 17 provides a map of pCP649. The complete sequence of pCP649 is provided below.

25 tagaactagtggatcccccgggctgcaggaattctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttc aaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaacagcggcgcaatggcggccgcatc tgatgtctttgcttggcgaatgttcatcttatftcttcctccctctcaataattttttcattctatccctxttctgtaaagtttattt^ ttttatcatcatgctttgaaaaaatatcacgataatatccattgttcte

30 gaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattxtcgttcttttct gtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtc tactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactctgaatttttttaaaaggagagggtaaag agtgagaagcaaaaaattgtggatctgaaattgatacactaatgcttttatatagggaaaaggtggtgaactactatggccaagcga attctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgc

35 gctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacat cgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtga tcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtca - if if Sli Uf 'MfV "-"If . GC821-2 ^

cgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctggtggtctcgccgccaccgctggc gcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgc tcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggc caacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacggaatgcctctcacaaggatccaagcc 5 gaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagtcgatttttacaagaattagctttatataatttctgttt ttctaaagttttatcagctacaaaagacagaaatgtattgcaatcttcaactaaatccatttgattctctccaatatgacgtttaataaattt ctgaaatacttgatttctttgttttttctcagtatacttttccatgttataacacataa^aacaactt aaagttgctxtttcccctttctatgtatgttttttactagtcatttaaaacgatacattaataggtacgaaaaagcaactt aaccagtcataccaataacttaagggtaactagcctcgccggcaatagttaccc^attatcaagataagaaagaaaaggatttttcg

10 ctacgctcaaatcctttaaaaaaacacaaaagaccacattttttaatg ggtctttattcttcaactøaagcacccattagttcaacaaa cgaaaattggataaagtgggatatttttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaag cagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattgga agagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaaca taaaacaagaaggatataaattttaccctgcatxtattttcttagtgacaagggtgataaactcaaatacagcttttagaactgg^

15 atagcgacggagagttaggttattgggataagttagagccactttøtacaatttø ctcctgtaaagaatgacttcaaagagttttatgatttatacc^ acc acctgaaaatgctttttctctttctattattccatggac^^ tacccattattacagcagga^aattcattaataaaggtaattcaatatatttaccg^ atcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtac

20 ttrttacagtcggttttctaatgtcactaacctgcccc^ aaccgacttctcctttttcgcttctttattccaattgctttattgacgttgagcctcggaacccttaacaatcccaaaacttgtcgaatggt cggcttaatagctcacgctatgccgacattcgtctgcaagtttagttaagggttcttctcaacgcacaataaattttctcggcataaatg cgtggtctaatttttatttttaataaccttgatagcaaaaaatgccattccaatacaaaaccacatacctataatcgacctgcaggaatt aattcctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcgga

25 tgcctgtctataaaattcccgatattggcttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatctta tttcttcctccctctcaataattttttcattctatcccttttrt^ gataatatccattgttctcacggaagcacacgcaggtcatttgaacgaatttxttcgacaggaatttgccgggactcaggagcattta acctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcg^ ggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaata

30 aattcacagaatagtcttttaagtaagtctactctgaatttttttatcaagcttatcgataccgtcgacctcgagggggggcccggtac ccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcac aattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgc gctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgc gtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag

35 gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtg gcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgctta ccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaaccc

40 ggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagtt GC821-2 ^

cttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaag agttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaa aggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttøagggattttggtcatgaga ttatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgaca gttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataa ctacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagc aataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgg gaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttg gtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcg gtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcatøattctcttactgtcatgccat ccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggc gtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaa ggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctg ggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttt tcaatattattgaagcatttatcagggttattgtctcatgagcggatacatattt^ cgcacatttccccgaaaagfgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatt^ accaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaag agtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatca ccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggg gaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggt cacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactg ttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggt aacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgga gctccaccgcggtggcggccgctc (SEQ ID NO: 144)

All constructs were confirmed by sequence analysis. PCR reactions were done using Hercules polymerase (Roche) as per the manufacturer's directions. pCP649 was transformed into B. subtilis comKpnbA and integrants selected on L agar containing chloramphenicol (5 μg/ml). The activity of the expressed perhydrolase was determined by the pNB activity assay as described herein. The results indicated that the perhydrolase was expressed and active

EXAMPLE 7 Expression of the Perhydrolase in Streptomyces. PC ^"i TΓ GC821-2 -

In this Example, experiments conducted to assess the expression of the perhydrolase in Streptomyces are described. To test expression of the perhydrolase in Streptomyces, a replicating plasmid was constructed with the phd gene being expressed from either the glucose isomerase (GIT) or the A4 promoter (See e.g., US/PCT / , filed November 18, 2004, herein incoφorated by reference). However, it is not intended that the present invention be limited to these specific promoters, as any suitable promoter will find use with the present invention. Also, although the strain used for perhydrolase expression in this Example was Streptomyces lividans TK-23, it is contemplated that any Streptomyces will find use in the present 10 invention. The Streptomyces strains were transformed and manipulated using methods known in the art (See e.g., Kieser et al, Practical h-β tnmycpis GftnRtir.s_; John Innes [2000]).

15 Construction of pSECGT-MSAT and pSECA4-MSAT Using standard methods known in the art, the phd coding sequence (See, Example 4) was cloned into pSECGT to place the gene under control of the GI promoter. Similarly, the gene was cloned in the same plasmid with the A4 promoter using methods 20 known in the art (See e.g., US/PCT / filed November 18, 2004, herein incoφorated by reference). Transformants were first selected in E. coli, verified by sequence analysis, and then transformed into S. lividans TK-23 using methods known in the art (See e.g., Kieser et al, [2000], supra). The correct clones expressed from the GI promoter and the A4 promoter were designated "pSECGT-MSAT" and "pSECA4-phd." 25 The sequence of pSECGT-MSAT is provided below, while Figure 18 provides a map of the plasmid. ctagagtcgaccacgcaggccgccaggtagtcgacgttgatctcgcagccgagcccggccggaccggcggcgctgagcgcg aggccgacggcgggacggccggcaccggtacgcggtggcgggtcgagttcggtgagcagcccaccggcgatcaggtcgtcg ff" GC821-2 ^ .?

acgagcgcggagacggtggcccgggtgagcccggtgacggcggcaactcccgcgcgggagagccgatctgtgctgtttgcc acggtatgcagcaccagcgcgagattatgggctcgcacgctcgactgtcggacgggggcactggaacgagaagtcaggcgag ccgtcacgcccttgacaatgccacatcctgagcaaataattcaaccactaaacaaatcaaccgcgtttcccggaggtaaccatggc caagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccc 5 cgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcac caccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcg acctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcgg tgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgcca ccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgta 10 cagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttc accgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacgggatccgcgagcggatc ggctgaccggagcggggaggaggacgggcggccggcggaaaagtccgccggtccgctgaatcgctccccgggcacggac gtggcagtatcagcgccatgtccggcatatcccagccctccgcatgccccgaattcggcgtaatcatggtcatagctgtttcctgtg tgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagcta 15 actcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgc ggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcg gtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcca gcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcg acgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcct 20 gttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatc tcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaac tatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatg taggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagc cagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagc 25 agattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgtt aagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatata tgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctg actccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctca ccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcc 30 agtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtg gtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaa aagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataa ttctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgacc gagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttc 35 ggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttg aatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatftgaatgtatttagaaaaat aaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctcttgacacatgcagctcccggagacg 40 gtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctg ψ< !,„,. 11 x GC821-2

gcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaa taccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagc tggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggcca gtaagcttgcatgcctgcaggagtggggaggcacgatggccgctttggtcgacctcaacgagacgatgaagccgtggaacgac 5 accaccccggcggccctgctggaccacacccggcactacaccttcgacgtctgatcatcactgacgaatcgaggtcgaggaac cgagcgtccgaggaacacaggcgcttatcggttggccgcgagattcctgtcgatcctctcgtgcagcgcgattccgagggaaac ggaaacgttgagagactcggtctggctcatcatggggatggaaaccgaggcggaagacgcctcctcgaacaggtcggaaggc ccacccttttcgctgccgaacagcaaggccagccgatccggattgtccccgagttccttcacggaaatgtcgccatccgccttgag cgtcatcagctgcataccgctgtcccgaatgaaggcgatggcctcctcgcgaccggagagaacgacgggaagggagaagacg 10 taacctcggctggccctttggagacgccggtccgcgatgctggtgatgtcactgtcgaccaggatgatccccgacgctccgagc gcgagcgacgtgcgtactatcgcgccgatgttcccgacgatcttcaccccgtcgagaacgacgacgtccccacgccggctcgc gatatcgccgaacctggccgggcgagggacgcgggcgatgccgaatgtcttggccttccgctcccccttgaacaactggttgac gatcgaggagtcgatgaggcggaccggtatgttctgccgcccgcacagatccagcaactcagatggaaaaggactgctgtcgct gccgtagacctcgatgaactccaccccggccgcgatgctgtgcatgaggggctcgacgtcctcgatcaacgttgtctttatgttgg 15 atcgcgacggcttggtgacatcgatgatccgctgcaccgcgggatcggacggatttgcgatggtgtccaactcagtcatggtcgt cctaccggctgctgtgttcagtgacgcgattcctggggtgtgacaccctacgcgacgatggcggatggctgccctgaccggcaat caccaacgcaaggggaagtcgtcgctctctggcaaagctccccgctcttccccgtccgggacccgcgcggtcgatccccgcata tgaagtattcgccttgatcagtcccggtggacgcgccagcggcccgccggagcgacggactccccgacctcgatcgtgtcgcc ctgagcgtccacgtagacgttgcgtgagagcaggactgggccgccgccgaccgcaccgccctcaccaccgaccgcgaccgc 20 gccatggccgccgccgacggcctggtcgccgccgccgcccgccggttcggcgcctgacccgaccaacccccgcggggcgc cggcacttcgtgctggcgccccgcccccacccaccaggagaccgaccatgaccgacttcgacggacgcctgaccgaggggac cgtgaacctggtccaggaccccaacggcggtggctggtccgcccactgcgctgagcccggttgcgactgggccgacttcgccg gaccgctcggcttccagggcctcgtggccatcgctcgccgacacacgcactgaccgcacgtcaaagccccgccggatacccg gcggggctctcttcggccctccaagtcacaccagccccaaggggcgtcgggagtggcggagggaacctctggcccgattggtg 25 ccaggattcccaccagaccaaagagcaacgggccggacttcgcacctccgacccgtccgctcccagactcgcgccccttagcc gggcgagacaggaacgttgctcgtgcccagagtacggagcgatgccgaggcattgccagatcggcccgccgggccccgctg ccactgcgggaccgcaattgcccacacaccgggcaaacggccgcgtatctactgctcagaccgctgccggatggcagcgaag cgggcgatcgcgcgtgtgacgcgagatgccgcccgaggcaaaagcgaacaccttgggaaagaaacaacagagtttcccgcac ccctccgacctgcggtttctccggacggggtggatggggagagcccgagaggcgacagcctctgggaagtaggaagcacgtc 30 gcggaccgaggctgcccgactgcggaaagccgcccggtacagccgccgccggacgctgtggcggatcagcggggacgccg cgtgcaagggctgcggccgcgccctgatggaccctgcctccggcgtgatcgtcgcccagacggcggccggaacgtccgtggt cctgggcctgatgcggtgcgggcggatctggctctgcccggtctgcgccgccacgatccggcacaagcgggccgaggagatc accgccgccgtggtcgagtggatcaagcgcggggggaccgcctacctggtcaccttcacggcccgccatgggcacacggacc ggctcgcggacctcatggacgccctccagggcacccggaagacgccggacagcccccggcggccgggcgcctaccagcga 35 ctgatcacgggcggcacgtgggccggacgccgggccaaggacgggcaccgggccgccgaccgcgagggcatccgagacc ggatcgggtacgtcggcatgatccgcgcgaccgaagtcaccgtggggcagatcaacggctggcacccgcacatccacgcgat cgtcctggtcggcggccggaccgagggggagcggtccgcgaagcagatcgtcgccaccttcgagccgaccggcgccgcgct cgacgagtggcaggggcactggcggtccgtgtggaccgccgccctgcgcaaggtcaaccccgccttcacgcccgacgaccg gcacggcgtcgacttcaagcggctggagaccgagcgcgacgccaacgacctcgccgagtacatcgccaagacccaggacgg 40 gaaggcgcccgccctcgaactcgcccgcgccgacctcaagacggcgaccggcgggaacgtcgccccgttcgaactcctcgg GC821-2 ^ ' ^~

acggatcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgctggagtggaacctctcgcgctg gcacgagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcggcagatgctcgggctcgacgg cggcgacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcggggagctgcgggccggggtcgccg tgaccgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccgggccgccgaaggcaaggacggc 5 aacgaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcggccgacacagtggtggtgctc acggcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgcgaggaagcaactgcacgccg acggcgagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgcccggcacatcgcccggctc gcaagtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgtacggcgggggtcggtgacgtacg cggcgacggcggccggggtcgaagccgcgggagtaatcctgggattactcgcccggggtcggccccgccggcacttcgtgca

10 ggcggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggccatgagcaccaccacccccaggga cgccgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcggccggagccgggactactgccg ccgctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgccgtggcgtcggcagtcgctcg ccgagatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcgggccgcaggtcagagcacggttcc gcccgctccggccctgccggacccccggctgcagctcgcccggccgccggtccccctgccgtccggcccgtcccagaggca

15 gcgtcggcggctcctgcctcccccgcccggcgccgaccgggacccgcaaaccccttgatccgctgtcgggggtgatcactacg gtgggtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggaccaggcgttagcggcagtgctggc cgcactgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgccgtggcggtcggcatggcgccc gccctcgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtccgccggttccgccggcatcttgc ccaccacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcgacggccgccacctcatcgcgg

20 cagcaggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcaccgcgacggcgggggtacggc gctccgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctccccgctgccctgtcggcgagc acaacggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgcccccgtcgtgtgcgcgggccg gtcggcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggccgcgcggcccgctcgcgg gccgccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcgatcct (SEQ ID NO: 145)

25 Figure 19 provides a map of pSEGT-phdA4, while the sequence is provided below: ctagagatcgaacttcatgttcgagttcttgttcacgtagaagccggagatgtgagaggtgatctggaactgctcaccctcgttggt ggtgacctggaggtaaagcaagtgacccttctggcggaggtggtaaggaacggggttccacggggagagagagatggccttg

30 acggtcttgggaaggggagcttcngcgcgggggaggatggtcttgagagagggggagctagtaatgtcgtacttggacaggga gtgctccttctccgacgcatcagccacctcagcggagatggcatcgtgcagagacagacccccggaggtaaccatggccaagc gaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgt gcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaa catcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctgg

35 tgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgt cacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgccaccgctg gcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgc gctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgag gccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacaatggggatccgcgagcggatcgg MiSI^'J k''' HMUt' GC821-2 ~\

ctgaccggagcggggaggaggacgggcggccggcggaaaagtccgccggtccgctgaatcgctccccgggcacggacgtg gcagtatcagcgccatgtccggcatatcccagccctccgcatgccccgaattcggcgtaatcatggtcatagctgtttcctgtgtga aattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaact cacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgg ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggta tcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagca aaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgtt ccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctca gttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcc agttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtUttttgUtgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgtta agggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatat gagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctga ctccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcac cggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcca gtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctøcaggcatcg^ tgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttøcatgatcccccatgttgtgcaaaaa agcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccg agttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcg gggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttta ctttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga atactcatactcttcctttttcaatattattgaagcatttatcaggg^ aacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaa ataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctcttgacacatgcagctcccggagacggt cacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctgg cttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaat accgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagct ggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccag taagcttgcatgcctgcaggagtggggaggcacgatggccgctttggtcgacctcaacgagacgatgaagccgtggaacgaca ccaccccggcggccctgctggaccacacccggcactacaccttcgacgtctgatcatcactgacgaatcgaggtcgaggaacc gagcgtccgaggaacacaggcgcttatcggttggccgcgagattcctgtcgatcctctcgtgcagcgcgattccgagggaaacg gaaacgttgagagactcggtctggctcatcatggggatggaaaccgaggcggaagacgcctcctcgaacaggtcggaaggcc cacccttttcgctgccgaacagcaaggccagccgatccggattgtccccgagttccttcacggaaatgtcgccatccgccttgagc gtcatcagctgcataccgctgtcccgaatgaaggcgatggcctcctcgcgaccggagagaacgacgggaagggagaagacgt aacctcggctggccctttggagacgccggtccgcgatgctggtgatgtcactgtcgaccaggatgatccccgacgctccgagcg cgagcgacgtgcgtactatcgcgccgatgttcccgacgatcttcaccccgtcgagaacgacgacgtccccacgccggctcgcg ;T/ *:tlr-fc*#' ^»'fc'll,j l' -,"n ii;; GC821-2 ^

atatcgccgaacctggccgggcgagggacgcgggcgatgccgaatgtcttggccttccgctcccccttgaacaactggttgacg atcgaggagtcgatgaggcggaccggtatgttctgccgcccgcacagatccagcaactcagatggaaaaggactgctgtcgctg ccgtagacctcgatgaactccaccccggccgcgatgctgtgcatgaggggctcgacgtcctcgatcaacgttgtctttatgttggat cgcgacggcttggtgacatcgatgatccgctgcaccgcgggatcggacggatttgcgatggtgtccaactcagtcatggtcgtcc 5 taccggctgctgtgttcagtgacgcgattcctggggtgtgacaccctacgcgacgatggcggatggctgccctgaccggcaatca ccaacgcaaggggaagtcgtcgctctctggcaaagctccccgctcttccccgtccgggacccgcgcggtcgatccccgcatatg aagtattcgccttgatcagtcccggtggacgcgccagcggcccgccggagcgacggactccccgacctcgatcgtgtcgccpt gagcgtccacgtagacgttgcgtgagagcaggactgggccgccgccgaccgcaccgccctcaccaccgaccgcgaccgcgc catggccgccgccgacggcctggtcgccgccgccgcccgccggttcggcgcctgacccgaccaacccccgcggggcgccg 10 gcacttcgtgctggcgccccgcccccacccaccaggagaccgaccatgaccgacttcgacggacgcctgaccgaggggaccg tgaacctggtccaggaccccaacggcggtggctggtccgcccactgcgctgagcccggttgcgactgggccgacttcgccgga ccgctcggcttccagggcctcgtggccatcgctcgccgacacacgcactgaccgcacgtcaaagccccgccggatacccggc ggggctctcttcggccctccaagtcacaccagccccaaggggcgtcgggagtggcggagggaacctctggcccgattggtgcc aggattcccaccagaccaaagagcaacgggccggacttcgcacctccgacccgtccgctcccagactcgcgccccttagccgg 15 gcgagacaggaacgttgctcgtgcccagagtacggagcgatgccgaggcattgccagatcggcccgccgggccccgctgcca ctgcgggaccgcaattgcccacacaccgggcaaacggccgcgtatctactgctcagaccgctgccggatggcagcgaagcgg gcgatcgcgcgtgtgacgcgagatgccgcccgaggcaaaagcgaacaccttgggaaagaaacaacagagtttcccgcacccc tccgacctgcggtttctccggacggggtggatggggagagcccgagaggcgacagcctctgggaagtaggaagcacgtcgcg gaccgaggctgcccgactgcggaaagccgcccggtacagccgccgccggacgctgtggcggatcagcggggacgccgcgt

20 gcaagggctgcggccgcgccctgatggaccctgcctccggcgtgatcgtcgcccagacggcggccggaacgtccgtggtcct gggcctgatgcggtgcgggcggatctggctctgcccggtctgcgccgccacgatccggcacaagcgggccgaggagatcacc gccgccgtggtcgagtggatcaagcgcggggggaccgcctacctggtcaccttcacggcccgccatgggcacacggaccggc tcgcggacctcatggacgccctccagggcacccggaagacgccggacagcccccggcggccgggcgcctaccagcgactg atcacgggcggcacgtgggccggacgccgggccaaggacgggcaccgggccgccgaccgcgagggcatccgagaccgga

25 tcgggtacgtcggcatgatccgcgcgaccgaagtcaccgtggggcagatcaacggctggcacccgcacatccacgcgatcgtc ctggtcggcggccggaccgagggggagcggtccgcgaagcagatcgtcgccaccttcgagccgaccggcgccgcgctcgac gagtggcaggggcactggcggtccgtgtggaccgccgccctgcgcaaggtcaaccccgccttcacgcccgacgaccggcac ggcgtcgacttcaagcggctggagaccgagcgcgacgccaacgacctcgccgagtacatcgccaagacccaggacgggaag gcgcccgccctcgaactcgcccgcgccgacctcaagacggcgaccggcgggaacgtcgccccgttcgaactcctcggacgg

30 atcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgctggagtggaacctctcgcgctggcac gagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcggcagatgctcgggctcgacggcggc gacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcggggagctgcgggccggggtcgccgtgac cgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccgggccgccgaaggcaaggacggcaac gaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcggccgacacagtggtggtgctcacg

35 gcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgcgaggaagcaactgcacgccgacg gcgagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgcccggcacatcgcccggctcgca agtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgtacggcgggggtcggtgacgtacgcgg cgacggcggccggggtcgaagccgcgggagtaatcctgggattactcgcccggggtcggccccgccggcacttcgtgcaggc ggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggccatgagcaccaccaccc^'ccagggacgc

40 cgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcggccggagccgggactactgccgccg F" T U tit U ^!' II" "' " if i "t" "i GC821-2 ^•- ^

ctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgccgtggcgtcggcagtcgctcgccg agatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcgggccgcaggtcagagcacggttccgcc cgctccggccctgccggacccccggctgcagctcgcccggccgccggtccccctgccgtccggcccgtcccagaggcagcgt cggcggctcctgcctcccccgcccggcgccgaccgggacccgcaaaccccttgatccgctgtcgggggtgatcactacggtgg gtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggaccaggcgttagcggcagtgctggccgca ctgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgccgtggcggtcggcatggcgcccgccct cgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtccgccggttccgccggcatcttgcccac cacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcgacggccgccacctcatcgcggcag caggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcaccgcgacggcgggggtacggcgctc 10 cgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctccccgctgccctgtcggcgagcacaa cggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgcccccgtcgtgtgcgcgggccggtcg gcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggccgcgcggcccgctcgcgggccg ccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcgatcct (SEQ ID NO: 146)

15 Two colonies of S. lividans TK-23 ρSECA4-phd were inoculated in 10 ml of TS medium + 50 ppm thiostrepton and incubated at 37°C with shaking at 200 φm for 2 days. Three mis of broth were used to inoculate 50 ml of Streptomyces Production medium 1 and the culture was incubated for 4 days at 37°C with shaking at 200 φm. A sample was taken to assay perhydrolase activity measurement as follows: 10 μls 20 of 20 mg/ml lysozyme were added to 200 μl of sample. After 1 hour of incubation at 37°C, samples were centrifuged and activity was measured using the pNB activity assay described herein. SDS-PAGE and Western blots were also prepared using both clones (pSECA4-ρhd and pSECGT-MSAT), as known in the art. Briefly, after SDS-PAGE, the proteins were transferred to PVDF membrane and Western blot analysis was conducted. 25 The perhydrolase was detected using an anti-perhydrolase polyclonal anti-sera (1 :500 dilution) prepared against purified perhydrolase protein by Covance. The blot was developed using the ECL kit from Amersham. The results indicated that Streptomyces lividans strains were capable of expressing active perhydrolase.

30 , . ,

GC821-2 - N r^,

EXAMPLE 8 Site-Scanning Mutagenesis of the M. smegmatis Perhydrolase Gene In this Example, experiments involving site-scanning mutagenesis of the M. smegmatis perhydrolase gene are described. In these experiments, the QuikChange® site- directed mutagenesis (QC; Stratagene) kit or the QuikChange® Multi Site-Directed mutagenesis (QCMS; Stratagene) kit was used to create site-saturation libraries at each codon in the entire M. smegmatis perhydrolase gene contained in the pMSAT-Ncol plasmid. Each perhydrolase codon was mutagenized by replacement with the NNG/C (NNS; 32 combinations) degenerate codon, which encodes for all 20 amino acids and one stop codon. In the case of the QC method, complementary overlapping primers were designed for each codon of interest with 18 bases flanking the NNS codon (See, Tables 8- 1 and 8-2). A comparison of cartridge purified versus unpurified primers (desalted only) revealed a better representation of amino acids in the libraries made with purified primers (15-19 amino acids versus 11-16 with unpurified primers). Thus, a majority of the libraries were created with the QC method and purified primers. A small number of the libraries were made using the QCMS method and a single 5' phosphorylated forward primer containing 18 bases flanking both sides of the NNS codon (See, Table 8-1), however this method resulted in a greater wild type background and fewer amino acid substitutions per site compared to the QC methods. Libraries "nsa301" and "nsa302" were made using the QCMS method, but a trinucleotide mix made up of a single codon for each of the 20 amino acids (i.e., rather than 32 possibilities encoded by NNS for the 20 amino acids) was incoφorated within the primers at the sites of interest.

it" Uf £"" tJ£ "'If".'-¹ '"'lⁱ'- t -Λ >» GC821-2 ^" ^

_- GC821-2 Ω

GC821-2 ^•^ C\

if l);3iUf^|r'ii" "'II' H GC821-2

if- ;: ffjlfφ IHJ»# GC821-2 1

GC821-2 n

-^. '^■ f K '•> "^•"ii'.''¹ "+ "¹" >» GC821-2 °^

1N201 bsa402F pacttcaccεagεccaacnnscεcεatctcεεεεtεεcc (SEO ID NO:347) 1R202 bsa403F ktcaccεaεeccaacaatnnsεatctcεεεεtgεccctc (SEO ED NO:348) JD203 bsa404F accεaεεccaacaatcgcnnsctcεεεεtεgccctcεcg (SEO ID NO:349) L204 bsa405F εaεgccaacaatcgcεatnnsεεgεtεεccctcεcεεaa (SEO ID NO:350) IG205 bsa406F εccaacaatcεcεatctcnnsgtεεccctcεcεgaacag (SEO ID NO:351) 1V206 bsa407F acaatcεcεatctcεεεnnsεccctcεcεεaacaεεtg (SEO ED NO:352) IA207 bsa408F aatcεcεatctcεεεεtεnnsctcεcεεaacaggtgcag (SEO ED NO:353) IL208 bsa409F kεcεatctcεεεgtggccnnsgcεεaacaεgtgcaεaεc (SEO ID NO:354) IA209 bsa410F εatctcεεεεtεεccctcnnsεaacaεεtεcaεaεcctε (SEO ID NO:355) E210 bsa411F ctcεεεεtεεccctcgcεnnscaεεtεcagaεcctεctg (SEO ED NO:356) 0211 bsa412F εεεεtεεccctcεcεεaannsεtgcaεagcctgctgtaa (SEO ID NO:357) IV212 bsa413F gtεεccctcgcgεaacaεnnscaεagcctgctgtaaaag (SEO ED NO:358) 213 bsa414F εccctcεcεεaacaεεtgnnsaεcctεctεtaaaaεgεc (SEO DP NO:359) IS214 hsa415F [ctcgcggaacaggtεcaεnnsctεctεtaaaaεεεcgaa (SEO ID NO:360) EL215 bsa416F εcεεaacaεεtεcagaεcnnsctεtaaaaεεεcgaattc (SEO EDNO:361) L216 hsa417F εaacaεεtεcaεaεcctεnnstaaaaεεεcgaattctεc (SEO ID NO:362)

U'Λ ^»»»%^•'^{• ,f»}lt^»iu ^■'„'^• ϊi

^ ^,f-^{if ;3 ILJ} " ^if ' ''' '"'''" ^|E * »' > GC821-2 - ι

P» iC/T, U l H-,s -U - ^ GC821-2 ^ o.

! Ibi ϋ ""if" ,•^■'^" fl" "l"

ilbiUHi",.

ψX if* ' "^• U 5a U »+,''^* "-it" \ ,m',"tl o GC821-2

IL iLlHt'l" ^• t-i _".: s GC821-2 (X

- C; t' t,» NI" ^Tψβ ,;; GC821-2 '"^

,,,,,_t ,_u, ,_t„_lt„ _ , „„,,„

GC821-2

>- ϋ ϋ ' .' ""»^{• <>} " ,.'- .!-- - \ GC821-2

QC Method to Create Site-Saturation Libraries The QC reaction consisted of 40.25 μL of sterile distilled HaO, 5 μL of PfuTurbo 1 Ox buffer from the kit, 1 μL dNTPs from the kit, 1.25 μL of forward primer (1 OOng/μL), 1.25 μL reverse primer (lOOng/μL), 0.25 μL of pMSAT-Ncol miniprep DNA as template (~50ng), and 1 μL of PfuTurbo from the kit, for a total of 50 μL. The cycling conditions were 95°C for lmin, once, followed by 19-20 cycles of 95^ΦC for 30 to 45 sec, 55°C for lmin, and 68°C for 5 to 8 min. To analyze the reaction, 5μL of the reaction was run on a 0.8% E-gel (Invitrogen) upon completion. Next, Dpnl digestion was carried out twice sequentially, with 1 μL and 0.5 μL of enzyme at 37°C for 2 to 8 hours. A negative confrol was carried out under similar conditions, but without any primers. Then, 1 μL of the -9p«I-digested reaction product was transformed into 50 μL of one-shot TOP 10 electrocompetent cells (Invitrogen) using a BioRad elecfroporator. Then, 300 μL of SOC provided with the TOP 10 cells (Invitrogen) were added to the elecfroporated cells and incubated with shaking for 1 hour before plating on LA plates containing lOppm kanamycin. The plates were incubated at 37°C overnight. After this incubation, 96 T » _" ^t! ϊ=» "' if •^{■■ ,f}"'f" , i if 1,;:. GC821-2 ^ v

colonies from each of the libraries (i.e., each site) were inoculated in 200μL of LB containing 10-50ppm of kanamycin in 96-well microtiter plates. The plates were frozen at -80°C after addition of glycerol to 20% final concentration, and they were used for high throughput sequencing at Genaissance with the M13F and M13R primers.

QCMS Method to Create Site-Saturation Libraries The QCMS reaction consisted of 19.25 μL of sterile distilled H20, 2.5 μL of lOx buffer from the kit, lμL dNTPs from the kit, lμL of 5' phosphorylated forward primer (lOOng/μL), 0.25 μL of pMSAT-Ncol miniprep DNA as template (~50ng), and lμL of the enzyme blend from the kit for a total of 25 μL. The cycling conditions were 95"C for lmin once, followed by 30 cycles of 95°C for lmin, 55^*C for lmin, and 68°C for 8 min. To analyze the reaction product, 5μL of the reaction were run on a 0.8% E-gel (Invitrogen) upon completion. Next, Dpήl digestion was carried out twice sequentially, with 0.5 μL of enzyme at 37°C for 2 to 8 hours. The controls, transformation, and sequencing was performed as for the QC method described above.

Details of Screening Plate Preparation Using a sterilized stamping tool with 96 pins, the frozen clones from each sequenced library plate were stamped on to a large LA plate containing lOppm kanamycin. The plate was then incubated overnight at 37°C. Individual mutant clones each representing each one of the 19 substitutions (or as many that were obtained) were inoculated into a Costar 96-well plate containing 195μL of LB made with 2 fold greater yeast extract and lOppm kanamycin. Each mutant clone for a given site was inoculated in quadruplicate. The plate was grown at 37°C and 225 φm shaking for 18 hrs in a humidified chamber. In a separate 96-well plate, 26μL of BugBuster (Novagen) with DNase were added to each well. Next, 125μL of the library clone cultures were added to the BugBuster-containing plate in corresponding wells and the plate was frozen at -80°C. U S O ⁱⁱ*'.- **^• W **'3K

GC821-2 ^

The plate was thawed, frozen and thawed again before use of the lysates in the peracid formation and peracid hydrolysis assays described herein.

Combinatorial Libraries and Mutants From the screening of the single site-saturation libraries, the important sites and substitutions were identified and combined in different combinatorial libraries. For example, libraries described in Table 8-3 were created using the following sites and substitutions:

L12C, Q, G

T25S, G, P

L53H, Q, G, S

S54V, L, A, P, T, R

A55G, T R67T, Q, N, G, E, L, F

K97R

V125S, G, R, A, P

F154Y

F196G

I'^ i i if , U ϋ» U 'HI- ^,■'^' - GC821-2 15

TABLE 8-3. Libraries Library Description Parent Method Template NSAA1 L12G S54(NNS) L12G QC NSAA2 S54V L12(NNS) S54V QC NSAA3 L12(NNS) S54(NNS) WT QCMS NSAB1 S54V T25(NNS) S54V QC NSAB2 S54V R67(NNS) S54V QC NSAB3 S54V V125(NNS) S54V QC NSAB4 L12I S54V T25(NNS) L12I S54V QC NSAB5 L12I S54V R67(NNS) L12I S54V QC NSAB6 L12I S54V V125(NNS) L12I S54V QC NSAC1 S54(NNS) R67(NNS) WT QCMS V125(NNS) .NSAC2 43 primer library, 10 sites S54V QCMS (lOOng total primers) NSAC3 same as nsaC2 but 300ng S54V QCMS total primers NSAC4 32 primer library, 8 sites S54V QCMS (lOOng total primers) NSAC5 same as nsaC4 but 300ng S54V QCMS total primers NSAC6 8 primers, 7 substitutions, S54V QCMS 5 sites (lOOng total primers) NSAC7 same as nsaC6 but 300ng S54V QCMS total primers *NNS indicates site-saturation library **A11 parent templates were derived from the pMSAT-NcoI plasmid and contained mutations at the indicated codons with in the M. smegmatis perhydrolase gene The QC or QCMS methods were used to create the combinations. The QC reaction was carried out as described above, with the exception being the template plasmid, which consisted of 0.25μL of miniprep DNA of the L12G mutant, S54V mutant, 10 or the L12I S54V double mutant plasmid derived from pMSAT-NcoI. The QCMS £/^'!^"/ U S O HV >'+0 H^>3 s GC821-2 O

reaction was also carried out as described above, with the exception of template and primers. In this case, 0.25 μL of the pMSAT-NcoI template were used for NSACl and NSAA3 or S54V template for NSAC2-C7 libraries. The NSAA3 and the NSACl libraries were made using 100 ng of each of the primers shown in the Table 8-4. The NSAC2, NSAC4, and NSAC6 libraries were made with a total of lOOng of all primers (all primers being equimolar), and NSAC3, NSAC5, NSAC7 libraries were made with a total of 300ng of all primers (all primers being approximately equimolar)

Table 8-4. Libraries Primer Libraries am? Primer Sequence NSACl S54NNS-FP gtgatcgaggagggactgnnsεcgcgcaccaccaacatc (SEO ID NO:579) πsrsACi R67NNS-FP acgaccccaccεatccεnnsctcaacggcεcgagctac (SEQ ID NO:580) NSACl IN125NNS-FP ctcaccaεcgcεgεcggcnnsggcaccacgtacccggca (SEO ED NO:581) INSAC2-C51 1L12C ctεtεtttcεεtεattccTGCacctggεεctgggtcccc (SEO ED NO:582) NSAC2-C7L12Q ctεtgtttcεεtgattccCAGacctεgεεctεεgtcccc (SEO ID NO:583) INSAC2-C5! 1L12I ctgtgtttcggtεattccATCacctggggctgggtcccc (SEO ID NO:584) NSAC2-C3 L12M ctεtεtttcggtgattccATGacctεgggctgggtcccc (SEO ID NO:585) NSAC2-C31 1L12T ctgtεtttcg tgattccACGacctggggctgggtcccc (SEQ DP NO:586) INSAC2-C5IT25S εtcεaaεacεεεgcacccAGCεaεcgεttcεcccccεac (SEO ID NO:587) JNSAC2-C5IT25G εtcεaaεacgεεgcacccGGCεaεcgεttcgcccccgac (SEO ED NO:588) INSAC2-C3 1T2SP gtcgaagacggggcacccCCGεagcggttcgcccccεac (SEO ID NO:589) INSAC2-C7IL53H gagεtgatcgaεgaεggaCACagcgcgcεcaccaccaac (SEO TD NQ-.590) NSAC2-C3 L53Q εaεεtgatcgaggagggaCAGagcεcgcεcaccaccaac (SEO ED NO: 591.) NSAC2-C3 1L53G gaggtgatcgaggagggaGGCaεcεcεcgcaccaccaac (SEO YD NQ-.592) INSAC2-C3 1L53S gaεεtεatcεaεgaεggaAGCagcεcεcεcaccaccaac (SEO ID NO:593) NSAC2-C71 1L53HS54V gaεgtgatcgaggagggaCACGTGgcgcgcaccaccaac (SEO ID NO:594) INSAC2-C3 L53QS54V gaεεtgatcgaεεaεggaCAGGTGεcεcεcaccaccaac (SEO ID NO:595) NSAC2-C3 JL53GS54V gaggtgatcgaggagggaGGCGTGεcgcgcaccaccaac (SEO ED NO:596) NSAC2-C3 IL53SS54V gagεtgatcgaggaεεεaAGCGTGεcgcgcaccaccaac (SEO ID NO:597) ^SAC2-C7 S54V εtεatcgaggaεgεactgGTGεcgcgcaccaccaacatc (SEO YD NO:598) INSAC2-C5 S54L εtgatcgaggagggactgCTGεcgcgcaccaccaacatc (SEO ID NO:599) INSAC2-C5 A55G atcgaεεagggactgagcGGCcgcaccaccaacatcgac (SEO ID NO:600) GC821-2 >

INSAC2-C5 A55T atcgaggagggactgagcACGcgcaccaccaacatcεac (SEO ID NO:601) 1NSAC2-C5] 1A55GS54V atcεagεaεεεactεGTGGGCcεcaccaccaacatcεac (SEO ID NO:602) lNSAC2-C5i A55TS54V atcgaggaggεactεGTGACGcgcaccaccaacatcεac (SEO ED NO:603) INSAC2-C5IR67T εacεaccccaccgatccgACGctcaacεεcgcgagctac (SEO ED NO:604) INSAC2-C5 R67Q gacgaccccaccgatccgCAGctcaacεεcgcεaεctac (SEO ID NO:605) INSAC2-C7IR67N εacgaccccaccgatccgAACctcaacεεcgcεagctac (SEO ID NQ-.606) INSAC2-C5! IK97R ctgggcaccaacgacaccCGCεcctacttccggcgcacc (SEO ID NO:607) ttiSAC2-C5j 1V125S ctcaccaεcgcgggcεgcAGCεεcaccacgtacccεgca (SEO ID NO:608) INSAC2-C7IV125G ctcaccaεcεcεεεcεεcGGCg caccacgtacccεgca (SEO ED NO:609) INSAC2-C5 V125R ctcaccaεcεcεggcεεcCGCggcaecacgtacccggca (SEO ED NO:610) INSAC2-C5 1V125A ctcaccagcgcgggcεgcGCGεεcaccacgtacccεgca (SEO ID NO:611) NSAC2-C5IV125P cteaccaεcεcεggcεgcCCGggcaccacεtacccggca (SEO ED NO:612) D SAC2-C3IF154Y ccctggttccagttgatcTACgagggcggcgagcagaag (SEO HP NO:613) INSAC2-C3IF196G gεcgtcεacεgaatccacGGCaccgaggccaacaatcgc (SEO E) NO:614) lNSAC2-C7IR67G-re εacεaccccaccgatccgGGCctcaacεgcεcgagctac (SEO ID NQ:615) INSAC2-C5lR67E-re εacεaccccaccgatccgGAGctcaacggcgcgagctac (SEO ID NO:616) NSAC2-C5 R67F-re εacεaccccaccgatccgTTCctcaacggcgcgagctac (SEQ ED NO:617) NSAC2-C51R67L-re εacgaccccaccgatccgCTGctcaacggcgcεagctac (SEO ID NO:618) INSAC2-C5 IS54P gtgatcgaggagggactgCCGgcgcgcaccaccaacatc (SEO ID NO:619) WSAC2-C5 S54R εtgatcgaggaεggactgCGCgcgcgcaccaccaacatc (SEO YD NO:620) NSAC2-C5 S54G εtεatcεaεgaεggactgGGCgcgcgcaccaccaacatc (SEO ID NO:621) INSAC2-C5 S54T εtεatcεaεεaεεεactεACGgcgcgcaccaccaacatc (SEO ID NO:622) NSAC2-C7I S54I εtεatcgaggagggactgATCgcgcgcaccaccaacatc (SEO ID NO:623) WSAC2-C5 'S54K ^gtεatcgaggagggactgAAGgcgcgcaccaccaacatc (SEO ID NO:624)

Screening of Combinatorial Libraries and Mutants For each of the NSAB1-B6 libraries, a 96-well plate full of clones was first sequenced. Once the sequencing results were analyzed, the mutants obtained for each library were inoculated in quadruplicate, similar to the site-saturation libraries described above. For the NSACl -C7 libraries, 96 colonies per/plate/library were initially inoculated, and each plate was screened without sequencing. Upon screening, some libraries looked better than others. Several plates for each of the NSACl, C2, C4, C6 libraries were screened. The "winners" from these single isolate screening plates were GC821-2 r

then streaked out for singles or directly screened in quadruplicate just like the site- saturation libraries (i.e., as described above). Only the "winners" identified were sequenced. EXAMPLE 9 5 Improved Properties of Multiply Mutated Perhydrolase Variants In this Example, experiments conducted to assess the properties of multiply- mutated perhydrolase variants are described. In these experiments, combinatorial mutants obtained from combinatorial libraries were tested in their performance in perhydrolysis, peracid hydrolysis and perhydrolysis to hydrolysis ratio. These parameters were 10 measured in the HPLC or ABTS assays described in Example 2, above. Combinatorial variants tested were:

L12I S54V, L12M S54T, 15 L12T S54V, L12Q T25S S54V, L53H S54V, S54P V125R, S54VV125G, 20 S54V F196G, S54V K97R V125G, and A55G R67T K97R V125G,

As is indicated in Table 9-1 below, all of these variants were better than wild type 25 enzyme in at least one of the properties of interest.

Table 9-1 Results for Multiple Variants Multiple Variant 1 Fold-Improvement in Property if, "f ^■ .•' ^»»ii • if ' „ GC821-2 ^ r^

EXAMPLE 10 PAF and PAD Assays of Perhydrolase Variants In this Example, assay results for PAF and PAD testing of perhydrolase variants are provided. The tests were conducted as described in Example 1, above. In addition, Tables are provided in which the protein expression of the variant was greater than wild- type under the same culture conditions (described herein). These results are indicated as the "protein performance index." Thus, a number greater than "1" in the protein performance index indicates that more protein was made for the particular variant than the wild-type. In the following Tables, "WT" indicates the wild-type amino acid residue; "Pos" indicates the position in the amino acid sequence; "Mut." and "Var" indicate the amino acid residue substituted at that particular position; "prot." indicates "protein; and "Perf. Ind" indicates the performance index.

-3 "' "- „

GC821-2

'^"ittⁱhlUⁱ4' 'NN:t S

GC821-2

-" ft ,. i .' .' ^f ."ϊft 'f •# " s ' ,.' it ,n ^•' " «

GC821-2

P ιj~I T, <U tx if— K^" ''^•«" ^•' . «* -^{l lC}-

GC821-2

llSGt4 4.G;iM ^>,

GC821-2

VU!51I v'^'4'04^'3S c^

GC821-2

^\

GC821-2

^lf - ^;" * ^t ""^,f" »^•' ^,t"^l" ^{( •}* '"»" • '^{;;v »} ^

GC821-2

p C T/ U S O ΗK ^' -

GC821-2

, < .„„ι , ,.' ^

GC821-2

The following Table provides variants with PAF results that were better than those observed for wild-type M. smegmatis perhydrolase. In this Table, the middle column indicates the amino acid residue in the wild-type perhydrolase (WT), followed by the position number and the variant amino acid in that position (Var).

Table 10-2. Variants with PAF Table 10-2. Variants with ϊ ^»AF Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/PosJ relative to WT PosJ relative to Pos Var — - WT — Pos Var WT - 2A002W 1.75 8F008G 1.09 2A002D 1.30 8F008H 1.02 2A002F 1.24 10D010L 3.97 2A002I 1.18 10D010W 3.18 2A002G 1.15 10D010K 2.13 2A002S 1.01 10D010Y 1.51 3K003Y 1.06 10D010T 1.47 3 K003I 1.05 10D010I 1.28 3 K003L 1.04 12L012Q 2.65 3 K003T 1.01 12L012C 2.29 3 K003H 1.01 12L012A 1.10 4R004Q 1.03 15G015A 1.54 5I005T 1.12 15G015S 1.05 5I005S 1.02 17V017G 1.17 6L006V 1.07 17V017R 1.10 6L006I 1.07 17V017A 1.01 6L006T 1.06 18P018Y 1.33 7C007K 2.69 18P018N 1.33 7C007Y 2.09 18P018C 1.26 7C007I 1.76 18P018E 1.22 7C007H 1.73 18P018V 1.19 7C007A 1.42 18P018R 1.16 7C007G 1.39 18P018Q 1.12 7C007M 1.13 18P018H 1.12 8F008R 1.43 18P018G 1.07 8F008V 1.18 19V019G 1.32

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF

Values Better Than WUd-Type Values Better Than Wild-Type Peracid Peracid - formation formation WT/PosJ relative to WT/PosJ relative to

Pos Var WT Pos Var WT 19V019S 1.24 26E026K 1.46 19V019R 1.03 26E026T 1.44 19V019L 1.00 26E026C 1.40 20E020W 2.94 26E026V 1.39 20E020G 2.36 26E026N 1.37 20E020T 2.22 26E026H 1.33 20E020L 2.20 26E026L 1.30 20E020H 2.17 26E026G 1.28 20E020V 2.11 26E026S 1.27 20E020S 2.01 26E026W 1.25 20E020C 1.57 27R027K 1.22 20E020N 1.40 28F028M 1.33 20E020A . 1.29 28F028A 1.27 20E020Q 1.27 28F028W 1.16 21 D021K 1.58 28F028L 1.09 21 D021W 1.55 28F028S 1.05 21 D021L 1.46 29A029W 1.91 21 D021A 1.46 29A029V 1.80 21 D021G 1.37 29A029R 1.76 21 D021Y 1.30 29A029Y 1.70 21 D021F 1.30 29A029G 1.60 21 D021S 1.24 29A029S 1.49 22G022A 1.55 29A029T 1.42 22G022T 1.03 29A029E 1.12 22G022S 1.02 29A029C 1.08 25T025G 1.86 30P030K 1.21 25T025S 1.60 30P030R 1.16 25T025A 1.33 30P030V 1.06 25T025I 1.02 30P030T 1.05 26E026M 2.00 30P030A 1.05 26E026A 1.93 30P030S 1.03 26E026R 1.48 30P030Q 1.01 U-/

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF

Values Better Than Wild-Type Values Better Than Wild-Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to

Pos Var WT Pos Var WT 30P030H 1.01 39A039W 1.23 30P030E 1.01 39A039V 1.21 31 D031W 1.83 39A039G 1.17 31 D031L 1.81 39A039R 1.17 31 D031T 1.45 39A039E 1.09 31 D031G 1.44 40Q040K 2.61 31 D031F 1.44 ^{■ ■} 40Q040I 2.58 31 D031N 1.34 40Q040W 2.39 31 D031V 1.28 . 40Q040L 2.14 31 D031A 1.24 40Q040T 2.01 31 D031R 1.22 40Q040R 1.89 31 D031S 1.15 40Q040Y 1.83 31 D031E 1.13 40Q040G 1.79 31 D031Q 1.07 40Q040S 1.57 32V032K 1.09 40Q040N 1.53 32V032R 1.05 40Q040D 1.16 33R033S 1.00 40Q040E 1.08 36G036I 1.32 41 Q041K 1.38 36G036K 1.27 41 Q041R 1.19 36G036L 1.24 41 Q041W 1.14 37V037S 1.40 41 Q041H 1.12 37V037I 1.26 41 Q041S 1.11 37V037A 1.25 41 Q041Y 1.09 37V037H 1.21 41 Q041V 1.07 37V037L 1.16 41 Q041A 1.03 37V037C 1.09 41 Q041L 1.00 37V037T 1.05 42L042K 2.46 39A039L 1.43 42L042W 2.06 39A039K 1.36 42L042H 1.92 39A039Y 1.36 42L042R 1.38 39A039I 1.26 42L042G 1.17 39A039T 1.26 42L042T 1.08 GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF

Values Better Than WUd-Type Values Better Than WUd-Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to

Pos Var WT Pos Var WT 42L042F 1.07 46F046G 1.02 43 G043A 1.49 46F046K 1.00 43 G043C 1.48 47E047R 2.45 43 G043K 1.42 47E047T 1.96 43 G043M 1.37 47E047P 1.36 43 G043Y 1.26 47E047S 1.28 43 G043E 1.25 47E047H 1.27 43 G043L 1.22 47E047G 1.20 43 G043R 1.22 47E047K 1.19 43G043S 1.18 47E047F 1.09 43 G043H 1.17 47E047I 1.03 43G043P 1.08 49I049G 1.34 44A044F 2.84 49I049H 1.27 44A044V 2.13 49I049S 1.24 44A044C 1.80 49I049K 1.23 44A044L 1.61 491049 V 1.20 44A044W 1.40 49I049L 1.14 44A044M 1.20 49I049Y 1.07 45D045K 1.34 49I049R 1.05 45D045T 1.27 49I049E 1.02 45D045R 1.16 49I049M 1.01 45D045W 1.15 50E050L 1.19 45D045S 1.13 50E050M 1.18 45D045G 1.13 50E050A 1.12 45D045H 1.1.3 51 E051V 1.47 45D045F 1.11 51 E051A 1.28 45D045L 1.05 51 E051G 1.22 45D045V 1.05 51 E051T 1.18 45D045Q 1.04 51 E051L 1.11 45D045A 1.04 51 E051I 1.07 46F046E 1.25 53 L053H 5.05 46F046D 1.17 53 L053Q 1.48 it-*¹- it. _H.-' ^■• '

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than WUd-Type Values Better Than WUd-Type Peracid Peracid formation formation WT/PosJ relative to WT PosJ relative to Pos Var WT Pos Var WT 53 L053G 1.32 62D062E 1.02 53 L053S 1.16 63P063G 1.71 53 L053T 1.02 63P063T 1.50 54S054P 5.20 63P063M 1.46 54S054I 4.78 63P063S 1.42 54S054V 4.72 63P063K 1.40 54S054A 3.46 63 P063A 1.35 54S054R 3.38 63 P063Y 1.35 54S054L 2.02 63P063W 1.35 54S054T 1.46 63P063V 1.31 54S054K 1.44 63P063R 1.31 54S054G 1.43 63P063F 1.25 54S054C 1.26 63 P063L 1.15 54S054Q 1.03 63P063Q 1.09 55A055G 1.69 64T064G 1.23 55A055T 1.69 64T064S 1.11 57T057S 1.63 65D065A 1.31 57T057R 1.61 65D065S 1.17 57T057V 1.28 65D065H 1.10 57T057I 1.19 66P066R 1.85 59N059W 1.13 66P066V 1.83 59N059R 1.09 66P066H 1.59 59N059T 1.07 66 P066I 1.59 59N059S 1.06 66 P066G 1.50 59N059Q 1.02 66 P066Q 1.46 60I060H 1.02 66P066T 1.41 60I060R 1.00 66P066S 1.39 61 D061H 1.44 66 P066Y 1.33 61 D061S 1.26 66P066L 1.14 61 D061R 1.11 66 P066N 1.12 61 D061I 1.08 67R067N 1.58 61 D061F 1.01 67R067G 1.39 &'•" if "+ "^•' " " »

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF

Pos Var WT Pos Var WT 67R067T 1.28 71 A071K 1.44 67R067F 1.26 71 A071R 1.40 67R067L 1.20 71 A071N 1.23 67R067Q 1.16 71 A071L 1.23 67R067W 1.07 71 A071F 1.13 67R067E 1.04 71 A071C 1.01 67R067P 1.01 72S072L 1.26 68L068E 1.44 72S072H 1.21 68L068W 1.21 72S072G 1.20 68L068I 1.13 72S072T 1.10 68L068G 1.09 72S072V 1.08 68L068V 1.09 72S072Y 1.07 68L068H 1.05 73Y073R 1.26 68L068T 1.03 73Y073Q 1.23 69N069V 1.99 73Y073S 1.17 69N069K 1.72 73Y073K 1.07 69N069R 1.49 74L074S 2.72 69N069I 1.47 74L074G 1.95 69N069H 1.36 74L074W 1.38 69N069T 1.35 75P075R 1.60 69N069L 1.30 75P075S 1.39 69N069S 1.21 ^" 75P075T 1.28 69N069G 1.20 75P075Q 1.21 69N069Q 1.07 75P075G 1.16 69N069W 1.05 75P075H 1.05 69N069C 1.05 75P075W 1.04 71 A071S 1.75 76S076P 1.23 71 A071T 1.70 77C077T 1.12 71 A071H 1.70 77C077V 1.05 71 A071G 1.59 77C077G 1.01 71 A071I 1.51 78L078G 4.98 71 A071E 1.45 78L078H 4.82 U i i LB 'Ml-,/ ""iHl f ^!'" " ~

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF

Values Better Than WUd-Type Values Better Than WUd-Type Peracid Peracid formation formation WT/Pos. relative to WT/PosJ relative to

Pos Var WT Pos Var WT 78L078E 3.01 82L082G 1.38 78L078N 2.68 82L082R 1.34 78L078T 1.87 82L082H 1.33 78L078Q 1.73 82L082K 1.19 78L078V 1.53 82L082T 1.18 78L078I 1.43 82L082I 1.17 78L078Y 1.39 82L082S 1.15 79A079H 1.93 82L082V 1.02 79A079L 1.80 83P083K 1.37 79A079I 1.59 83P083G 1.31 79A079M 1.50 83P083H 1.27 79A079N 1.48 83P083R 1.19 79A079Q 1.47 83P083S 1.17 79A079R 1.47 84L084K 1.10 79A079W 1.27 84L084H 1.01 79A079T 1.17 85D085Q 3.09 79A079E 1.12 85D085R 2.38 80T080C 1.31 85D085S 2.28 80T080V 1.23 85D085H 1.55 80T080G 1.16 85D085N _. 1.54 80T080A 1.00 85D085G 1.41 81 H081K 1.52 85D085T 1.33 81 H081L 1.23 85D085E 1.12 81 H081N 1.17 85D085F 1.01 81 H081G 1.17 86L086A 1.38 81 H081A 1.15 86L086C 1.16 81 H081C 1.13 86L086G 1.15 81 H081W 1.13 88I088H 1.20 81 H081V 1.10 88I088T 1.03 81 H081F 1.10 88I088G 1.01 81 H081S 1.04 90M090T 1.27 82L082P 1.46 90M090I 1.13 ^' 8 * j it ", >

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than WUd-Type Values Better Than WUd-Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to Pos Var WT Pos Var WT 90M090V 1.08 103T103K 1.09 90M090S 1.06 103T103I 1.08 90M090L 1.02 103T103L 1.05 91 L091G 1.21 104P104H 2.84 91 L091T 1.06 104P104T 2.70 92G092V 1.49 104P104G 2.67 92G092S 1.26 104P104V 2.59 93T093Y 5.26 104P104S 2.48 93T093F 3.52 104P104I 2.43 93T093A 1.38 104P104W 2.05 93T093C 1.08 104P104C 1.95 95D095E 2.04 104P104E 1.84 96T096S 1.04 104P104F 1.79 97K097R 2.80 104P104N 1.62 97K097Q 1.14 104P104R 1.62 98A098L 2.22 104P104Q 1.34 98A098H 2.09 104P104M 1.09 98A098I 2.05 105L105P 1.71 98A098Y 2.02 105L105C 1.56 98A098S 1.73 105L105F 1.30 98A098T 1.72 105L105W 1.28 98A098G 1.57 105L105G 1.08 98A098C 1.30 106D106K 1.28 98A098N 1.24 106D106L 1.20 98A098D 1.11 106D106G 1.18 98A098P 1.10 106D106H 1.09 100F100W 1.08 106D106E 1.08 100F100E 1.01 106D106T 1.06 101 R101K 1.24 106D106I 1.04 103T103W 1.26 106D106F 1.02 103T103Y 1.19 106D106C 1.01 103T103G 1.11 107I107E 2.55 l!. ' ',>•^•- '„ "„ h^ ._#

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF

Values Better ThanWUd- ^■Type Values Better Than WUd-Type Peracid Peracid formation formation WT/PosJ relative to WT/Pos./ relative to

Pos Var WT Pos Var WT 107I107S 2.04 115V115G 1.09 107I107N 1.81 115V115I 1.05 107I107G 1.76 115V115Y 1.03 107I107V 1.00 116T116G 1.10 108A108L 1.41 116T116A 1.01 108A108T 1.05 117Q117H 2.33 10 L109N 1.52 117Q117T 2.23 109L109W 1.30 117Q117Y 2.23 109L109Q 1.18 117Q117W 2.16 109L109Y 1.16 117Q117V 2.15 109L109I 1.05 117Q117G 2.08 109L109D 1.00 117Q117A 2.05 111M111K 1.98 117Q117S 1.95 111M111I 1.95 117Q117F 1.57 111M111L 1.55 117Q117R 1.56 111M111T 1.49 117Q117M 1.54 111M111F 1.47 117Q117E 1.15 111M111V 1.47 118V118Y 1.25 111M111Y 1.43 118V118K 1.13 111M111S 1.03 118V118G 1.08 112S112L 1.03 120T120S 1.09 112S112H 1.00 121S121L 1.35 113V113L 1.50 121S121W 1.33 113V113H 1.34 121S121R 1.26 113V113K 1.19 121S121K 1.24 113V113R 1.13 121S121G 1.20 113V113Y 1.11 121S121C 1.18 113V113F 1.05 121S121N 1.14 113V113Q 1.03 121S121T 1.13 115V115W 1.23 121S121A 1.12 115V115T 1.15 121S121V 1.12 115V115L 1.12 122A122H 1.14 Saλ i IfL- »

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than WUd-Type Values Better Than WUd-Type Peracid Peracid formation formation WT PosJ relative to WT/PosJ relative to Pos Var WT Pos Var WT 122A122I 1.13 127T127H 1.57 122A122T 1.08 127T127V 1.07 122A122K 1.08 127T127I 1.06 122A122V 1.04 127T127S 1.05 122A122S 1.03 128T128L 1.06 123 G123D 1.73 128T128K 1.06 123 G123V 1.40 130P130T 1.19 123 G123P 1.32 130P130H 1.17 123 G123E 1.13 130P130K 1.16 123 G123T 1.06 130P130G 1.16 123G123H 1.00 130P130S 1.16 124G124L 1.92 130P130V 1.15 124G124I 1.85 130P130W 1.15 124G124T 1.64 130P130I 1.12 124G124H 1.59 130P130L 1.12 124G124V 1.44 130P130R 1.11 124G124F 1.32 130P130F 1.08 124G124S 1.27 130P130E 1.00 124G124Y 1.23 131 A131L 1.83 124G124R 1.14 131 A131R 1.76 124G124Q 1.12 131 A131H 1.72 125V125G 2.95 131 A131G 1.66 125V125S 1.94 131 A131W 1.61 125V125A 1.69 131 A131V 1.59 125V125P 1.50 131 A131P 1.52 125V125R 1.30 131 A131Y 1.50 125V125D 1.24 131 A131S 1.48 125V125Y 1.08 131 A131E 1.36 125V125I 1.01 131 A131D 1.31 126G126T . 1.58 131 A131Q 1.29 126G126P 1.17 132P132Y 1.57 126G126L 1.17 132P132S 1.13 i if

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than WUd-Type Values Better Than WUd-Type Peracid Peracid formation formation WT PosJ relative to WT/PosJ relative to Pos Var WT Pos Var WT 133 K133Y 1.12 142L142K 1.60 133K133L 1.05 142L142F 1.05 133K133H 1.02 143A143K 3.16 134V134G 1.71 143A143H 2.90 134V134T 1.25 143A143L 2.51 134V134N 1.18 143A143V 2.45 134V134S 1.16 143A143W 2.27 134V134L 1.13 143A143T 2.18 134V134I 1.12 143A143R 2.15 136V136T 1.13 143A143S 1.77 137V137M 1.22 143A143Q 1.74 137V137L 1.09 143A143F 1.56 137V137T 1.08 143A143P 1.53 137V137A 1.07 143A143G 1.48 137V137G 1.02 143A143D 1.45 138S138I 1.15 143A143E 1.43 138S138G 1.05 143A143C 1.39 140P140A 1.90 143A143N 1.30 140P140T 1.74 144P144Y 2.34 140P140S 1.31 144P144K 2.09 141 P141L 2.32 144P144H 1.94 141 P141I 2.29 144P144F 1.82 141 P141H 2.07 144P144R 1.76 141 P141V 1.96 144P144S 1.69 141 P141T 1.84 144P144T 1.46 141 P141S 1.70 144P144G 1.45 141 P141R 1.65 144P144D 1.45 141 P141G 1.64 144P144N 1.44 141 P141Q 1.39 144P144L 1.43 141 P141N 1.32 144P144Q 1.37 141 P141A 1.10 144P144M 1.24 142L142W 2.41 144P144A 1.09

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than WUd-Type Values Better Than WUd-Type Peracid Peracid formation formation WT PosJ relative to WT PosJ relative to Pos Var WT Pos Var WT 145M145L 1.72 151 Q151K 1.07 145M145F 1.49 151 Q151H 1.06 145M145R 1.15 151 Q151S 1.05 145M145W 1.15 151 Q151C 1.05 145M145C 1.02 151 Q151Y 1.01 145M145T 1.01 152L152V 1.22 147H147A 1.28 152L152K 1.21 147H147S 1.26 152L152R 1.20 147H147T 1.20 152L152W 1.18 147H147P 1.12 152L152T 1.12 147H147E 1.11 152L152S 1.12 148P148V 2.43 152L152Y 1.09 148P148K 1.79 152L152H 1.09 148P148L 1.64 152L152G 1.08 148P148A 1.64 152L152E 1.08 148P148R 1.51 152L152Q 1.07 148P148T 1.50 152L152D 1.07 148P148Y 1.46 152L152I 1.04 148P148S 1.46 152L152C 1.00 148P148E 1.42 153 I153K 1.62 148P148F 1.37 153 I153H 1.46 148P148Q 1.33 153I153T 1.27 148P148D 1.03 153 I153L 1.27 150F150L 1.29 153 I153F 1.23 150F150E 1.23 153I153A 1.19 151 Q151D 1.47 154F154Y 1.32 151 Q151R 1.36 155E155T 1.49 151 Q151P 1.35 155E155R 1.47 151 Q151A 1.29 155E155L 1.31 151 Q151T 1.24 155E155Y 1.27 151 Q151M 1.24 155E155K 1.23 151 Q151E 1.14 155E155G 1.17 USQty> M3N # r^

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than WUd-Type Values Better Than WUd-Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to Pos Var WT Pos Var WT 155E155S 1.08 158E158T 1.45 155E155D 1.08 158E158P 1.41 155E155F 1.07 158E158N 1.41 156G156P 1.44 158E158M 1.39 156G156T 1.15 158E158I 1.38 156G156K 1.10 158E158D 1.35 156G156M 1.09 159Q159R 1.15 156G156C 1.07 159Q159C 1.13 156G156N 1.07 159Q159S 1.10 156G156R 1.05 159Q159D 1.09 156G156H 1.04 159Q159A 1.08 156G156S 1.02 159Q159M 1.07 157G157T 1.74 159Q159P 1.06 157G157R 1.51 159Q159L 1.02 157G157S 1.30 161 T161R 3.61 157G157K 1.28 161T161Y 2.40 157G157F 1.27 161 T161H 1.82 157G157V 1.23 161 T161W 1.41 157G157H 1.14 161T161I 1.40 157G157I 1.11 161 T161V 1.27 158E158H 2.40 161 T161L 1.25 158E158K 2.08 161 T161Q 1.04 158E158F 2.06 162T162K 1.22 158E158R 1.99 162T162R 1.17 158E158Y 1.77 162T162W 1.15 158E158W 1.77 162T162Y 1.03 158E158L 1.59 162T162H 1.02 158E158S 1.57 163 E163L 1.50 158E158V 1.52 163E163Y 1.41 158E158Q 1.49 163 E163H 1.32 158E158C 1.46 163 E163G 1.25 158E158A 1.45 163 E163W 1.21 . I »^»|{" ' ,

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF

Values Better Than WUd- ^■Type Values Better Than WUd-Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to

Pos Var WT Pos Var WT 163 El 63 V 1.13 167V167H 1.03 163 E163R 1.12 168Y168G 1.89 163 E163S 1.12 168Y168T 1.51 163E163A 1.11 168Y168V 1.19 163 E163C 1.11 169S169Y 1.26 163E163F 1.07 169S169R 1.24 165A165R 1.70 169S169K 1.21 165A165K 1.35 169S169I 1.16 165A165F 1.23 169S169T 1.15 165A165Q 1.21 169S169L 1.08 165A165V 1.21 169S169C 1.03 165A165Y 1.20 169S169Q 1.02 165A165T 1.18 170A170K 1.71 165A165I 1.17 170A170G 1.59 165A165P 1.14 170A170I 1.59 165A165L 1.08 170A170S 1.47 165A165G 1.05 170A170F 1.44 165A165N 1.01 170A170T 1.40 165A165S 1.00 170A170E 1.28 166R166Y 1.29 170A170D 1.27 166R166L 1.27 70A170N 1.21 166R166I 1.26 170A170V 1.20 166R166W 1.25 170A170C 1.15 166R166H 1.20 170A170Q 1.15 166R166T 1.19 170A170L 1.05 166R166V 1.17 170A170W 1.04 166R166K 1.17 170A170M 1.03 166 R166S 1.16 171 L171K 2.05 166 R166G 1.15 171 L171H 1.67 167V167T 1.13 171 L171T 1.54 167 VI 671 1.08 171 L171I 1.53 167V167Y 1.07 171 L171S 1.43 f-T OS€i'lv i,f ' &t

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better ThanWUd- ■Type Values Better Than WUd- ■Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to Pos Var WT Pos Var WT 171L171F 1.30 175M175W 1.25 171L171G 1.26 176K176W 1.19 171L171Y 1.20 176K176T 1.04 171L171V 1.02 176K176Y 1.04 172A172I 1.70 176K176V 1.04 172A172S 1.59 176K176G 1.01 172A172W 1.43 178P178L 1.82 172A172G 1.41 178P178Y 1.38 172A172V 1.40 178P178K 1.34 172A172T 1.25 178P178W 1.14 172A172L 1.20 178P178G 1.09 172A172C 1.20 179F179L 1.15 173S173Y 1.19 179F179Y 1.05 173S173K 1.17 180F180L 1.30 173S173W 1.16 180F180I 1.20 173S173L 1.15 180F180V 1.14 173S173R 1.09 180F180Y 1.12 173S173H 1.07 180F180W 1.11 173S173T 1.06 180F180K 1.08 174F174G 1.60 180F180T 1.01 174F174P 1.54 181D181A 1.35 174F174Q 1.42 181D181K 1.33 174F174C 1.32 181D181Y 1.29 174F174S 1.16 181D181W 1.26 174F174L 1.05 181D181L 1.25 175M175T 2.21 181D181R 1.23 175M175G 2.04 181D181S 1.21 175M175V 1.93 181D181Q 1.14 175M175L 1.61 181D181E 1.10 175M175Q 1.56 181D181G 1.09 175M175R 1.55 181D181C 1.09 175M175N 1.39 181D181P 1.03 -

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF

Values Better Than WUd ^■Type Values Better Than WUd-Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to

Pos Var WT Pos Var WT 181 D181T 1.02 187S187R 1.04 182A182T 1.14 187S187G 1.03 184S184Y 1.06 187S187F 1.02 184S184F 1.05 188T188Y 1.48 184S184T 1.04 188T188V 1.22 184S184H 1.02 188T188S 1.16 185V185K 1.37 188T188I 1.13 185V185Y 1.37 188T188H 1.11 185V185W 1.36 188T188R 1.01 185V185H 1.30 189D189L 1.30 185V185L 1.23 189D189H 1.25 185V185R 1.15 189D189W 1.09 185V185G 1.12 190G190W 1.88 185V185T 1.11 190G190K 1.01 185V185S 1.09 191 V191Y 1.32 185V185I 1.07 191 V191H 1.30 185V185F 1.02 191 V191W 1.20 186I186G 1.86 191 V191S 1.20 186I186T 1.51 191 V191K 1.17 186I186A 1.46 191 V191I 1.14 186I186S 1.39 191 V191F 1.13 1861186V 1.28 191 V191R 1.05 186I186L 1.17 191 V191L 1.04 186I186F 1.01 196F196H 1.77 187S187K 1.45 196F196L 1.77 187 S187Y 1.43 196F196C 1.74 187S187I 1.38 196F196M 1.65 187S187L 1.37 196F196G 1.59 187 S187W 1.30 196F196S 1.58 187S187H 1.29 196F196Y 1.41 187 S187V 1.23 196F196V 1.40 187S187T 1.12 196F196I 1.32 ■ C: ,I* ' - -H. ^• ,'₃

GC821-2

Table 10-2. Variants with PAF Table 10-2. Variants with PAF Values Better Than WUd- ■Type Values Better Than WUd-Type Peracid Peracid formation formation WT/PosJ relative to WT/PosJ relative to Pos Var WT Pos Var WT 196F196W 1.01 201N201G 1.08 197T197L 1.21 202R202W 1.97 198E198R 1.82 202R202F 1.89 198E198I 1.80 202R202E 1.69 198E198V 1.60 202R202H 1.64 198E198W 1.59 202R202T 1.55 198E198L 1.57 202R202S 1.49 198E198P 1.52 202R202A 1.48 198E198Y 1.48 202R202C 1.44 198E198C 1.38 202R202M 1.43 198E198F 1.37 202R202L 1.43 198E198Q 1.28 202R202G 1.39 198E198T 1.25 202R202I 1.33 198E198N 1.24 203D203L 2.42 198E198M 1.18 203 D203R 2.23 198E198S 1.06 203 D203I 1.99 199A199C 1.77 203 D203W 1.99 199A199K 1.72 203D203F 1.92 199A199E 1.56 203D203H 1.84 199A199L 1.38 203D203C 1.78 199A199T 1.33 203D203S 1.66 199A199R 1.33 203 D203V 1.66 199A199V 1.32 203D203G 1.63 199A199D 1.31 203D203Q 1.60 199A199H 1.27 203D203A 1.53 199A199Y 1.24 203D203E 1.34 199A199F 1.23 203 D203N 1.05 199A199S 1.20 199A199G 1.14 199A199M 1.07 201 N201Y 1.29 201 N201F 1.16 _*- ••' _{'" ""} ■_"'• .

GC821-2

The following Table, provides variants with a PAF PI greater than 1.

CT/mSO - t (7%

GC821-2

Table 10-4 provides variants with PAF PI values greater than 2.0. _,

IK^' it. U .■' ii ,!' "ii if., if "' II" •• » " _'»■ ' '»

GC821-2

^, '

GC821-2

The following Table provides PAD assay results for various variants.

.„ -"ii" .-^• ii" τ .. >

GC821-2

-^"L , if,, i' . ii ^«.- ii ,.^• it - ' ll i

GC821-2

μ t ,. r^

GC821-2

GC821-2

Table 10-5. PAD Assay Results

PAD Perf. Position WT/Pos/ Variant iMutation Ind.

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

The following Table provides variants that are better than wild-type at degrading peracids (i.e., the performance index for the variant is better than the wild-type).

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/PosJVar.PAD PI Pos. WT/PosJVar.PAD PI 1 M001I 1.19 5I005M 1.09 1M001L 2.11 5I005E 1.59 2A002D 1.05 5I005L 1.63 2A002R 1.17 51005 A 1.88 2A002W 1.17 5I005C 2.47 2A002P 1.17 5I005D 3.11 2A002Q 1.29 6L006C 1.22 2A002E 1.38 6L006M 1.44 3K003T 1.03 6L006A 1.99 3K003S 1.17 7C007A 1.03 3K003Q 1.19 7C007H 1.37 3K003R 1.29 7C007I 1.48 3K003Y 1.39 7C007E 1.63 3K003M 1.44 7C007K 2.95 3K003P 1.45 8F008M 1.11 3K003C 1.52 8F008L 1.31 3K003L 1.84 8F008A 1.33 3K003H 1.89 8F008C 4.01 3K003A 2.14 10D010L 2.04 3K003I 2.44 13T013I 1.05 3K003E- 3.51 13T013E 1.09 3K003G 3.74 13T013L 1.47 4R004D 1.18 13T013M 1.47 4R004C 1.34 13T013C 1.55 4R004P 1.44 13T013A 1.88 4R004A 1.64 13T013N 2.61 GC821-2

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/PosJVar.PAD PI Pos. WT/PosJVar.PAD PI 13T013P 2.73 21 D021K 1.80 16W016K 1.03 21 D021Y 2.01 16W016I 1.06 22G022I 1.03 16W016Y 1.09 22G022T 1.16 16W016L 1.16 22G022E 1.19 17V017S 1.04 22G022L 1.35 18P018N 1.42 22G022P 1.36 18P018Q 3.26 22G022Q 1.44 18P018R 3.97 22G022A 1.66 18P018C 4.16 23A023H 1.04 18P018Y 4.17 23A023L 1.30 18P018V 4.85 24P024C 1.04 18P018E 4.87 24P024K 1.36 18P018G 4.96 24P024L 1.51 18P018H 6.05 26E026M 1.10 18P018L 7.40 26E026H 1.19 20E020D 1.14 26E026D 1.39 20E020S 1.18 26E026A 1.45 20E020H 1.20 26E026K 1.47 20E020T 1.25 26E026L 1.71 20E020V 1.27 27R027I 1.41 20E020A 1.28 27R027K 1.55 20E020W 1.30 27R027L 2.60 20E020N 1.34 27R027A 2.78 20E020P 1.43 28F028E 1.04 20E020Q 1.56 28F028W 1.17 20E020C 1.76 28F028C 1.21 21 D021S 1.11 28F028Y 1.36 21 D021E 1.39 28F028M 1.37 21 D021F 1.41 28F028A 1.48 21 D021W 1.44 28F028L 2.02 21 D021L 1.57 28F028D 2.07 21 D021A 1.75 29A029C 1.15 21 D021G 1.76 30P030H 1.08 GC821-2

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/PosJVar.PAD PI Pos. WT/PosJVar.PAD PI 30P030G 1.09 33R033N 1.30 30P030R 1.14 33R033A 1.32 30P030L 1.17 33R033C 1.73 30P030E 1.24 33R033G 2.63 30P030Y 1.31 33R033K 2.72 30P030I 1.38 33R033L 2.90 30P030K 1.39 34W034P 1.21 30P030S 1.49 34W034M 1.22 30P030T 1.64 34W034C 1.49 30P030V 1.74 34W034A 2.29 31 D031V 1.08 35T035M 2.72 31 D031T 1.11 35T035A 3.85 31 D031Q 1.13 35T035C 4.72 31 D031W 1.14 35T035I 5.38 31 D031G 1.16 35T035E 5.73 31 D031A 1.18 36G036C 1.06 31 D031S 1.23 36G036A 1.07 31 D031F 1.39 36G036H 1.10 31D031R 1.49 36G036K 1.71 31 D031N 1.55 36G036I 1.81 31 D031L 1.61 36G036L 2.49 32V032S 1.09 36G036D 2.50 32V032N 1.61 37V037I 1.04 32V032W 1.71 37V037L 1.16 32V032Q 1.74 37V037S 1.49 32V032G 2.65 37V037N 1.52 32V032M 3.41 37V037C 1.63 32V032I 3.51 37V037A 2.00 32V032A 3.64 37V037P 2.10 32V032E 3.92 38L038V 1.12 32V032D 4.19 39A039W 1.02 32V032L 4.72 39A039Y 1.13 32V032K 4.73 40Q040N 1.00 33R033S 1.01 40Q040I 1.10 GC821-2

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/Pos/Var.PAD PI Pos. WT/PosJVar.PAD PI 40Q040E 1.28 47E047K 1.06 40Q040R 1.48 47E047G 1.10 40Q040L 1.49 47E047I 1.15 40Q040D 1.59 48V048Q 1.39 40Q040S 1.65 48V048F 1.42 40Q040T 1.81 48V048A 1.63 40Q040Y 2.02 48V048M 1.79 40Q040G 2.17 48V048C 2.25 40Q040W 2.59 48V048L 2.29 40Q040K 3.64 48V048P 3.08 41 Q041G 1.09 491049 Y 1.02 41 Q041H 1.14 49I049M 1.02 41 Q041R 1.27 49I049L 1.03 41 Q041K 1.61 49I049G 1.12 41 Q041L 1.92 49I049K 1.26 41 Q041A 2.58 49I049A 1.87 42L042F 1.02 50E050P 1.02 42L042P 1.34 50E050M 1.04 42L042K 1.41 50E050G 1.11 42L042C 1.43 50E050D 1.22 43G043A 1.07 50E050A 1.23 43G043L 1.82 51 E051T 1.17 43 G043E 1.88 51 E051M 1.20 44A044C 1.92 51 E051D 1.28 45D045F 1.04 51 E051G 1.34 46F046C 1.16 51 E051K 2.00 46F046A 1.25 51 E051A 2.72 46F046E 1.31 52G052W 2.47 46F046D 1.39 53 L053H 1.70 46F046M 1.42 54S054N 1.29 46F046K 1.46 54S054P 1.30 46F046P 1.50 54S054A 1.41 46F046L 1.54 55A055N 1.05 47E047L 1.02 55A055K 1.08 GC821-2

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/Pos/Var.PAD PI Pos. WT/Pos/Var.PAD PI 55A055C 1.26 63P063Q 1.05 57T057S 1.01 63P063W 1.11 57T057G 1.05 63P063G 1.22 58T058L 1.12 63P063L 1.23 58T058H 1.49 63P063T 1.32 59N059Q 1.86 64T064G 1.08 59N059T 5,63 64T064M 1.09 59N059S 7.32 64T064A 1.20 59N059K 8.21 64T064L 1.22 59N059E 9.88 66P066S 1.02 59N059V 9.97 66P066T 1.10 59N059G 10.00 69N069D 1.11 59N059F 10.23 69N069A 1.13 59N059A 10.44 69N069Q 1.14 59N059Y 11.14 69N069C 1.20 59N059C 11.23 69N069L 1.20 59N059D 11.72 69N069S 1.42 59N059W 12.80 69N069T 1.43 59N059L 14.74 69N069H 1.52 60I060G 1.04 69N069K 1.59 601060 V 1.06 69N069V 1.73 60I060H 1.07 69N069I 1.75 60I060Y 1.19 70G070L 1.01 61 D061P 1.13 70G070A 1.41 61 D061Q 1.16 70G070H 1.90 61 D061L 1.20 71 A071K 1.01 61 D061G 1.25 71 A071M 1.11 61 D061S 1.35 72S072F 1.15 61 D061R 1.59 72S072G 1.76 61 D061I 1.66 72S072M 2.13 61 D061H 1.67 72S072C 2.18 61 D061K 1.72 72S072H 2.48 63P063K 1.02 72S072N 2.85 63P063V 1.04 72S072A 3.52 GC821-2

Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than WUd-Type Than WUd-Type Pos. WT/PosJVar.PAD PI Pos. WT/PosJVar.PAD PI 73Y073M 1.13 80T080C 1.15 73Y073C 1.20 80T080S 1.40 73Y073A 1.40 80T080G 1.50 74L074F 1.13 81 H081N 1.00 74L074M 1.21 81 H081L 1.03 74L074A 2.90 81 H081W 1.09 75P075E 1.19 81 H081C 1.09 75P075L 1.19 81 H081A 1.45 75P075W 1.31 81 H081M 1.54 75P075Y 1.32 82L082M 1.06 75P075V 1.39 83P083C 1.01 75P075C 1.42 83P083R 1.09 75P075D 2.09 83P083N 1.10 76S076C 1.06 83P083K 1.16 76S076T 1.11 83P083E 1.26 76S076A 1.11 83P083M 1.88 76S076H 1.11 83P083A 2.36 76S076P 1.20 84L084F 1.01 76S076V 1.35 84L084G 1.01 76S076K 1.53 85D085R 1.03 76S076M 1.61 85D085A 1.09 76S076D 1.94 85D085H 1.24 76S076E 2.09 85D085E 1.25 76S076G 2.15 85D085C 1.50 76S076L 4.70 85D085G 1.60 77C077T 1.03 85D085F 1.98 77C077D 1.05 86L086C 2.44 78L078T 1.10 86L086A 3.32 78L078I 1.11 87V087P 1.64 78L078G 1.38 87V087C 2.22 78L078H 1.57 87V087L 4.30 80T080V 1.01 88I088M 1.09 80T080Q 1.07 88I088P 3.51 80T080A 1.11 89I089L 1.22 GC821-2

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/PosJVar.PAD PI Pos. WT/PosJVar.PAD PI 89I089A 1.83 104P104V 1.02 89I089P 1.91 104P104H 1.03 90M090C 1.09 104P104N 1.44 90M090E 1.15 104P104C 1.83 90M090A 1.41 104P104E 1.97 90M090D 2.88 104P104I 2.05 91 L091I 1.05 104P104M 2.24 91 L091C 1.27 105L105Q 1.04 91 L091A 1.45 105L105H 1.23 91 L091D 1.47 105L105R 1.25 92G092C 2.05 105L105G 1.40 93T093A 1.05 105L105W 1.71 96T096F 1.24 105L105F 1.73 96T096G 1.28 105L105C 1.92 96T096L 1.93 106D106S 1.02 96T096M 2.53 106D106W 1.07 96T096C 3.76 106D106E 1.09 96T096A 4.20 106D106C 1.10 98A098Y 1.15 106D106A 1.13 98A098P 1.26 106D106H 1.18 98A098N 1.40 106D106K 1,24 98A098C 1.42 106D106T 1.38 98A098L 1.47 106D106F 1.45 98A098D 2.19 106D106G 1.45 100F100C 1.28 106D106V 1.68 100F100T 1.42 107I107L 1.04 100F100N 1.45 107I107S 1.33 100F100A 2.02 107I107C 1.41 100F100M 2.19 107I107T 1.53 101 R101L 1.12 108A108S 1.00 102R102Q 1.19 108A108G 1.13 102R102Y 1.29 108A108L 2.56 102R102L 1.64 108A108K 2.97 102R102A 1.79 110G110A 1.01 GC821-2

Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than WUd-Type Than WUd-Type Pos. WT/PosJVar.PAD PI Pos. WT/Pos/Var.PAD PI 110G110D 1.40 115V115Y 2.07 110G110C 1.43 115V115D 2.21 110G110E 1.76 115V115P 2.21 110G110F 2.29 115V115W 2.48 111 M111C 1.01 116T116N 1.05 111 M111A 1.02 116T116C 1.05 111 M111I 1.03 116T116H 1.08 111 M111Y 1.06 116T116M 1.39 111 M111W 1.23 117Q117F 1.02 111 M111N 1.31 117Q117R 1.05 112S112L 1.00 117Q117T 1.10 112S112E 1.16 117Q117H 1.12 113V113M 1.06 117Q117Y 1.13 113V113Q 1.11 117Q117P 1.13 113V113R 1.11 117Q117E 1.21 113V113P 1.14 117Q117A 1.73 113V113N 1.22 117Q117M 1.89 113V113A 1.31 118V118L 1.05 114L114T 1.05 118V118C 1.14 114L114A 1.07 118V118Y 1.34 114L114G 1.14 118V118Q 1.50 114L114C 1.14 119L119A 1.02 114L114I 1.17 120T120V 1.07 114L114M 1.28 120T120S 1.07 115V115C 1.08 120T120K 1.09 115V115S 1.14 120T120M 1.22 115V115Q 1.15 120T120L 1.26 115V115A 1.19 120T120N 1.42 115V115T 1.28 120T120E 1.53 115V115L 1.30 120T120I 1.56 115V115M 1.32 120T120Y 1.61 115V115R 1.63 121 S121E 1.04 115V115F 1.69 121 S121N 1.06 115V115G 1.76 121 S121Q 1.09 GC821-2

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/Pos/Var.PAD PI Pos. WT/Pos/Var.PAD PI 121 S121T 1.26 132P132Y 4.78 121 S121L 1.49 132P132G 4.98 121 S121A 1.55 132P132S 5.05 121 S121V 1.59 132P132C 5.68 121 S121C 1.64 132P132A 6.08 122A122L 1.02 132P132Q 6.15 123G123K 1.12 133K133Y 1.44 123G123A 1.19 133K133L 1.92 123G123Y 1.24 134V134C 1.37 123G123M 1.38 134V134G 1.42 123G123L 1.38 134V134S 1.44 123G123W 1.39 134V134L 1.45 125V125G 1.09 134V134A 1.64 126G126M 1.17 134V134P 1.71 126G126D 1.22 134V134M 1.89 127T127A 1.10 134V134N 2.80 128T128M 1.06 135L135D 2.90 128T128H 1.08 136V136T 1.13 128T128V 1.15 136V136L 1.13 128T128P 1.16 136V136C 1.23 128T128W 1.23 136V136A 1.60 128T128S 1.27 137V137M 1.13 128T128A 1.31 137V137L 1.27 128T128Q 1.34 137V137C 1.42 128T128N 1.36 137V137A 1.46 128T128K 1.57 138 S138G 1.11 128T128R 1.70 138S138C 1.18 128T128F 1.71 138S138A 1.28 128T128L 1.72 138S138N 1.31 128T128Y 1.81 138 S138P 1.39 131 A131R 1.04 140P140C 1.07 132P132N 1.05 140P140A 1.83 132P132L 2.24 140P140H 2.25 132P132E 3.02 140P140F 2.89 GC821-2

Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than WUd-Type Than WUd-Type Pos. WT/Pos/Var.PAD PI Pos. WT/Pos/Var.PAD PI 140P140G 3.11 147H147D 1.18 141P141A 1.08 147H147P 1.21 143A143C 1.07 147H147N 1.25 143A143E 1.13 147H147L 1.29 143A143D 1.22 147H147M 1.44 143A143L 1.28 148P148V 1.04 143A143H 1.36 148P148A 1.06 143A143K 1.37 148P148T 1.09 144P144M 1.01 148P148E 1.19 144P144F 1.08 148P148G 1.20 144P144Q 1.08 148P148S 1.21 144P144K 1.09 148P148R 1.25 144P144R 1.14 148P148K 1.30 144P144L 1.15 148P148D 1.34 144P144D 1.38 148P148Y 1.37 144P144N 1.49 148P148L 1.39 144P144H 1.60 148P148F 1.50 144P144Y 1.65 149W149H 1.01 146P146N 1.00 150F150Y 1.07 146P146G 1.04 150F150H 1.18 146P146R 1.06 150F150L 1.30 146P146M 1.23 151 Q151P 1.91 146P146A 1.36 151 Q151E 2.07 146P146Y 1.44 151 Q151K 2.19 146P146F 1.53 151 Q151H 2.19 146P146H 1.57 151 Q151S 2.25 146P146C 1.69 151 Q151R 2.32 146P146L 2.00 151 Q151T 2.37 147H147Q 1.03 151 Q151C 2.55 147H147W 1.05 151 Q151Y 2.75 147H147K 1.06 151 Q151D 2.81 147H147E 1.10 151 Q151A 2.93 147H147Y 1.12 151 Q151M 6.36 147H147C 1.17 152L152M 1.10 GC821-2

Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than WUd-Type Than WUd-Type Pos. WT/Pos/Var.PAD PI Pos. WT/Pos/Var.PAD PI 152L152C 1.14 156G156H 1.40 152L152E 1.23 156G156Y 1.40 152L152A 1.29 156G156T 1.53 152L152Y 1.37 156G156M 1.62 152L152W 1.55 156G156D 1.62 153I153V 1.15 157G157I 1.33 153I153A 1.49 157G157F 1.42 153I153L 1.50 157G157K 1.47 153I153T 1.62 157G157H 1.57 153I153S 1.66 158E158H 1.01 153I153F 1.75 158E158P 1.19 153I153P 1.87 158E158Q 1.24 153I153H 2.00 158E158S 1.27 153I153K 2.44 158 El 58 A 1.28 154F154Y 4.96 158E158R 1.29 155E155S 1.12 158E158W 1.31 155E155G 1.12 158E158C 1.37 155E155T 1.19 158E158N 1.58 155E155D 1.24 158E158M 1.73 155E155K 1.33 158E158F 1.77 155E155N 1.79 158E158K 1.88 155E155L 2.07 158E158L 1.96 155E155A 2.59 158E158Y 2.48 155E155P 2.60 159Q159H 1.48 155E155Y 2.65 160K160N 1.12 155E155M 2.91 160K160A 1.14 156G156S 1.04 160K160R 1.15 156G156K 1.11 160K160D 1.19 156G156E 1.14 160K160C 1.29 156G156R 1.21 160K160Q 1.41 156G156A 1.21 160K160M 1.47 156G156P 1.29 160K160P 1.66 156G156C 1.37 161 T161L 1.16 156G156N 1.38 161 T161V 1.24 GC821-2

Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than WUd-Type Than WUd-Type Pos. WT/Pos/Var.PAD PI Pos. WT/Pos/Var.PAD PI 161 T161Q 1.50 165A165R 1.29 161 T161M 1.72 165A165Q 1.32 161T161Y 2.62 165A165T 1.32 162T162R 1.23 165A165P 1.34 162T162G 1.82 165A165C 1.42 162T162S 2.01 165A165L 1.55 162T162W 2.04 165A165M 1.56 162T162I 2.21 165A165D 1.69 162T162Q 2.45 166R166W 1.08 162T162Y 2.89 166R166F 1.10 162T162K 3.13 166R166K 1.20 162T162F 3.23 166R166N 1.21 162T162M 3.49 166R166Y 1.22 162T162C 3.57 166R166M 1.29 162T162L 3.59 166R166I 1.39 162T162N 3.84 166 R166P 1.50 162T162H 3.91 166R166L 1.50 162T162P 4.37 166R166A 1.51 163 E163N 1.00 166R166D 1.55 163E163C 1.08 166R166H 1.56 163E163D 1.08 167V167I 1.00 163E163A 1.79 167V167S 1.86 163 E163Y 1.89 167V167H 2.11 163 E163L 1.94 167V167Y 2.15 164L164Q 1.01 167V167R 2.25 164L164V 1.02 167V167Q 2.41 164L164S 1.11 167V167T 2.47 164L164M 1.26 167V167L 2.56 164L164N 1.31 167V167G 2.83 164L164R 1.61 167V167M 3.84 164L164P 2.41 167V167A 4.99 165A165G 1.07 167V167C 5.37 165A165V 1.13 167V167D 5.54 165A165N 1.20 167V167P 6.08 GC821-2

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/Pos/Var.PAD PI Pos. WT/Pos/Var.PAD PI 168Y168F 5.17 172A172D 1.42 168Y168L 5.39 172A172Y 1.76 169S169Y 1.10 173 S173T 1.29 169S169A 1.13 173S173H 1.49 169S169R 1.19 173S173I 2.22 169S169K 1.27 173 S173F 2.30 169S169Q 1.37 173 S173R 2.47 169S169C 1.38 173 S173V 2.54 169S169M 1.40 173 S173E 2.65 169S169L 1.47 173 S173P 2.66 169S169I 1.53 173S173A 2.72 170A170C 1.06 173S173M 3.01 170A170E 1.17 173S173K 3.01 170A170F 1.17 173 S173C 3.07 170A170N 1.17 173S173Y 3.54 170A170M 1.28 173S173W 3.67 170A170D 1.32 173 S173L 3.86 170A170P 1.33 174F174H 1.05 171 L171H 1.07 174F174K 1.17 171 L171G 1.33 174F174P 1.46 171 L171Y 1.35 174F174Y 1.66 171L171T 1.36 174F174L 1.83 171 L171V 1.39 174F174A 2.09 171 L171I 1.42 174F174M 2.20 171 L171K 1.53 175M175N 1.02 171 L171A 1.66 175M175E 1.43 171 L171C 1.73 176K176C 1.01 171 L171S 1.76 176K176R 1.03 171 L171Q 1.93 176K176E 1.08 171 L171F 1.97 176K176W 1.16 171 L171M 2.22 176K176D 1.18 171 L171N 2.79 176K176A 1.19 172A172M 1.06 176K176F 1.28 172A172L 1.22 176K176V 1.33 GC821-2

Table 10-6. Variants with Table 10-6. Variants with Peracid Degradation Greater Peracid Degradation Greater Than WUd-Type Than WUd-Type Pos. WT/Pos/Var.PAD PI Pos. WT/Pos/Var.PAD PI 176K176M 1.33 184S184Q 1.16 178P178K 1.70 184S184I 1.21 178P178T 2.28 184S184V 1.25 178P178V 2.70 184S184F 1.27 178P178G 2.95 184S184K 1.61 178P178S 3.06 184S184A 1.69 178P178Q 3.64 184S184M 1.77 178P178M 3.87 184S184E 1.86 178P178E 4.15 184S184N 1.93 178P178A 4.39 184S184L 2.00 178P178D 6.44 184S184D 2.24 178P178Y 6.91 184S184C 2.39 178P178L 7.15 185V185F 1.20 179F179G 1.16 185V185Q 1.41 179F179V 1.17 185V185M 1.46 179F179Y 1.47 186I186L 1.14 179F179E 1.80 186I186M 1.38 179F179L 1.89 186I186A 1.79 180F180W 1.81 186I186D 4.29 180F180C 1.94 187S187K 1.16 180F180I 2.11 187S187D 1.40 180F180L 2.13 187S187G 1.46 180F180A 2.70 187S187L 1.46 180F180Y 2.99 187S187H 1.51 180F180N 3.05 187S187I 1.58 180F180V 3.24 187S187N 1.59 180F180M 4.36 187S187C 1.67 181 D181A 1.23 187S187A 1.72 183 G183P 1.02 187S187M 1 87 183 G183R 1.09 188T188N 1.69 183 G183Y 1.45 188T188E 1.97 183 G183L 1.50 189D189A 1.18 183 G183C 1.99 189D189T 1.21 184S184Y 1.09 189D189I 1.27 GC821-2

Table 10-6. Variants with Table 10-6. Variants with

Peracid Degradation Greater Peracid Degradation Greater

Than WUd-Type Than WUd-Type

Pos. WT/Pos/Var.PAD PI Pos. WT/Pos/Var.PAD PI 189D189L 1.30 197T197A 1.42 190G190C 1.17 197T197M 2.38 190G190Y 1.39 198E198T 1.16 190G190P 1.86 198E198S 1.18 190G190D 2.02 198E198F 1.21 190G190H 2.92 198E198V 1.44 190G190A 3.42 198E198Q 1.46 190G190M 5.54 198E198A 1.46 191 V191T 1.03 198 El 981 1.48 191 V191R 1.91 198E198L 1.54 191 V191K 2.17 198E198N 1.67 191 V191F 2.75 198E198P 1.72 191 V191C 2.81 198E198Y 1.77 191 V191Y 4.34 198E198W 1.78 191 V191L 4.69 198E198C 1.83 191 V191A 5.06 198E198M 1.86 191 V191E 5.46 198E198R 1.88 191 V191Q 5.83 199A199F 1.15 191 V191D 6.03 199A199H 1.15 191 V191M 7.34 199A199R 1.17 193 G193S 1.60 199A199T 1.22 193G193E 3.15 199A199E 1.31 193 G193Q 4.29 199A199D 1.33 193 GI 93 V 5.21 199A199V 1.45 195H195P 1.16 199A199K 1.53 195H195M 1.28 199A199Y 1.59 195H195K 1.33 199A199L 1.65 195H195Y 1.49 199A199C 2.45 195H195E 1.70 201N201D 1.64 195H195D 1.93 202R202M 1.76 196F196I 1.12 202R202G 1.82 196F196L 1.17 202R202S 1.84 196F196C 1.18 202R202C 1.93 197T197H 1.24 202R202A 1.97 GC821-2

Table 10-6. Variants with Peracid Degradation Greater Than WUd-Type Pos. WT/Pos/Var.PAD PI 202R202I 1.99 202R202E 2.05 202R202L 2.05 202R202T 2.06 202R202H 2.09 202R202F 2.16 202R202W 2.52 203D203Q 1.03 203D203S 1.13 203D203I 1.19 203D203N 1.28 203D203G 1.33 203D203F 1.34 203D203H 1.54 203D203P 1.71 203D203R 1.77 203D203A 1.96 203D203L 2.08 203D203C 2.09

The following Table provides variants that exhibited peracid degradation that was less than wild-type.

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 1 M001V 0.94 2A002S 0.66 2A002Y 0.46 2A002G 0.84 2A002N 0.59 2A002F 0.93 2A002V 0.60 3K003V 0.84 2A002I 0.61 4R004L 0.01 2A002T 0.61 4R004V 0.08 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 4R004L 0.15 8F008S 0.01 4R004W 0.48 8F008R 0.46 4R004G 0.79 8F008H 0.64 4R004S 0.91 8F008G 0.65 4R004E 0.97 8F008T 0.77 4R004Y 0.98 8F008K 0.83 4R004H 0.99 8F008P 0.83 4R004Q 0.99 8F008V 0.85 4R004T 1.00 8F008Y 0.90 5I005G 0.01 8F008N 0.96 5I005N 0.01 9G009H 0.01 5I005P 0.01 9G009T 0.01 5I005R 0.01 10D010W 0.01 5I005F 0.15 10D010K 0.01 5I005S 0.37 10D010Y 0.01 5I005H 0.63 10D010T 0.01 5I005T 0.72 10D010I 0.01 51005 V 0.92 10D010V 0.01 6L006S 0.01 10D010S 0.01 6L006K 0.01 10D010G 0.01 6L006G 0.01 10D010R 0.01 6L006H 0.01 10D010A 0.01 6L006R 0.01 10D010M 0.01 6L006W 0.01 10D010N 0.01 6L006E 0.01 10D010P 0.01 6L006Q 0.01 10D010E 0.15 6L006V 0.35 11 SOI IT 0.01 6L006T 0.35 11 SOI IV 0.01 6L006I 0.82 11 SOI ID 0.01 7C007S 0.01 11 SOI IE 0.01 7C007R 0.01 11 S011F 0.01 7C007Y 0.54 11 S011G 0.01 7C007M 0.68 11 S011L 0.01 7C007G 0.69 11 S011Q 0.01 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 11 SOUR 0.01 14W014E 0.15 11 S011H 0.33 14W014F 0.22 11 SOI IK 0.40 14W014A 0.27 li son A 0.53 14W014Y 0.66 11 SOI 11 0.56 15G015C 0.01 12L012V 0.01 15G015N 0.01 12L012S 0.01 15G015D 0.01 12L012G 0.01 15G015E 0.01 12L012R 0.01 15G015P 0.01 12L012D 0.01 15G015A 0.61 12L012P 0.01 15G015S 0.63 12L012W 0.02 16W016S 0.01 12L012T 0.06 16W016G 0.01 12L012A 0.07 16W016H 0.01 12L012K 0.13 16W016T 0.01 12L012H 0.16 16W016R 0.01 12L012F 0.17 16W016N 0.01 12L012Q 0.22 16W016P 0.15 12L012C 0.22 16W016Q 0.31 12L012N 0.66 16W016M 0.37 13T013Q 0.51 16W016A 0.55 13T013V 0.63 16W016D 0.57 13T013S 0.68 16W016E 0.65 13T013G 0.77 16W016V 0.88 14W014I 0.01 17V017A 0.68 14W014S 0.01 17V017E 0.75 14W014G 0.01 17V017G 0.84 14W014K 0.01 17V017K 0.84 14W014V 0.01 17V017F 0.85 14W014L 0.01 17V017T 0.86 14W014T 0.01 17V017Y 0.88 14W014R 0.01 17V017R 0.94 14W014N 0.01 17V017P 0.96 14W014P 0.01 17V017I 0.99 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 17V017L 1.00 24P024T 0.66 18P018S 0.07 24P024A 0.68 19V019P 0.01 24P024G 0.76 19V019M 0.12 24P024I 0.85 19V019R 0.34 24P024R 0.91 19V019Q 0.40 24P024H 0.97 19V019A 0.55 25T025P 0.01 19V019G 0.56 25T025H 0.01 19V019S 0.57 25T025L 0.01 19V019E 0.62 25T025R 0.01 19V019Y 0.70 25T025M 0.01 19V019D 0.79 25T025E 0.01 19V019L 0.91 25T025D 0.01 19V019K 0.97 25T025K 0.13 20E020L 0.73 25T025W 0.14 20E020G 0.78 25T025I 0.35 21 D021P 0.86 25T025G 0.43 22G022K 0.01 25T025C 0.51 22G022W 0.23 25T025V 0.51 22G022R 0.56 25T025S 0.58 22G022V 0.85 25T025A 0.86 22G022S 0.98 26E026S 0.28 23A023R 0.28 26E026T 0.40 23A023S 0.34 26E026W 0.47 23A023G 0.35 26E026N 0.48 23A023F 0.44 26E026R 0.81 23A023V 0.60 26E026G 0.87 23A023Q 0.73 26E026C 0.94 23A023P 0.73 26E026V 0.97 23A023W 0.80 26E026P 0.99 23A023M 0.95 27R027W 0.01 23A023Y 0.96 27R027T 0.01 24P024S 0.61 27R027P 0.48 24P024Q 0.65 27R027C 0.58 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 27R027S 0.69 35T035Q 0.01 27R027G 0.84 35T035N 0.01 27R027E 0.93 35T035R 0.01 27R027V 0.94 35T035V 0.34 28F028G 0.01 36G036S 0.26 28F028P 0.39 36G036T 0.33 28F028V 0.53 36G036V 0.38 28F028S 0.70 36G036M 0.54 29A029V 0.44 36G036N 0.56 29A029T 0.47 36G036W 0.68 29A029S 0.55 36G036Q 0.71 29A029Y 0.59 36G036R 0.90 29A029P 0.62 37V037T 0.81 29A029R 0.73 37V037H 0.96 29A029W 0.74 37V037W 0.98 29A029M 0.77 38L038K 0.01 29A029G 0.80 38L038G 0.01 29A029E 0.84 38L038E 0.01 29A029D 1.00 38L038P 0.01 30P030M 0.79 38L038Q 0.01 30P030Q 0.91 38L038R 0.01 30P030A 0.92 38L038D 0.12 31 D031E 0.88 38L038S 0.29 32V032P 0.01 38L038A 0.63 32V032R 0.72 38L038C 0.72 33R033V 0.94 39A039S 0.01 34W034R 0.01 39A039G 0.30 34W034E 0.01 39A039N 0.43 34W034Q 0.04 39A039R 0.64 34W034S 0.08 39A039I 0.71 34W034T 0.15 39A039P 0.74 34W034V 0.73 39A039T 0.79 34W034G 0.88 39A039M 0.81 34W034I 0.94 39A039E 0.83 GC821-2

Table 10-7. Variants with Table 10-7. Variants with

Peracid Degradation Results Peracid Degradation Results

Less than WUd-Type Less than WUd-Type

Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var. PAD PI 39A039C 0.92 44A044R 0.01 39A039K 0.96 44A044E 0.03 39A039L 0.97 44A044V 0.50 39A039V 0.98 44A044F 0.80 40Q040P 0.01 44A044W 0.85 41 Q041V 0.01 44A044M 0.98 41 Q041S 0.22 44A044L 0.99 41 Q041P 0.66 45D045S 0.38 41 Q041Y 0.70 45D045T 0.44 41 Q041W 0.88 45D045R 0.49 42L042W 0.01 45D045V 0.50 42L042H 0.01 45D045P 0.53 42L042T 0.01 45D045Q 0.57 42L042Q 0.28 45D045W 0.58 42L042S 0.45 45D045H 0.78 42L042R 0.64 45D045L 0.78 42L042I 0.66 45D045M 0.78 42L042V 0.73 45D045G 0.84 42L042M 0.74 45D045A 0.84 42L042G 0.76 45D045C 0.84 43G043S 0.23 45D045K 0.87 43G043P 0.31 46F046T 0.43 43G043V 0.33 46F046W 0.63 43G043Q 0.48 46F046S 0.66 43 G043R 0.59 46F046V 0.79 43 G043C 0.73 46F046I 0.88 43 G043I 0.77 46F046G 0.94 43 G043K 0.86 47E047P 0.36 43 G043M 0.88 47E047R 0.62 43 G043Y 0.94 47E047N 0.63 43 G043H 0.96 47E047S 0.63 44A044S 0.01 47E047M 0.70 44A044Y 0.01 47E047A 0.76 44A044T 0.01 47E047F 0.76 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 47E047C 0.77 52G052F 0.01 47E047T 0.84 52G052I 0.07 47E047D 0.98 52G052P 0.24 47E047H 0.99 52G052L 0.24 48V048R 0.01 52G052Q 0.28 48V048S 0.42 52G052R 0.35 48V048G 0.87 52G052E 0.55 48V048N 0.98 52G052A 0.79 48V048E 0.99 53L053R 0.01 49I049P 0.16 53L053W 0.01 49I049R 0.29 53L053P 0.01 49I049W 0.68 53L053D 0.01 49I049H 0.74 53L053E 0.19 49I049S 0.79 53 L053K 0.24 49I049E 0.88 53L053S 0.26 49I049V 0.97 53 L053G 0.33 50E050R 0.01 53L053V 0.65 50E050W 0.14 53L053I 0.66 50E050V 0.43 53L053Q 0.72 50E050I 0.58 53L053T 0.84 50E050S 0.65 54S054F 0.01 50E050Q 0.91 54S054W 0.01 50E050L 0.97 54S054H 0.01 51 E051R 0.01 54S054K 0.08 51 E051I 0.04 54S054I 0.12 51 E051W 0.17 54S054Y 0.12 51 E051V 0.37 54S054G 0.17 51 E051Q 0.76 54S054L 0.26 51 E051L 0.93 54S054V 0.29 52G052H 0.01 54S054E 0.30 52G052S 0.01 54S054T 0.33 52G052V 0.01 54S054R 0.35 52G052T 0.01 54S054M 0.48 52G052M 0.01 54S054Q 0.53 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var. PAD PI Pos WT/Pos/Var.PAD PI 54S054D 0.65 58T058V 0.96 54S054C 0.88 58T058S 0.96 55A055V 0.01 59N059R 0.01 55A055I 0.01 59N059M 0.01 55A055P 0.01 59N059P 0.01 55A055W 0.01 60I060P 0.32 55A055Y 0.18 60I060D 0.66 55A055R 0.25 60I060C 0.67 55A055T 0.42 60I060M 0.68 55A055G 0.73 60I060A 0.79 55A055L 0.87 60I060R 0.81 55A055S 0.87 60I060L 0.91 55A055H 0.92 60I060E 0.92 56R056C 0.01 60I060K 0.96 56R056G 0.01 60I060S 1.00 56R056T 0.01 61 D061F 0.70 56R056E 0.01 61 D061A 0.71 56R056Q 0.01 61 D061C 0.85 56R056S 0.12 61 D061Y 0.95 56R056L 0.24 61 D061V 0.97 56R056N 0.27 61 D061N 1.00 56R056A 0.69 62D062T 0.01 57T057R 0.01 62D062I 0.01 57T057P 0.01 62D062V 0.01 57T057N 0.25 62D062H 0.01 57T057C 0.40 62D062W 0.01 57T057Y 0.55 62D062S 0.01 57T057H 0.61 62D062L 0.01 57T057A 0.65 62D062G 0.01 57T057L 0.76 62D062R 0.01 57T057V 0.87 62D062M 0.01 57T057I 0.87 62D062P 0.01 58T058M 0.03 62D062Q 0.01 58T058A 0.36 62D062A 0.11 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 62D062C 0.49 66P066F 0.67 62D062E 0.60 66P066Y 0.70 63P063A 0.60 66P066D 0.72 63P063R 0.80 66P066I 0.84 63P063S 0.90 66P066V 0.89 63P063M 0.91 66P066H 0.95 63P063F 0.93 66P066L 0.99 63P063Y 0.95 67R067F 0.01 64T064R 0.11 67R067W 0.02 64T064D 0.64 67R067P 0.04 64T064W 0.69 67R067E 0.11 64T064Q 0,87 67R067V 0.12 64T064C 0.88 67R067Q 0.13 64T064P 0.94 67R067L 0.16 64T064H 0.96 67R067A 0.22 64T064N 0.98 67R067T 0.32 64T064S 0.99 67R067N 0.33 65D065V 0.20 67R067G 0.41 65D065R 0.22 67R067K 0.99 65D065H 0.40 68L068G 0.01 65D065Y 0.42 68L068A 0.01 65D065P 0.42 68L068M 0.03 65D065S 0.47 68L068C 0.06 65D065W 0.50 68L068S 0.07 65D065T 0.50 68L068N 0.10 65D065G 0.52 68L068E 0.13 65D065I 0.62 68L068H 0.22 65D065A 0.72 68L068Q 0.25 66P066N 0.38 68L068F 0.25 66 P066Q 0.42 68L068T 0.32 66?066G 0.44 68L068P 0.35 66 P066R 0.51 68L068D 0.44 66P066C 0.52 68L068Y 0.45 66P066A 0.56 68L068R 0.47 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var. PAD PI Pos WT/Pos/Var.PAD PI 68L068V 0.51 71 A071C 0.99 68L068W 0.56 72S072Y 0.07 68L068I 0.73 72S072W 0.34 69N069Y 0.17 72S072P 0.56 69N069W 0.55 72S072Q 0.66 69N069P 0.59 72S072L 0.70 69N069R 0.83 72S072R 0.74 69N069G 0.98 72S072D 0.80 70G070M 0.01 72S072V 0.83 70G070T 0.01 72S072E 0.93 70G070P 0.01 72S072T 0.97 70G070V 0.01 73Y073P 0.01 70G070C 0.01 73Y073R 0.26 70G070R 0.01 73Y073L 0.50 70G070Y 0.01 73Y073G 0.51 70G070K 0.01 73Y073H 0.52 70G070N 0.01 73Y073I 0.64 70G070Q 0.01 73Y073S 0.68 70G070F 0.01 73Y073V 0.74 70G070I 0.27 73Y073N 0.76 70G070E 0.33 73Y073D 0.80 70G070S 0.64 73Y073Q 0.87 71 A071P 0.01 73Y073K 0.94 71 A071N 0.61 74L074S 0.01 71 A071D 0.65 74L074G 0.57 71 A071G 0.68 74L074V 0.61 71 A071S 0.69 74L074I 0.64 71 A071R 0.77 74L074W 0.67 71 A071H 0.78 74L074Y 0.86 71 A071I 0.79 75P075M 0.30 71 A071T 0.79 75P075R 0.46 71 A071E 0.81 75P075Q 0.61 71 A071L 0.84 75P075S 0.63 71 A071F 0.99 75P075T 0.69 GC821-2

Table 10-7. Variants with Table 10-7. Variants with

Peracid Degradation Results Peracid Degradation Results

Less than WUd-Type Less than WUd-Type

Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 75P075I 0.74 79A079I 0.67 75P075H 0.86 79A079S 0.78 75P075K 0.88 79A079G 0.92 75P075G 0.93 79A079P 0.94 76S076W 0.01 79A079L 0.96 76S076Y 0.18 80T080W 0.01 76S076F 0.46 80T080L 0.01 76S076Q 0.90 80T080K 0.01 77C077Y 0.01 80T080R 0.01 77C077R 0.01 80T080E 0.01 77C077W 0.01 80T080P 0.01 77C077F 0.01 80T080H 0.05 77C077G 0.18 80T080Y 0.11 77C077L 0.73 80T080I 0.15 77C077S 0.76 80T080N 0.53 77C077V 0.80 81 H081R 0.01 77C077A 0.91 81 H081Y 0.14 78L078E 0.01 81 H081K 0.56 78L078N 0.01 81 H081S 0.69 78L078M 0.48 81 H081V 0.71 78L078Q 0.52 81 H081P 0.72 78L078C 0.78 81 H081Q 0.75 78L078Y 0.81 81 H081G 0.80 78L078V 0.83 81 H081F 0.90 79A079H 0.01 82L082R 0.01 79A079F 0.01 82L082S 0.01 79A079C 0.03 82L082W 0.01 79A079Q 0.27 82L082V 0.19 79A079E 0.27 82L082G 0.31 79A079N 0.28 82L082T 0.38 79A079M 0.28 82L082H 0.47 79A079R 0.32 82L082I 0.51 79A079W 0.53 82L082K 0.51 79A079T 0.60 82L082P 0.52 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 82L082A 0.98 86L086H 0.01 83P083T 0.01 86L086S 0.01 83P083V 0.19 86L086R 0.01 83P083L 0.21 86L086E 0.01 83P083H 0.61 86L086Q 0.01 83P083W 0.62 86L086W 0.08 83P083G 0.68 86L086V 0.12 83P083S 0.79 86L086T 0.28 83P083Q 0.82 86L086G 0.70 83P083D 0.83 86L086Y 0.82 83P083F 0.99 86L086P 0.99 84L084W 0.01 87V087S 0.01 84L084V 0.42 87V087G 0.01 84L084P 0.43 87V087Y 0.01 84L084T 0.44 87V087R 0.01 84L084A 0.45 87V087K 0.01 84L084Q 0.52 87V087D 0.01 84L084S_. 0.55 87V087F 0.10 84L084R 0.57 87V087T 0.15 84L084N 0.67 87V087A 0.17 84L084K 0.79 87V087M 0.75 84L084D 0.85 88I088H 0.01 84L084I 0.87 88I088T 0.01 84L084H 0.99 88I088G 0.01 85D085I 0.10 88I088N 0.01 85D085L 0.24 88I088Q 0.01 85D085V 0.25 89I089H 0.01 85D085W 0.34 89I089S 0.01 85D085P 0.54 89I089G 0.01 85D085Y 0.55 89I089W 0.01 85D085S 0.68 89I089Q 0.01 85D085T 0.71 89I089E 0.01 85D085N 0.78 89I089F 0.75 85D085Q 0.99 89I089V 0.82 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var. PAD PI Pos WT/Pos/Var.PAD PI 89I089T 0.90 94N094M 0.03 90M090S 0.01 94N094C 0.07 90M090W 0.01 94N094Y 0.12 90M090G 0.01 94N094G 0.53 90M090P 0.01 94N094A 0.74 90M090V 0.08 94N094P 0.79 90M090T 0.15 94N094S 0.88 90M090R 0.36 95D095E 0.75 90M090I 0.66 96T096I 0.01 90M090Q 0.77 96T096W 0.01 90M090L 0.98 96T096Y 0.01 91 L091G 0.01 96T096R 0.14 91 L091T 0.01 96T096V 0.59 91 L091Q 0.01 96T096S 0.79 91 L091E 0.01 96T096P 0.89 91 L091S 0.43 97K097Q 0.01 91 L091V 0.79 97K097G 0.01 91 L091M 0.88 97K097I 0.01 92G092V 0.01 97K097W 0.01 92G092S 0.01 97K097L 0.01 92G092E 0.01 97K097V 0.01 92G092F 0.01 97K097Y 0.01 93T093Q 0.01 97K097S 0.01 93T093Y 0.03 97K097T 0.01 93T093D 0.23 97K097M 0.22 93T093S 0.49 97K097A 0.23 93T093F 0.54 97K097P 0.27 93T093C 0.95 97K097R 0.59 94N094L 0.01 98A098T 0.27 94N094T 0.01 98A098G 0.56 94N094V 0.01 98A098S 0.65 94N094H 0.01 98A098I 0.65 94N094R 0.01 98A098H 0.92 94N094W 0.01 99Y099R 0.29 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 99Y099V 0.31 103T103Y 0.01 99Y099S 0.37 103T103G 0.01 99Y099W 0.57 103T103K 0.01 99Y099H 0.59 103T103I 0.01 99Y099I 0.61 103T103L 0.01 99Y099G 0.70 103T103H 0.01 99Y099P 0.81 103T103A 0.01 99Y099A 0.82 103T103V 0.01 99Y099L 0.86 103T103S 0.01 100F100W 0.01 103T103C 0.01 100F100K 0.01 103T103R 0.01 100F100D 0.01 103T103N 0.01 100F100E 0.15 103T103F 0.01 100F100S 0.85 103T103P 0.01 101 R101W 0.01 104P104R 0.01 101 R101K 0.07 104P104W 0.23 101 R101Q 0.11 104P104T 0.33 101 R101V 0.44 104P104S 0.53 101 R101D 0.80 104P104Q 0.85 101 R101Y 0.80 104P104F 0.86 101 R101P 0.86 104P104G 0.98 101 R101N 0.92 105L105V 0.01 101 R101C 0.95 105L105E 0.53 101 R101I 0.96 105L105S 0.61 101 R101F 0.97 105L105Y 0.62 102R102W 0.01 105L105T 0.64 102R102F 0.23 105L105P 0.90 102R102G 0.27 106D106R 0.56 102R102C 0.36 106D106Q 0.62 102R102V 0.61 106D106P 0.63 102R102D 0.68 106D106N 0.64 102R102P 0.89 106D106M 0.86 102R102S 0.96 106D106I 0.92 103T103W 0.01 106D106L 1.00 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 107I107E 0.01 110G110P 0.22 107I107G 0.01 110G110I 0.23 107I107F 0.01 110G110S 0.30 107I107Q 0.01 110G110Q 0.34 107I107R 0.01 110G110R 0.48 107I107P 0.32 110G110H 0.73 107I107Y 0.52 110G110N 0.77 107I107A 0.80 110G110M 0.82 107I107N 0.93 111 M111R 0.01 107I107V 0.97 111 M111S 0.14 108A108E 0.61 111 M111H 0.19 108A108Q 0.73 111 M111G 0.32 108A108T 0.87 111 M111P 0.57 108A108V 0.95 111 M111E 0.67 109L109W 0.01 111 M111L 0.67 109L109D 0.11 111 M111K 0.71 109L109I 0.14 111 M111T 0.76 109L109E 0.19 111 M111F 0.78 109L109R 0.21 111 M111D 0.79 109L109H 0.22 111 M111V 0.93 109L109Q 0.22 112S112Y 0.01 109L109F 0.32 112S112R 0.01 109L109A 0.32 112S112P 0.01 109L109S 0.38 112S112H 0.38 109L109P 0.43 112S112V 0.48 109L109G 0.51 112S112M 0.56 109L109V 0.54 112S112W 0.58 109L109M 0.63 112S112K 0.68 109L109N 0.66 112S112T 0.72 109L109T 0.79 112S112N 0.85 109L109Y 0.83 112S112F 0.88 110G110T 0.01 112S112A 0.94 110G110W 0.01 113V113S 0.57 110G110Y 0.01 113V113G 0.58 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 113V113K 0.72 118V118K 0.01 113V113H 0.76 118V118W 0.01 113V113W 0.80 118V118E 0.01 113V113L 0.85 118V118R 0.07 113V113T 0.86 118V118P 0.22 113V113D 0.87 118V118D 0.40 113V113E 0.94 118V118I 0.55 113V113C 0.94 118V118G 0.56 113V113F 0.96 118V118S 0.82 113V113Y 0.98 118V118A 0.85 114L114H 0.01 118V118T 0.92 114L114E 0.01 118V118M 0.93 114L114Q 0.12 118V118F 1.00 114L114P 0.28 119L119G 0.01 114L114S 0.55 119L119S 0.01 114L114V 0.60 119L119F 0.01 114L114N 0.77 119L119R 0.01 115V115I 0.99 119L119P 0.01 116T116Y 0.47 119L119T 0.10 116T116V 0.57 119L119N 0.11 116T116R 0.62 119L119V 0.15 116T116L 0.68 119L119W 0.20 116T116W 0.75 119L119C 0.24 116T116I 0.76 119L119D 0.28 116T116Q 0.77 119L119E 0.32 116T116P 0.84 119L119I 0.43 116T116G 0.90 119L119H 0.46 116T116E 0.91 119L119Y 0.56 116T116A 0.95 120T120P 0.01 116T116S 0.96 120T120H 0.50 117Q117W 0.71 120T120R 0.60 117Q117V 0.76 120T120A 0.66 117Q117G 0.79 120T120Q 0.78 117Q117S 0.87 120T120C 0.92 GC821-2

Table 10-7. Variants with Table 10-7. Variants with

Peracid Degradation Results Peracid Degradation Results

Less than WUd-Type Less than WUd-Type

Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var. PAD PI 121 S121P 0.38 124G124M 0.01 121 S121R 0.70 124G124W 0.01 121 S121W 0.77 124G124P 0.01 121 S121K 0.78 124G124A 0.03 121 S121G 0.99 124G124Q 0.21 122A122G 0.01 124G124T 0.32 122A122D 0.06 124G124V 0.33 122A122F 0.15 124G124R 0.41 122A122H 0.17 124G124L 0.54 122A122R 0.40 124G124S 0.56 122A122S 0.43 124G124Y 0.56 122A122K 0.45 124G124N 0.60 122A122E 0.47 124G124D 0.64 122A122T 0.52 124G124C 0.67 122A122P 0.55 124G124F 0.95 122A122I 0.65 125V125W 0.25 122A122N 0.70 125V125E 0.39 122A122Q 0.74 125V125R 0.47 122A122W 0.86 125V125C 0.54 122A122V 0.89 125V125D 0.54 122A122M 0.94 125V125P 0.62 123 G123C 0.30 125V125F 0.63 123G123Q 0.31 125V125S 0.79 123 G123T 0.54 125V125Y 0.81 123 G123E 0.56 125V125A 0.93 123 G123V 0.59 125V125I 0.94 123 G123R 0.60 126G126I 0.01 123G123N 0.71 126G126V 0.18 123 G123H 0.74 126G126Y 0.23 123 G123F 0.80 126G126L 0.54 123 G123P 0.81 126G126A 0.55 123 G123D 0.84 126G126E 0.60 124G124I 0.01 126G126P 0.67 124G124H 0.01 126G126T 0.74 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 126G126R 0.76 130P130G 0.01 126G126N 0.85 130P130S 0.01 126G126S 0.90 130P130L 0.09 126G126C 0.98 130P130E 0.22 127T127L 0.01 130P130W 0.28 127T127E 0.01 130P130V 0.37 127T127Q 0.15 130P130I 0.41 127T127I 0.20 130P130A 0.44 127T127H 0.60 130P130F 0.48 127T127D 0.62 130P130R 0.53 127T127M 0.64 130P130K 0.55 127T127C 0.65 130P130C 0.64 127T127V 0.68 130P130M 0.76 127T127G 0.71 131 A131W 0.01 127T127P 0.77 131 A131D 0.40 127T127S 0.83 131 A131Y 0.48 128T128D 0.66 131 A131L 0.59 129Y129W 0.01 131 A131S 0.68 129Y129G 0.01 131 A131P 0.71 129Y129K 0.01 131 A131Q 0.74 129Y129V 0.01 131 A131V 0.78 129Y129T 0.14 131 A131H 0.82 129Y129A 0.17 131A131G 0.87 129Y129R 0.18 131 A131E 0.97 129Y129M 0.21 132P132V 0.01 129Y129D 0.23 132P132T 0.01 129Y129L 0.27 132P132W 0.01 129Y129N 0.53 132P132F 0.01 129Y129P 0.59 132P132I 0.01 129Y129C 0.61 132P132H 0.01 129Y129S 0.69 132P132R 0.01 129Y129F 0.71 132P132D 0.01 130P130T 0.01 133K133C 0.01 130P130H 0.01 133 K133A 0.10 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var. PAD PI Pos WT/Pos/Var.PAD PI 133K133V 0.23 137V137I 0.70 133K133G 0.31 137V137T 0.93 133K133H 0.31 138S138I 0.35 133K133M 0.33 138S138V 0.69 133K133T 0.39 139P139S 0.01 133K133I 0.45 139P139G 0.01 133K133Q 0.52 139P139R 0.01 133 K133S 0.58 139P139C 0.01 133K133F 0.59 139P139D 0.01 133K133P 0.71 139P139E 0.01 133K133E 0.76 139P139F 0.01 133K133R 0.83 139P139H 0.01 133K133W 0.99 139P139I 0.01 134V134Q 0.79 139P139K 0.01 134V134T 0.86 139P139N 0.01 134V134I 0.89 139P139Q 0.01 135L135T 0.01 139P139T 0.01 135L135W 0.01 139P139V 0.01 135L135K 0.01 140P140T 0.01 135L135S 0.01 140P140S 0.01 135L135F 0.01 140P140V 0.01 135L135G 0.01 140P140W 0.01 135L135R 0.01 140P140I 0.01 135L135P 0.01 140P140Y 0.01 135L135Q 0.17 140P140Q 0.01 135L135V 0.43 140P140R 0.01 135L135E 0.63 141 P141R 0.01 135L135M 0.78 141 P141G 0.01 136V136P 0.01 141 P141S 0.02 136V136E 0.20 141 P141T 0.12 136V136N 0.40 141 P141V 0.16 137V137N 0.01 141 P141Q 0.37 137V137G 0.26 141 P141I 0.38 137V137S 0.29 141 P141L 0.65 GC821-2

Table 10-7. Variants with Table 10-7. Variants with

Peracid Degradation Results Peracid Degradation Results

Less than WUd-Type Less than WUd-Type

Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 141 P141H 0.79 145M145F 0.77 141 P141N 0.97 145M145P 0.78 142L142W 0.01 145M145S 0.78 142L142I 0.28 145M145T 0.79 142L142S 0.31 145M145A 0.79 142L142Q 0.33 145M145Y 0.82 142L142V 0.33 145M145C 0.93 142L142P 0.44 146P146W 0.68 142L142F 0.54 146P146T 0.76 142L142A 0.56 146P146V 0.77 142L142K 0.66 146P146S 0.96 142L142C 0.70 147H147S 0.75 143A143W 0.01 147H147T 0.84 143A143P 0.39 147H147I 0.92 143A143G 0.42 147H147V 0.92 143A143S 0.63 147H147R 0.94 143A143F 0.68 147H147A 0.98 143A143Q 0.81 148P148Q 0.98 143A143N 0.82 149W149R 0.01 143A143T 0.97 149W149E 0.01 143A143R 0.99 149W149P 0.01 143A143V 0.99 149W149C 0.12 144P144G 0.62 149W149I 0.24 144P144A 0.79 149W149A 0.31 144P144T 0.81 149W149S 0.33 144P144S 0.92 149W149Q 0.40 145M145W 0.01 149W149T 0.44 145M145G 0.26 149W149G 0.45 145M145E 0.48 149W149M 0.49 145M145I 0.53 149W149F 0.50 145M145Q 0.57 149W149L 0.64 145M145L 0.61 149W149Y 0.75 145M145V 0.63 150F150P 0.32 145M145R 0.69 150F150N 0.36 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 150F150G 0.46 155 El 55 V 0.47 150F150V 0.51 155 El 551 0.65 150F150A 0.54 155E155Q 0.69 150F150T 0.58 156G156I 0.01 150F150W 0.62 156G156F 0.73 150F150M 0.63 156G156W 0.90 150F150E 0.73 156G156L 0.94 150F150C 0.78 156G156V 0.97 150F150I 0.78 157G157R 0.01 150F150K 0.85 157G157P 0.01 151 Q151L 0.01 157G157S 0.19 151 Q151V 0.01 157G157V 0.40 151 Q151F 0.01 157G157C 0.61 151 Q151I 0.01 157G157E 0.84 151 Q151W 0.32 157G157M 0.85 152L152I 0.61 157G157A 0.87 152L152P 0.61 157G157D 0.94 152L152T 0.69 157G157T 0.99 152L152Q 0.76 158 El 58V 0.89 152L152G 0.77 158E158D 0.89 152L152S 0.84 158E158T 0.91 152L152D 0.86 158E158I 0.94 152L152V 0.88 159Q159A 0.28 152L152R 0.91 159Q159C 0.31 152L152K 0.91 159Q159P 0.49 152L152H 0.92 159Q159D 0.63 153 I153N 0.89 159Q159L 0.70 154F154T 0.01 159Q159G 0.72 154F154G 0.01 159Q159S 0.73 154F154V 0.01 159Q159R 0.74 154F154S 0.29 159Q159M 0.84 154F154Q 0.97 159Q159E 0.97 155E155R 0.01 160K160W 0.01 155E155F 0.23 160K160G 0.30 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 160K160H 0.57 166 R166T 0.74 160K160S 0.70 166R166V 0.76 160K160L 0.95 166R166G 0.91 160K160I 1.00 166R166S 0.95 161 T161R 0.01 168Y168G 0.01 161 T161H 0.01 168Y168T 0.01 161 T161W 0.01 168Y168V 0.01 161T161N 0.01 168Y168I 0.01 161 T161G 0.43 168Y168C 0.01 161 T161C 0.56 168Y168Q 0.01 161 T161S 0.57 169S169P 0.89 161 T161I 0.98 169S169T 0.97 163E163F 0.27 170A170I 0.44 163E163R 0.49 170A170S 0.47 163E163V 0.55 170A170G 0.62 163E163P 0.77 170A170T 0.72 163E163G 0.80 170A170V 0.74 163E163H 0.82 170A170K 0.83 163E163S 0.85 170A170W 0.83 163E163W 0.98 170A170L 0.85 164L164Y 0.01 170A170Q 0.89 164L164A 0.01 170A170Y 0.89 164L164D 0.01 171 L171R 0.01 164L164E 0.01 172A172K 0.01 164L164G 0.01 172A172R 0.01 164L164H 0.12 172A172E 0.01 164L164F 0.86 172A172Q 0.18 164L164C 0.91 172A172V 0.39 164L164T 0.99 172A172W 0.45 165A165I 0.59 172A172P 0.58 165A165K 0.82 172A172I 0.58 165A165Y 0.84 172A172T 0.71 165A165S 0.94 172A172N 0.76 165A165F 1.00 172A172G 0.84 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 172A172S 0.85 180F180K 0.01 172A172C 0.86 180F180T 0.01 174F174W 0.01 180F180R 0.01 174F174Q 0.46 180F180S 0.01 174F174C 0.48 180F180G 0.01 174F174R 0.52 180F180Q 0.01 174F174S 0.61 181 D181Y 0.01 174F174T 0.64 181 D181W 0.01 174F174V 0.67 181 D181L 0.01 174F174G 0.91 181 D181T 0.01 175M175P 0.08 181 D181V 0.01 175M175A 0.66 181 D181R 0.22 175M175Y 0.72 181 D181K 0.47 175M175G 0.75 181D181G 0.52 175M175W 0.76 181 D181S 0.55 175 Ml 75 V 0.81 181 D181Q 0.60 175M175Q 0.83 181 D181P 0.66 175M175L 0.86 181 D181E 0.72 175M175R 0.86 181 D181C 0.85 175M175T 0.90 182A182I 0.01 176K176S 0.72 182A182R 0.01 176K176G 0.73 182A182Q 0.01 176K176P 0.78 182A182P 0.01 176K176L 0.92 182A182T 0.11 176K176Y 0.93 182A182N 0.53 176K176N 0.94 182A182S 0.85 176K176T 0.97 182A182G 0.94 176K176Q 0.97 182A182C 0.99 178P178W 0.02 183 G183S 0.01 179F179Q 0.01 183 G183Q 0.01 179F179S 0.34 183 G183V 0.01 179F179W 0.86 183 G183F 0.19 179F179H 0.93 183 G183H 0.95 179F179N 0.95 183 G183D 0.99 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos Var.PAD PI Pos WT/Pos/Var.PAD PI 184S184T 0.60 188T188F 0.01 184S184H 0.74 188T188Y 0.09 184S184G 0.82 188T188I 0.10 184S184P 0.85 188T188V 0.15 185V185W 0.01 188T188L 0.42 185V185H 0.01 188T188M 0.75 185V185G 0.01 188T188G 0.79 185V185D 0.01 188T188C 0.87 185V185S 0.53 188T188S 0.91 185V185Y 0.58 188T188A 0.95 185 VI 851 0.63 189D189F 0.37 185V185R 0.79 189D189R 0.39 185V185K 0.79 189D189N 0.57 185V185C 0.83 189D189V 0.71 185V185E 0.88 189D189W 0.76 185V185T 0.91 189D189E 0.77 185V185L 0.93 189D189G 0.80 186I186G 0.01 189D189S 0.81 186I186S 0.01 189D189M 0.88 186I186R 0.01 189D189C 0.94 186I186P 0.01 189D189H 0.95 186I186T 0.23 189D189P 0.97 1861186V 0.48 190G190V 0.01 186I186F 0.76 190G190S 0.01 187S187P 0.01 190G190Q 0.29 187S187T 0.23 190G190W 0.41 187S187Q 0.35 190G190R 0.51 187S187W 0.52 190G190K 0.57 187S187R 0.55 190G190L 0.82 187S187V 0.58 191 V191H 0.01 187S187F 0.65 191 V191W 0.01 187S187Y 0.80 191 V191S 0.01 188T188H 0.01 191 V191G 0.01 188T188R 0.01 191 V191N 0.01 GC821-2

Table 10-7. Variants with Table 10-7. Variants with Peracid Degradation Results Peracid Degradation Results Less than WUd-Type Less than WUd-Type Pos WT/Pos/Var.PAD PI Pos WT/Pos/Var.PAD PI 191 V191I 0.02 195H195V 0.60 192D192S 0.01 195H195Q 0.96 192D192P 0.01 195H195A 0.98 192D192F 0.01 196F196H 0.01 192D192H 0.01 196F196G 0.01 192D192I 0.01 196F196S 0.01 192D192Q 0.01 196F196Q 0.01 192D192R 0.01 196F196W 0.38 192D192T 0.01 196F196P 0.39 192D192V 0.01 196F196V 0.68 192D192W 0.01 196F196M 0.71 192D192N 0.15 196F196Y 0.97 192D192C 0.56 197T197R 0.01 193G193H 0.01 197T197L 0.65 193 G193C 0.01 197T197S 0.75 193 G193T 0.01 197T197G 0.81 193G193N 0.01 197T197I 0.84 194I194S 0.01 197T197C 0.86 194I194A 0.01 197T197V 0.89 194I194C 0.01 197T197N 0.91 194I194P 0.01 199A199M 0.93 194I194F 0.01 199A199S 0.99 194I194W 0.01 199A199G 0.99 194I194R 0.01 201N201Y 0.01 194I194Y 0.01 201 N201T 0.01 194I194G 0.04 201 N201V 0.01 194I194L 0.58 201 N201R 0.01 1941194V 0.78 201N201S 0.06 195H195S 0.08 201N201H 0.10 195H195C 0.10 201 N201G 0.30 195H195L 0.18 201N201L 0.35 195H195N 0.22 201N201F 0.67 195H195R 0.24 201N201E 0.72 195H195F 0.40 203D203V 0.50 GC821-2

Table 10-7. Variants with Peracid Degradation Results Less than WUd-Type Pos WT/Pos/Var.PAD PI 203D203W 0.52 203D203E 0.90 The following Table provides variants that have protein performance indices

("Prot. PI") better than wild-type.

Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than WUd- PI Values Better Than WUd- Type Type Pos WT/Pos/Var. Prot. PI Pos WT/Pos/Var. Prot. PI 2 A002Y 1.61 17V017A 1.21 2A002N 1.30 17V017E 1.11 2A002I 1.25 17V017F 1.09 2A002V 1.18 17V017I 1.08 2A002T 1.17 17V017K 1.06 2A002S 1.15 17V017T 1.03 5I005M 1.29 18P018C 2.56 7C007A 1.22 18P018H 2.50 7C007G 1.07 18P018L 2.50 7C007M 1.03 18P018E 2.47 8F008N 1.23 18P018G 2.47 8F008M 1.05 18P018N 2.35 8F008G 1.03 18P018V 2.30 8F008P 1.01 18P018Q 2.13 11 S011H 1.06 18P018R 2.01 11 SOU A 1.04 18P018Y 1.68 11 S01 ID 1.03 18P018S 1.05 11 S01 IE 1.01 19V019G 1.39 11 S011Q 1.01 19V019A 1.23 12L012N 1.06 19V019E 1.10 12L012Q 1.05 19V019Q 1.07 13T013V 1.17 19V019K 1.03 14W014Y 1.02 19V019M 1.00 16W016Y 1.02 20E020G 1.11 GC821-2

Table 10-8. Sites with Protein Table 10-8. Sites with Protein PI Values Better Than WUd- PI Values Better Than WUd- Type Type Pos WT/Pos/Var. Prot. PI Pos WT/Pos/Var. Prot. PI 20E020P 1.08 30P030H 1.05 20E020A 1.08 30P030Y 1.04 20E020N 1.01 32V032M 1.11 20E020V 1.01 32V032A 1.10 22G022A 1.07 32V032I 1.08 22G022I 1.03 32V032Q 1.03 23A023F 1.03 32V032L 1.01 24P024T 1.43 35T035C 1.16 24P024G 1.34 3 G036C 1.09 24P024S 1.31 36G036N 1.08 24P024H 1.15 36G036Q 1.07 24P024I 1.11 36G036S 1.06 24P024L 1.06 36G036A 1.00 25T025C 1.37 37V037N 1.09 25T025V 1.30 39A039V 1.18 25T025G 1.27 39A039E 1.03 25T025A 1.23 46F046A 1.05 25T025I 1.19 46F046C 1.01 25T025P 1.10 47E047I 1.02 25T025M 1.04 54S054A 1.33 29A029G 1.22 54S054C 1.21 29A029P 1.07 54S054E 1.16 29A029M 1.06 54S054D 1.08 29A029D 1.06 54S054H 1.06 29A029V 1.05 54S054N 1.01 29A029S 1.05 54S054M 1.01 29A029T 1.02 55A055N 1.12 29A029E 1.02 55A055S 1.08 30P030E 1.20 56R056Q 1.02 30P030A 1.15 58T058V 1.13 30P030S 1.12 60I060A 1.20 30P030L 1.07 60I060M 1.14 30P030Q 1.06 60I060V 1.06 30P030K 1.06 60I060L 1.02 GC821-2

Table 10-8. Sites with Protein Table 10-8. Sites with Protein

PI Values Better Than WUd- PI Values Better Than WUd-

Type Type

Pos WT/Pos/Var. Prot. PI Pos WT/Pos/Var. Prot. PI 61 D061A 1.41 67R067A 1.39 61 D061N 1.12 67R067V 1.24 61 D061V 1.10 67R067P 1.04 61 D061Y 1.03 67R067F 1.01 61 D061Q 1.02 68L068A 1.07 61 D061L 1.00 68L068V 1.01 62D062A 1.06 68L068G 1.00 62D062M 1.06 69N069C 1.18 63P063S 1.17 69N069G 1.06 63P063Y 1.12 69N069D 1.05 63P063M 1.09 69N069S 1.03 63P063Q 1.08 70G070A 1.08 63P063A 1.06 72S072L 1.07 63P063V 1.06 72S072A 1.06 63P063R 1.02 72S072Y 1.03 63P063T 1.02 73Y073N 1.25 64T064Q 1.13 73Y073Q 1.20 64T064M 1.07 73Y073C 1.18 64T064R 1.05 73Y073D 1.09 64T064C 1.05 73Y073V 1.08 64T064S 1.03 73Y073M 1.05 66P066Q 1.91 73Y073L 1.03 66P066G 1.78 74L074I 1.45 66 P066N 1.62 74L074Y 1.19 66P066C 1.51 74L074V 1.18 66 P066I 1.51 74L074A 1.01 66 P066R 1.26 75P075M 1.22 66P066H 1.23 75P075S 1.18 66 P066V 1.12 75P075T 1.10 66P066Y 1.08 75P075Y 1.08 66 P066 A 1.03 75P075C 1.06 66P066F 1.02 75P075Q 1.04 67R067Q 1.60 75P075L 1.02 67R067L 1.46 75P075E 1.00 GC821-2

Table 10-8. Sites with Protein Table 10-8. Sites with Protein

PI Values Better Than WUd- PI Values Better Than WUd-

Type Type

Pos WT/Pos/Var. Prot. PI Pos WT/Pos/Var. Prot. PI 76S076W 1.06 96T096G 1.03 77C077L 1.44 97K097A 1.11 77C077V 1.33 97K097R 1.02 77C077A 1.20 98A098S 1.17 77C077S 1.19 98A098T 1.03 77C077T 1.18 98A098N 1.01 78L078I 1.06 99Y099S 1.45 78L078V 1.04 99Y099L 1.39 79A079C 1.16 99Y099H 1.30 79A079E 1.12 99Y099A 1.29 79A079S 1.09 99Y099V 1.28 79A079Q 1.05 99Y099G 1.23 79A079M 1.04 99Y099W 1.20 79A079R 1.02 99Y099I 1.11 80T080S 1.12 100F100M 1.20 80T080E 1.02 100F100N 1.12 8OTO80Q 1.02 100F100W 1.06 82L082G 1.24 100F100S 1.02 82L082R 1.15 101 R101L 1.33 82L082V 1.14 101 R101N 1.11 82L082S 1.13 101 R101Q 1.03 82L082P 1.11 101 R101D 1.02 82L082M 1.07 102R102Q 1.09 82L082K 1.03 103T103G 1.20 82L082A 1.00 103T103S 1.14 83 P083G 1.01 103T103H 1.14 84L084V 1.23 103T103N 1.07 86L086Q 3.66 103 T103K 1.05 89I089V 1.09 103T103P 1.01 89I089L 1.07 104P104S 1.44 93T093Q 2.03 104P104V 1.40 96 T096A 1.32 104P104E 1.37 96T096V 1.12 104P104C 1.34 96T096S 1.05 104P104N 1.32 GC821-2

Table 10-8. Sites with Protein Table 10-8. Sites with Protein

PI Values Better Than WUd- PI Values Better Than WUd-

Type Type

Pos WT/Pos/Var. Prot. PI . Pos WT/Pos/Var. Prot. PI 104P104T 1.29 113V113N 1.01 104P104G 1.25 114L114C 1 10 104P104Q 1.24 114L114A 1 03 104P104H 1.11 114L114M 1 00 104P104I 1.07 115V115I 1 14 104P104M 1.01 115V115C 1 14 105L105Y 1.18 115V115A 1 11 105L105H 1.07 115V115M 1 05 105L105G 1.07 115V115L 1 02 105L105C 1.05 116T116N 1 68 105L105Q 1.03 116T116H 1 48 105L105T 1.00 116T116G 1 44 105L105P 1.00 116T116C 1 30 106D106E 1.02 116T116E 1 29 107I107S 1.05 116T116Q 1 29 1071107V 1.04 116T116M 1 28 107I107C 1.00 116T116S 1 24 108A108G 1.15 116T116Y 1 09 108A108S 1.14 116T116A 1 08 108A108T 1.08 116T116R 1 03 109L109E 1.24 116T116L 1 03 109L109I 1.21 117Q117S 1 13 109L109D 1.15 117Q117H 1 12 109L109N 1.13 117Q117E 1 10 109L109F 1.11 117Q117T 1 06 109L109Q 1.08 117Q117A 1 03 109L109A 1.07 118V118C 1 28 109L109H 1.06 118V118A 1 20 109L109V 1.06 118V118I 1 01 109L109M 1.00 119L119C 1 18 110G110S 1.01 119L119A 1 18 112S112N 1.09 119L119N 1 14 112S112E 1.05 119L119I 1 .06 113V113C 1.06 119L119S 1 .05 GC821-2

Table 10-8. Sites with Protein Table 10-8. Sites with Protein

PI Values Better Than WUd- PI Values Better Than WUd-

Type Type

Pos WT/Pos/Var. Prot. PI Pos WT/Pos./Var.Prot. PI 119L119V 1.04 124G124C 1.07 119L119E 1. 04 124G124Q 1.02 119L119R 1 00 125V125I 1.05 120T120S 1 35 126G126N 1.04 120T120E 1 19 126G126E 1.02 120T120C 1 14 126G126A 1.02 120T120K 1 12 127T127A 1.10 120T120N 1 10 127T127S 1.08 120T120A 1 09 127T127V 1.06 120T120H 1 07 127T127C 1.04 120T120Q 1 05 127T127G 1.04 120T120Y 1 01 127T127D 1.03 120T120L 1 00 127T127E 1.03 121 S121N 1 17 127T127M 1.02 121 S121L 1 12 128T128N 1.29 121 S121A 1 10 128T128M 1.28 121 S121C 1 09 128T128Q 1.24 121 S121G 1 07 128T128A 1.23 121 S121R 1 06 128T128H 1.19 121 S121K 1 04 128T128P 1.18 121 S121E 1 01 128T128D 1.14 121 S121Q 1 01 128T128K 1.10 122A122N 1 11 128T128S 1.07 122A122L 1 07 128T128V 1.05 122A122P 1 07 128T128R 1.03 122A122M 1 06 128T128F 1.01 122A122V 1 05 129Y129F 1.44 122A122S 1 05 129Y129C 1.42 122A122E 1 04 129Y129A 1.39 122A122I 1 04 129Y129D 1.35 122A122Q 1 02 129Y129M 1.28 124G124M 1 .36 129Y129N 1.24 124G124A 1 .20 129Y129L 1.22 124G124N 1 .18 129Y129P 1.11 GC821-2

Table 10-8. Sites with Protein Table 10-8. Sites with Protein

PI Values Better Than WUd- PI Values Better Than WUd-

Type Type

Pos WT/Pos/Var. Prot. PI Pos WT/Pos/Var. Prot. PI 129Y129G 1.10 149W149L 1.06 129Y129S 1.08 150F150A 1.70 129Y129W 1.01 150F150M 1.69 129Y129V 1.00 150F150N 1.52 130P130G 1.11 150F150C 1.41 130P130E 1.08 150F150P 1.38 130P130K 1.05 150F150K 1.33 130P130A 1.03 150F150E 1.32 130P130M 1.03 150F150T 1.27 133K133Q 1.13 150F150V 1.26 133K133S 1.02 150F150W 1.26 133K133A 1.01 150F150Y 1.24 133K133R 1.01 150F150I 1.19 133K133E 1.01 150F150L 1.14 135L135M 1.01 150F150G 1.13 136V136L 1.03 150F150H 1.09 138S138A 1.44 151 Q151K 1.04 138S138C 1.17 153I153N 1.04 138S138G 1.09 157G157A 1.00 141 P141A 1.13 159Q159E 1.14 141 P141G 1.02 159Q159A 1.13 142L142I 1.05 159Q159G 1.03 143A143G 1.17 161 T161C 1.01 145M145I 1.16 162T162C 1.17 145M145L 1.07 162T162I 1.16 147H147L 1.09 162T162H 1.08 147H147C 1.04 162T162L 1.05 149W149G 1.39 162T162F 1.05 149W149A 1.35 162T162Y 1.03 149W149M 1.32 164L164M 1.09 149W149S 1.28 164L164V 1.08 149W149F 1.27 165A165G 1.14 149W149Y 1.15 165A165Q 1.05 149W149Q 1.10 165A165S 1.05 GC821-2

Table 10-8. Sites with Protein Table 10-8. Sites with Protein

PI Values Better Than WUd- PI Values Better Than WUd-

Type Type

Pos WT/Pos/Var. Prot. PI Pos WT/Pos/Var. Prot. PI 166R166M 1.26 184S184G 1.15 166R166K 1 19 184S184D 1.15 166R166G 1 19 184S184C 1.14 166R166N 1 16 184S184Q 1.09 166R166D 1 16 184S184H 1.07 166R166A 1 12 184S184N 1.03 166R166L 1 08 184S184V 1.03 166R166T 1 04 184S184K 1.02 167V167L 1 13 185 VI 851 1.03 167V167H 1 12 186I186M 1.11 167V167G 1 08 188T188C 2.04 167V167M 1 04 188T188I 1.85 167V167I 1 04 188T188L 1.76 167V167S 1 04 188T188M 1.60 167V167C 1 01 188T188V 1.53 168Y168F 1 28 188T188S 1.52 168Y168L 1 27 188T188R 1.41 170A170C 1 02 188T188A 1.40 171 L171I 1 16 188T188G 1.32 172A172C 1 09 188T188N 1.24 172A172G 1 07 191 V191C 1.04 175M175Y 1 35 194I194L 1.32 175M175L 1 19 194I194C 1.17 175M175W 1 14 194I194A 1.15 175M175N 1 11 194I194W 1.12 175M175R 1 02 1941194V 1.03 176K176R 1 06 194I194Y 1.01 176K176Q 1 02 196F196L 1.09 178P178E 1 05 201 N201H 1.49 182A182C 1 03 183 G183S 1 08 184S184E 1 39 184S184A 1 31 184S184M 1 25 GC821-2

The following Table provides variants that have a PAD PI that is greater than 1.5, a PAF that is greater than or equal to 0.1 , and a protein PI that is greater than or equal to 0.1

GC821-2

GC821-2

GC821-2

The following Table provides variants with a PAD PI that is less than 0.5, a PAF that is greater than or equal to 0.1 , and a protein PI that is greater than or equal to 0.1.

GC821-2

GC821-2

GC821-2

In addition to the assay results described above, various mutations were found to result in unstable protein such that perhydrolase protein was not expressed. Thus, in contrast to the substitutions that resulted in enhanced expression as compared to wild- type, there were some substitutions that are not as favorable, at least under the conditions used herein. However, it is not intended that the present invention exclude these substitutions, as it is contemplated that these substitutions, taken alone or in combination will find use in alternative embodiments of the present invention.

GC821-2

The following Table provides performance indices obtained in PAF and PAD assays for various variants, as well as the protein performance index. GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

GC821-2

EXAMPLE 11 Cloning and Expression of a Sinorhizobium meliloti RSM02162 M. smegmatis Perhydrolase Homologue In this Example, cloning and expression of a S. meliloti perhydrolase homologue are described. The sequences used in cloning and expression are provided below. The gene RSM02162 (SEQ ID NO:625) was synthesized by DNA2.0. The gene was given the designation "G00355" and was provided cloned into the commercially available vector, pDRTvΕ (InvivoGen). The gene was amplified by PCR from this clone using the primer set G00355rbsF/ G00355R, Tag DNA polymerase (Roche) as per the manufacturer's directions, with G00355 as the template (10 ng/50 μl reaction) and 10 picomoles (per 50 μl reaction) of each primer. The amplification was carried out in an MJ Research PCR machine using 30 cycles of (1 minute at 95°C; 1 minute at 55°C; and 1 minute at 72°C). The amplification of the correct size fragment was confirmed by agarose gel electrophoresis. The fragment was cloned directly into pCR2.1TOPO (Invitrogen) and transformed into E. coli Top 10 cells (Invitrogen). Transformants were selected on L agar containing carbenicillin (100 μg ml) at 37°C. The correct construct was confirmed by sequence analysis and designated "pMC355rbs." Figure 20 provides a map of this plasmid.

Primer sequences:

G00355rbsF

5'-ggccctaacaggaggaattaaccatggtggaaaaacgttccgttctgtgc-3' (SEQ ID NO:626)

G00355R

5'-Gcgcgcttagaacagagccgctactttgtcagc-3' (SEQ ID NO:627)

Gene sequence (including stop codon) of RSM02162 :

5'- GC821-2

atggtggaaaaacgttccgttctgtgctttggtgattctctgacttggggctggattccggtgaaagagagctccccaactctgcgtt acccatacgaacagcgttggaccggtgctatggctgcacgtctgggtgatggttaccacatcattgaagaaggcctgtccgctcgt actactagcctggacgacccaaacgacgctcgtctgaacggctctacctacctgccgatggctctggcttctcacctgccactgga tctggtaatcattatgctgggtaccaacgacaccaaaagctactttcatcgtaccccatacgagattgccaacggcatgggtaaact ggtaggtcaggtcctgacctgtgcaggtggtgttggtacgccttatccagcaccgaaagtcctggtggttgcacctccaccactgg caccaatgccagatccgtggttcgaaggtatgttcggcggtggttacgagaaatctaaggaactgtccggtctgtacaaagcactg gctgatttcatgaaagtggagttcttcgcagcgggtgattgtatctccaccgacggtatcgacggtatccacctgagcgctgaaacc aacatccgcctgggtcatgctattgctgacaaagtagcggctctgttctaa-3' (SEQ ID NO:625)

G00355 Protein sequence:

MVEKRSNLCFGDSLTWGWffVKESSPTLRYPYEQRWTGAMAARLGDGYHIIEEG LS ARTTSLDDPΝDARLΝGSTYLPMALASHLPLDLVΠMLGTΝDTKSYFHRTPYEIA ΝGMGKLNGQNLTCAGGVGTPYPAPKVLWAPPPLAPMPDPWFEGMFGGGYEKS, KELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETΝIRLGHAIADKVAALF

(SEQ ID ΝO:628)

Complete sequence of pDRIVEG00355: gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagc gggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttg tgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctctaatacgactcactataggg aaagctcggtaccacgcatgctgcagacgcgttacgtatcggatccagaattcgtgattttagaacagagccgctøctttgtcagca atagcatgacccaggcggatgttggtttcagcgctcaggtggataccgtcgataccgtcggtggagatacaatcacccgctgcga agaactccactttcatgaaatcagccagtgctttgtacagaccggacagttccttagatttctcgtaaccaccgccgaacataccttc gaaccacggatctggcattggtgccagtggtggaggtgcaaccaccaggactttcggtgctggataaggcgtaccaacaccacc tgcacaggtcaggacctgacctaccagtttacccatgccgttggcaatctcgtatggggtacgatgaaagtagcttttggtgtcgttg gtacccagcataatgattaccagatccagtggcaggtgagaagccagagccatcggcaggtaggtagagccgttcagacgagc gtcgtttgggtcgtccaggctagtagtacgagcggacaggccttcttcaatgatgtggtaaccatcacccagacgtgcagccatag caccggtccaacgctgttcgtatgggtaacgcagagttggggagctctctttcaccggaatccagccccaagtcagagaatcacc aaagcacagaacggaacgtttttccaccataatctgaattcgtcgacaagcttctcgagcctaggctagctctagaccacacgtgtg ggggcccgagctcgcggccgctgtattctatagtgtcacctaaatggccgcacaattcactggccgtcgttttacaacgtcgtgact gggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatggaaattgtaagcgttaatattttgttaaaattcgcgttaaatt^ taaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttg ttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggccc GC821-2

actacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgat ttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctg gcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcaggtggcactttt cggggaaatgtgcgcggaacccctatttgtttattWctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatg cttcaatøatattgaaaaaggaagagtatgagtattcaacarltccgtgtcgcccttattccctttt^ gctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaaca gcggutøgatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggc cgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaa gcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgac aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggc gaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggccctt ccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaa gccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcc tcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaa gtgaagatcctttttgataatctcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaac gggaaacgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggc aatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaat gatgttacagatgagatggtcagactaaactggctgacggaatttatgcc^ tgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgat gcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtattt^ caatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaa atgcataaacn^gccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacg aggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctcct tcattacagaaacggctttttcaaaaatatggtattgataatcctga ^ gaattaattcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttctt^ gatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgttt caactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccactt caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttacc gggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgga gcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggac aggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcc tgtcgggmcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacg cggcctt ttacggttcctggccttttgctggcclittgctcacatgttctttcctgcgttatccc cctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaaga

(SEQ ID NO:629)

Complete sequence pMC355rbs: GC821-2

agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaag cgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgtt gtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttggtaccgagctcggatcca ctagtaacggccgccagtgtgctggaattcgcccttggccctaacaggaggaattaaccatggtggaaaaacgttccgttctgtgc tttggtgattctctgacttggggctggattccggtgaaagagagctccccaactctgcgttacccatacgaacagcgttggaccggt gctatggctgcacgtctgggtgatggttaccacatcattgaagaaggcctgtccgctcgtactactagcctggacgacccaaacga cgctcgtctgaacggctctacctacctgccgatggctctggcttctcacctgccactggatctggtaatcattatgctgggtaccaac gacaccaaaagctactttcatcgtaccccatacgagattgccaacggcatgggtaaactggtaggtcaggtcctgacctgtgcag gtggtgttggtacgccttatccagcaccgaaagtcctggtggttgcacctccaccactggcaccaatgccagatccgtggttcgaa ggtatgttcggcggtggttacgagaaatctaaggaactgtccggtctgtacaaagcactggctgatttcatgaaagtggagttcttc gcagcgggtgattgtatctccaccgacggtatcgacggtatccacctgagcgctgaaaccaacatccgcctgggtcatgctattgc tgacaaagtagcggctctgttctaagcgcgcaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatct agagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtt acccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaac agttgcgcagcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtga ccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagct ctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgta gtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgtt^ acactcaaccctatctcggtctattch^gatttataagggattttgccgatttcggcctøttggttaa atttaacgcgaattttaacaaaattcagggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacg gtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgca gtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggta aggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaaga gacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggct atgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaag accgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgc agctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatccca ccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgacc accaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatc aggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcga tgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatca ggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccg ctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttcc gtgtcgcccttattcccttttttgcggcartttgccttcctgttttt agttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtli cgccccgaagaacgttttccaa tgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacact attctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctg ccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaac atgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg GC821-2

cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatg gaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcg tgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatata tactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctn^ agttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagca gagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcg ctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggata aggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaac aggagagcgcacgagggagcttccagggggaaacgcctggtatcrttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagca^ ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgcto^ gccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag (SEQ ID NO:630)

Expression of the Homologue from pMC355rbs To express the S. meliloti RSM02162 protein from the plasmid pMC355rbs (See,

Figure 20, for a map of this plasmid), a single colony was inoculated into a 5 mis of L broth containing 100 μg/ml carbenicillin and grown overnight at 37°C with shaking at 200 rpm. Lysates were prepared by pelleting the cells from 1 ml of the overnight culture by centrifugation and lysed with BugBuster (Novagen). The supernatants were assayed using the pNA activity assay, perhydrolysis assay, and a pNC6 assay (to test its ability to hydrolyze carbon chains longer than C4), as described herein.

Assay Results The following Table (Table 11-1) provides a comparison of the hydrolysis activity of pNA by G00355 as compared to the M. smegmatis perhydrolase

Table 11-1. pNA Hydrolysis Activity GC821-2

*Rate is absorbance units/min read at 405 nm in a spectrophotometer.

The following Table (Table 11-2) provides a comparison of the perhydrolysis of triacetin by G00355 compared to the M. smegmatis perhydrolase.

The following Table (Table 11-3) provides a comparison of pNC6 hydrolysis by

G00355 compared to the M. smegmatis perhydrolase.

*Rate is absorbance units/min read at 405 nm in a spectrophotometer. GC821-2

As these results indicate, the homologue RSM02162 from S. meliloti identified by amino acid sequence homology to the M. smegmatis perhydrolase demonstrated similar, albeit less perhydrolysis activity than the M. smegmatis perhydrolase. However, this enzyme exhibited different substrate specificity, as it was able to hydrolyze pNC6, while the wild-type M. smegmatis perhydrolase cannot. The results of the pNC6 hydrolysis assay indicated that certain positions/substitutions provided an improvement in the ability of the enzyme to utilize longer chain substrates The positions and substitutions identified in preliminary screens are provided in the following Table. It is not intended that the present invention be limited to these specific positions and substitutions, as it is contemplated that additional positions and/or substitutions will also provide improved activity on longer chain substrates.

EXAMPLE 12 Amplification of Genes Encoding M. smegmatis Perhydrolase Homologues from Environmental Isolates GC821-2

In this Example, methods used to amplify genes encoding M. smegmatis perhydrolase homologues from environmental isolates are described. Organisms from soil samples that were positive for the transesterification reaction were purified to single colonies. To amplify the genes by PCR, the degenerate primer sets 1 AF/5AR and leF/5iR were used in a PCR reaction containing isolated chromosomal DNA from 8 environmental strains exhibiting the transesterification reaction. The PCR reaction was carried out using Taq DNA polymerase (Roche) as per the manufacturer's protocol, with 1 μg of chromosomal DNA added as template and 10 picomoles of each primer in a 50μl reaction. The reaction was carried out for 30 cycles of (1 minute at 95°C; 1 minute at 50°C, and 1 minute at 72°C). Since the partial coding sequence of the perhydrolase gene from Mycobacterium parafortuitum was already isolated, the same strain was used as a positive control. The strains were designated as: 2G, 2D, 9B, 14B, 18D, 19C, 20A. As indicated below, 20 A was typed as Mycobacterium parafortuitum, and 9B is Mycobacterium gilvum. Based on protein homology, it was inferred that 2D is also M. parafortuitum and 14B is M. gilvum.

Primer Sequences

1AF: 5'-gccaagcgaattctgtgtttcggngaytcnyt-3' (SEQ ID NO:631)

5AR:

5'-cgattgttcgcctcgtgtgaartgnrtnccrtc-3' (SEQ ED NO:632) leF:

5'-acggtcctgtgctttggngaytcnyt-3' (SEQ ID NO:633)

5iR:

5'-ccgctggtcctcatctggrtgntcnccrtc-3' (SEQ ID NO:634) Amplification with the above primer sets was expected to yield bands of approximately 500 bp. In all cases except 2G, the 1 AF/5AR primer set produced a band GC821-2

of the expected size. In the case of 19C, both primer sets produced bands of the expected size. The ~500 bp bands were purified from agarose gels using a gel purification kit (Qiagen) and analyzed by sequencing. While the strains 2G and 19C yielded bands of the expected size with both primer sets they were not the fragments encoding the M. smegmatis perhydrolase homologue.

Partial Sequences of 2D Perhydrolase Homologue and Protein:

Gene: 5 '- attctgtgtttcggggattccttgacgtggggatggatccctgtcgaagaaggtgtgcccaccgagcggttcccgcgtga cgtccggtggaccggcgtgctggccgacctgctgggcgaccgctacgaggtgatcgaggaaggcctgtcggcgcgcacca ccaccgccgacgacccggccgacccccggctcaacggttcgcagtatctgccgtcgtgtctggccagccatctgccgctg gacctggtgatcctgatgctcggcatcaacgacaccaaggcgaattttggccgcaccccgttcgacatcgccaccggtat gggagtgcttgccacgcaggtgctcaccagcgccggtggcgtggggaccagctatcccgcgccgcaggtgctgatcgtgg cgccgccgccgctgggcgagctgccccacccctggttcgacctggtgttctccggcggccgtgagaagaccgccgagttg gcccgcgtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggctcggtgatcagcaccgacggcgt ggacggcacccacttcacacgaggcgaaacaatcga (SEQ ID NO:635)

Protein: ILCFGDSLTWGWIPNEEGNPTERFPRDNRWTGNLADLLGDRYENIEEGLSARTTT ADDPADPRLΝGSQYLPSCLASHLPLDLNILMLGIΝDTKAΝFGRTPFDIATGMGVL ATQVLTSAGGNGTSYPAPQNLINAPPPLGELPHPWFDLNFSGGREKTAELARVYS ALASFMKNPFFDAGSNISTDGVDGTHFTRGETI (SEQ ID ΝO:636)

Partial Sequences of 9B Perhydrolase Homologue and Protein:

Gene: 5'-taccgtcgatgtgtggcctcgtgtgaagtgggtgccgttgccaagcgaattctgtgtttcggggattcgttgacgtgggg ctggatcccggtcgaggaaggtgtacccacccaacgttttccgaagcgggtgcgctggaccggggtgctggccgacgaac tgggtgctggctatgaggttgtcgaggaggggttgagcgcgcgcaccaccaccgctgacgaccctaccgatccccggctg aacggctcggactacctccccgcatgcctggccagccacctgccgctggacctggtgatcctgatgctcgggaccaacga caccaaggcgaatctgaatcgcacacccgtcgacatcgccagcggaatgggcgtcctggccacccaggtgctcaccagcg cgggcggggtcggcaccagctacccggccccgcaggtgttgatcgtggcaccgccgccgctggccgagatgccgcacccg tggttcgagctggtcttcgacggcggccgggagaagaccgcccaactggcccgggtgtacagcgcgctggcgtcgttcat gaaggtgccgttcttcgacgccggatcggtgatcagcaccgacggtgtcgacggcacccacttcacacgaggcgaaacaa tcgaccgg (SEQ ID NO:637) GC821-2

Protein:

GGRCNASCENGANAKRE CFGDSLTWGWIPNEEGNPTQRFPKRNRWTGNLADEL

GAGYEVNEEGLSARTTTADDPTDPRLΝGSDYLPACLASHLPLDLVILMLGTΝDTK

AΝLΝRTPNDIASGMGNLATQNLTSAGGNGTSYPAPQVLINAPPPLAEMPHPWFEL

NFDGGREKTAQLARNYSALASFMKVPFFDAGSNISTDGVDGTHFTRGETIDR

(SEQ ID ΝO:638)

Partial Sequences of 14B Perhydrolase Homologue and Protein:

Gene: 5'- attctgtgtttcggagattcgttgacgtggggctggatcccggtcgaggaaggtgtacccacccaacgttttccgaagcg ggtgcgctggaccggggtgctggccgacgaactgggtgctggctatgaggttgtcgaggaggggttgagcgcgcgcacca ccaccgctgacgaccctaccgatccccggctgaacggctcggactacctccccgcatgcctggccagccacctgccgctg gacctggtgatcctgatgctcgggaccaacgacaccaaggcgaatctgaatcgcacacccgtcgacatcgccagcggaat gggcgtcctggccacccaggtgctcaccagcgcgggcggggtcggcaccagctacccggccccgcaggtgttgatcgtgg caccgccgccgctggccgagatgccgcacccgtggttcgagctggtcttcgacggcggccgggagaagaccgcccaactg gcccgggtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggatcggtgatcagcaccgacggtgt cgacggcacccacttcacacgagg (SEQ ID NO:639)

Protein:

ILCFGDSLTWGWTPNEEGNPTQRFPKRNRWTGNLADELGAGYEWEEGLSARTT TADDPTDPRLΝGSDYLPACLASHLPLDLNILMLGTΝDTKAΝLΝRTPNDIASGMGV

LATQNLTSAGGVGTSYPAPQNLIVAPPPLAEMPHPWFELVFDGGREKTAQLARN YSALASFMKNPFFDAGSNISTDGNDGTHFTR (SEQ ED ΝO:640)

Partial Sequences of 20A Perhydrolase Homologue and Protein:

Gene:

5'- ttgccaagcggaattctgtgtttcggggattctttgacgtggggatggatccctgtcgaagaaggtgtgcccaccgagcg gttcccgcgtgacgtccggtggaccggcgtgctggccgacctgctgggcgaccgctacgaggtgatcgaggaaggcctgt cggcgcgcaccaccaccgccgacgacccggccgacccccggctcaacggttcgcagtatctgccgtcgtgtctggccagc catctgccgctggacctggtgatcctgatgctcggcatcaacgacaccaaggcgaattttggccgcaccccgttcgacat cgccaccggtatgggagtgcttgccacgcaggtgctcaccagcgccggtggcgtggggaccagctatcccgcgccgcagg tgctgatcgtggcgccgccgccgctgggcgagctgccccacccctggttcgacctggtgttctccggcggccgtgagaag accgccgagttggcccgcgtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggctcggtgatcag caccgacggcgtggacggcacccacttcacacgaggcgaaacaatcga-3' (SEQ ID NO:641) GC821-2

Protein:

LPSGILCFGDSLTWGWIPNEEGNPTERFPRDVRWTGVLADLLGDRYEVIEEGLSA RTTTADDPADPRLΝGSQYLPSCLASHLPLDL MLGIΝDTKAΝFGRTPFDIATGM GVLATQVLTSAGGVGTSYPAPQVLIVAPPPLGELPHPWFDLVFSGGREKTAELAR VYSALASFMKNPFFDAGSVISTDGVDGTHFTRGETI (SEQ ID ΝO:642)

Identification of the Natural Isolates To type the environmental isolates used in this Example, plates of the purified strains were sent to MIDI for 16S rRNA typing. 20A is Mycobacterium parafortuitum, 9B is Mycobacterium gilvum. By protein homology we infer that 2D is also M. parafortuitum and 14B is M. gilvum.

EXAMPLE 13 Sequence and Taxonomic Analyses of Perhydrolase Homologues In this Example, sequence and taxonomic analyses of M. smegmatis perhydrolase homologues are provided

Taxonomic Assignment The basic "List of 60" protein sequences accessed from public databases and used for construction of primer sets for screening of metagenomic libraries (BRAIN) was converted into a document illustrating the microbial taxonomic origins of the proteins, as described below. This information was used to produce the following alignment. 1 50 MSAT (1) MAKRILCFGDSLUWGWVPVEDGAPU-ERFAPDVRWUG 14B natural isolate (1) ILCFGDSLTWGWIPVEEGVPT-QRFPKRTOWTG 20A (1) LPSGILCFGDS TWGWIPVEEGVPT-ERFPRDVRWTG 2D natural isolate (1) ILCFGDS TWGWIPVEEGVPT-ERFPRDVRWTG 9B Natural Isolate (1) -GGRCVASCEVGAVAKRI CFGDS TWGWIPVEEGVPT-QRFPKRVRWTG M. parafortuitum COl (1) MAKRILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG Sm-RSM05666 (1) MKTVLCYGDSLTWGYDATGSG RHALEDRWPS GC821-2

At-Q8UAC0 (1) —MKTVIAFGDSLTWGADPATG L--RHPVEHRWPD At-Q8UFG4 _(!) -MVKSVLCFGDSLTWGSNAETGG RHSHDD PS M091_M4aEll ₍1₎ —MKTILAYGDSLTYGANPIPGGP RHAYEDR PT M1-RMO00301 (1) MAGGTRLDECTGERMKTV CYGDS TWGYNAEGG RHA EDRWPS P.dejongeii RVM04532 (1) MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG Q92XZ1 Sinorhizobium meliloti (1) MEETVARTVLCFGDSNTHGQVPGRGPLDR YRREQRWGG Q98MY5 Mesorhizobium loti (1) MKTVLCYGDSLTWGYNAEGG RHALEDRWPS RSM02162_Sm {1) MVEKRSVLCFGDSLTWGWIFVKESSPT-LRYPYEQRWTG S261_M2aA12 {1) MKNILAFGDSLTWGFVAGQDAR HPFETRWPN

Smal993 Sinorhizobium meliloti (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG Consensus (1) KTILCFGDSLTWGWIPV EG P RHP E RW G 51 100 MSAT (37) VLAQQLGADFEVIE—EGLSARUUNIDDPUDPRL-NGASYLPSCLAUHLP 14B natural isolate (33) VLADELGAGYEWE—EGLSARTTTADDPTDPRL-NGSDYLPACLASHLP 20A (37) VLADLLGDRYEVIE—EGLSARTTTADDPADPRL-NGSQYLPSCLASHLP 2D natural isolate (33) VLADLLGDRYEVIE—EGLSARTTTADDPADPRL-NGSQYLPSCLASHLP 9B Natural Isolate (49) VLADELGAGYEWE—EGLSARTTTADDPTDPRL-NGSDYLPACLASHLP M. parafortuitum COl (37) VLADLLGDRYEVIE--EGLSARTTTAEDPADPRL-NGSQYLPSCLASHLP • Sm-RSM05666 (32) VLQKALGSDAHVIA--EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAP At-Q8UAC0 (32) VLE-ΛELAGKAKVHP--EGI.GGRTTCYDDHAGPACRNGARALEVA1-SCHMP At-Q8UFG4 (33) VLQKALGSDVHVIFTHEGLGGRTTAYDDHTGDCDRNGARLLPTJ-LHSHAP M091_M4aEll (33) ALEQGLGGKARVIA--EGLGGRTTVHDDWFANADRNGARVLPTLLESHSP M1-RMLO00301 (45) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP P. ejongeii RVM04532 (37) VIAKALGAGFRVIE--EGQNGRTTVHEDPLNICR-KGKDYLPACLESHKP Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE—EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHAP Q98MY5 Mesorhizobium loti (31) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP RSM02162_Sm (39) AMAARLGDGYHIIE—EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP S261_M2aA12 (32) ALAAGLGGKARVIE--EGQNGRTTVFDDAATFESRNGSVALPLLLISHQP Smal993 Sinorhizobium meliloti (50) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTY1-PMALASHLP Consensus (51) VLA LGG Y VIE EGLSGRTT DDP D L NGS YLPT LASHLP 101 150 MSAT (84) LDLVIIMLGUNDUKAYFRRUPLDIA--LGMSVLVUQVLUSAGGVGUUYPA 14B natural isolate (80) LDLVILMLGTNDTKANLNRTPVDIA--SGMGVLATQVLTSAGGVGTSYPA 20A (84) LDLVILMLGINDTKANFGRTPFDIA—TGMGVLATQVLTSAGGVGTSYPA 2D natural isolate (80) LDLVILMLGINDTKANFGRTPFDIA—TGMGVLATQVLTSAGGVGTSYPA 9B Natural Isolate (96) LDLVILMLGTNDTKANLNRTPVDIA—SGMGVLATQVLTSAGGVGTSYPA M. parafortuitum COl (84) LDLVILMLGTNDTKANFGRTPFDIA—TGMGVLATQVLTSAGGVGTSYPA Sm-RSM05666 (80) LDLIVFMLGSNDM PIIHGTAFGAV—KGIERLVNLVRRHDWPTETE-EG At-Q8ϋAC0 (80) LDLVIIMLGTNDIKPVHGGRAEAAV—SGMRRLAQIVETFIYKPREA—V At-Q8UFG4 (83) DMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHV QVSDW-EA M091_M4aEll (81) LDLIVIMLGTNDIKPHHGRTAGEAG—RGMARLVQIIRGHYAGRMQD—E M1-RMLO00301 (93) IDLIVIMLGANDMKPWIHGNPVAAK—QGIQRLIDIVRGHDYPFDWP—A P. ejongeii RVM04532 (84) LDLVILMLGTNDLKSTFNVPPGEIA—AGAGVLGRMILAGDAGPENR--P Q92XZ1 Sinorhizobium meliloti (87) LDLIIIMLGTNDLKRRFNMPPSEVA--MGIGCLVHDIRELSPGRTGN—D Q98MY5 Mesorhizobium loti (79) IDLIVIMLGANDMKPWIHGNPVAAK—QGIQRLIDIVRGHDYPFDWP—A GC821-2

RSM02162_Sm (86) LDLVIIMLGTNDTKSYFHRTPYEIA— GMGKLVGQVLTCAGGVGTPYPA S261_M2aA12 (80) LDLVIIMLGTNDIKFAARCRAFDAS— GMERLIQIVRSANYMKGYK—I

Smal993 Sinorhizobium meliloti ( 97) LDLVIIMLGTNDTKSYFHRTPYEIA—NGMGKLVGQVLTCAGGVGTPYPA Consensus (101) LDLVIIMLGTNDMKA RTP DIA GMGRLV VLT AGGVG A 151 200 MSAT (132) PKVLWSPPPLAPM-PHPWFQLIF-EGGEQKUUELARVYSALASFMKVPF 14B natural isolate (128) PQVLIVAPPPLAEM-PHPWFELVF-DGGREKTAQLARVYSALASFMKVPF 20A (132) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF ^•2D natural isolate (128) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF 9B Natural Isolate (144) PQVLIVAPPPI^AEM-PHPWFELVF-DGGREKTAQLARVYSALASFMKVPF M. parafortuitum COl (132) PQVLIVAPPPLGEL-PHP FDLVF-SGGREKTAELARVYSALASFMKVPF Sm-RSM05666 (127) PEILIVSPPPLCET—ANSAFAAMFAGGVEQSAMLAPLYRDLADELDCGF At-Q8UAC0 (126) PKLLIVAPPPCVAG PGGEPAG-GRDIEQSMRLAPLYRKLAAELGHHF At-Q8UFG4 (132) PDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDCGF M091_M4aEll (127) PQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHF M1-RML000301 (139) PQILIVSPPWSRT—ENADFREMFAGGDEASKQLAPQYAALADEVGCGF P. ejongeii RVM04532 (130) PQLLLMCPPKVRDLSAMPDLDAKI-PHGAARSAEFPRHYKAQAVALKCEY Q92XZ1 Sinorhizobium meliloti (133) PEIMIVAPPPMLED—LKEWESIF-SGAQEKSRKLALEFEIMADSLEAHF Q98MY5 Mesorhizobium loti (125) PQILIVSPPWSRT—ENADFREMFAGGDEASKQLAPQYAALADEVGCGF RSM02162_Sm (134) PKVLVVAPPPLAPM-PDPWFECMF-GGGYEKSKELSGLYKALADFMKVEF S261_M2aA12 (126) PEILIISPPSLVPT—QDEWFNDLWGHAIAESKLFAKHYKRVAEELKVHF

Smal993 sinorhizobium meliloti (145) PKVLWAPPPIAPM-PDPWFEGMF-GGGYEKSrøLSGLYKALADFMKVEF Consensus (151) PQVLIVAPPPL EM P FE VF GG EKS LARVY ALAD MKV F 201 241 MSAT (180) FDAGSVISUDGVDGIHFUEANNRDLGVALAEQVRSLL (SEQ ID NO:643) 14B natural isolate (176) FDAGSVISTDGVDGTHFTR (SEQ ID NO: 644) 20A (180) FDAGSVISTDGVDGTHFTRGETI (SEQ ID NO: 645) 2D natural isolate (176) FDAGSVISTDGVDGTHFTRGETI (SEQ ID NO: 646) 9B Natural Isolate (192) FDAGSVISTDGVDGTHFTRGETIDR (SEQ ID NO:647) M. parafortuitum COl (180) FDAGSVISTDGVDGIHFTRGEQST (SEQ ID NO: 648) Sm-RSM05666 (175) FDGGSVARTTPIDGVHLDAENTRAVGRGLEPWRMMLGL— (SEQ ID NO: 649) At-Q8UAC0 (172) FDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG (SEQ ID NO: 650) At-Q8UFG4 (182) FDAGSVARTTPVDGVHLDAENTRAIGRGLEPWRMMLGL— (SEQ ID NO: 651) M091_M4aEll (177) FDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL— (SEQ ID NO: 652) M1-RMLO00301 (187) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVML (SEQ ID NO: 653) P.dejongeii RVM04532 (179) FNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG (SEQ ID NO: 654) Q92XZ1 Sinorhizobium meliloti (180) FDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA1SEQ ID NO: 655) Q98MY5 Mesorhizobium loti (173) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVMLEL— (SEQ ID NO: 656) RSM02162_Sm (182) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF (SEQ ID NO: 657) S261_M2aA12 (174) FDAGTVAVADKTDGGHLDAVNTKAIGVALVPWKSILAL— (SEQ ID NO: 658)

Smal993 Sinorhizobium meliloti (193) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF (SEQ ID NO: 659) Consensus (201) FDAGSVISTD VDGIHLDA T IG AL VR LL (SEQ ID NO:660) GC821-2

The alignment tree from the CLUSTALW alignment (which approximates to a phylogenetic tree) suggests 3 or 4 groupings. From this alignment, a hypothetical protein sequence was constructed from the consensus sequence. Where no consensus existed the site was filled with the Per amino acid; gaps were ignored. This provided a Per-consensus sequence:

1 TILCFGDSLT WGWIPVEEGA PTERHPPEVR WTGVLAQQLG GDYEVIEEGL 51 SGRTTNIDDP TDPRLNGSSY LPTCLASHLP LDLVIIMLGT NDMKAYFRRT 101 PLDIALGMGR LVTQVLTSAG GVGTTYPAPQ VLIVAPPPLA EMPHPWFELV 151 FEGGEEKSTE LARVYSALAD FMKVPFFDAG SVISTDGVDG IHLDAANTRD 201 IGVALAEQVR SLL (SEQ ID NO:661) This consensus sequence was used for a BLASTP search against a non-redundant database. This search identified 55 hits. The majority of the 'hits' were GDSL or GDSI type molecules covering a wide range of microbial diversity. However, only the first 14 'hits' had e- values and bit- values in the reliable range. At first sight, this appeared to provide further molecules with a GDSL/N - G/ARTT motif, but this was found to be due to differences in coding (Swiss Prot vs GenBank) The screening of 3 environmental libraries (at BRAIN) resulted in 10 clones with a GDSL motif. A further 2 clones were derived from the BRAIN library. The following Table (Table 13-1) lists the clones and indicates their activity.

GC821-2

M40cD4 Strongest hit: arylesterase of Brucella melitensis (46% identical). Motifs: GDSL - GAND; GQTT instead of GRTT. Sequence alignment against the core list of organisms places it close to Caulobacter vibrioides and Brucella melitensis in the alpha- Proteobacteria.

M44aA5 Strongest hit:Acyl-CoA thioesterase of Pseudomonas aeruginosa (43% identical). Motifs: GDSL - GGND; no GRTT or equivalent. Sequence alignment against the core list of organisms places it close to Pseudomonas sp in the gamma- Proteobacteria.

GC821-2

M2bBll Strongest hit: arylesterase of Brucella melitensis. Motifs: GDSL - GAND; no

GRTT or equivalent. Sequence alignment against the core list of organisms shows no strong association placing it between the alpha- and g tma-Proteobacteria.

M2aA12 Strongest hit: arylesterase of Agrobacterium tumefaciens (42% identical)

Motifs: GDSL - GRTT - GTND. Sequence alignment against the core list of organisms places it close to Agrobacterium tumefaciens in the εdp a-Proteobacteria.

GC821-2

M75bA2 Strongest hit: incomplete. BLAST search revealed nothing significant. Motifs: GDSL - GTND; no GRTT or equivalent. Sequence alignment against the core list of organisms shows no convincing associations. The closest neighbors appear to be the Vibrio - Aeromonas groups of the gaxa a-Proteobacteria.

M70aE8 Strongest hit: acyl-CoA thioesterase from E. coli (30% identical), and aryl esterase hydrolase from Vibrio mimicus (27% identical). Based on incomplete sequence GDSL-type esterase (BRAIN) from Neisseria meningitidis (50% identical). Motifs: GDSL - GGND; no GRTT - replaced with GRTN. Sequence alignment against the core list of organisms shows the closest association to Neisseria meningitidis, a member of the beta-Proteobacteria.

GC821-2

M4aEll Strongest hit: arylesterase from Agrobacterium tumefaciens (59% identity) Motifs: GDSL - GRTT - GTND. Sequence alignment against the core list of organisms shows the closest association to members of the alp a-Proteobacteria such as Agrobacterium.

Estll4 Strongest hit: phosphatidylcholine sterol acyltransferase from Aeromonas hydrophila (gamma-Proteobacteria) (30% identical). Motifs: GDSL - GPND; no GRTT GC821-2

but GATT may be an equivalent. Sequence alignment against the core list of organisms shows the closest association to Acidophilium sp. and Aeromonas/Vibrio within the gamma-Proteobacteria.

EstlOS Strongest hit: Pseudomonas aeruginosa outer membrane esterase, and hypothetical protein Pseudomonas putida (27% identical). Motifs: GDSL - GAND, no GRTT or equivalent. Sequence alignment against the core list of organisms shows the closest association to members of the gamma-Proteobacteria. )

GC821-2

An overall alignment of these clones/sequences (here shown underlined) indicates that they are scattered throughout the alignment tree of strains indicating that the metagenomic screening has provided a variety of sequences and not a limited diversity.

-2

GC821-2

Gene Mining for GRTT - Type Esterases (clones with perhydrolase activity)

Sinorhizobium meliloti Smal 993 -hypothetical protein Sme Motifs: GDSL - ARTT - GTND Sinorhizobium meliloti .Q92XZ 1 -hypothetical protein Sme Motifs: GDSN - GRTT - GTND

Mesorhizobium loti Q98MY5-arylesterase_Mlo Motifs:GDSL - GRTT - GAND

Moraxella bovis AAK53448 (lipase)

Motifs: GDSL - GSND, no GRTT or equivalent in this sequence order.

(perhydrolase activity low, questionable sequence) Agrobacterium tumefaciens Q8UACO Motifs: GDSL - GRTT - GTND

Agrobacterium tumefaciens Q8UFG4 Motifs: GDSL - GRTT - GTND

Mesorhizobium loti RMLO00301 Motifs: GDSL - GRTT - GAND

Sinorhizobium meliloti RSM05666 Motifs: GDSL - GRTT - GSND

(this clone was inactive for perhydrolase activity; and probably represents a false negative)

Sinorhizobium meliloti RSM02162 Motifs: GDSL - ARTT - GTND

Prosthecobacter dejongeii RVM05432 Motifs: GDSN - GRTT - GTND GC821-2

A GDSxi -x₂RTT - Gx₃ND motif characterizes the active clones/sequences, where: Xi = L orN X2 = A or G X^ T or A or S The Moraxella bovis AAK53448 sequence does not fit this pattern and is excluded from the alignment analysis provided below:

Multiple Sequence Alignment of Active Clones/Sequences 1 50 ACT MSMEG (1) MAKRILCFGDSLUWGWVPVEDGAPU-ERFAPDVRWUG Q98MY5 Mesorhizobium loti (1) ___MKTVLCYGDSLTWGYNAEGGR HALEDRWPS

Smal993 Sinorhizobium meliloti (1) rølNSHSWRTLMVEKRSV CFGDSLTWG IPVKESSPT-LRYPYEQRWTG Q92XZ1 Sinorhizobium meliloti (1) MEETVARTVLCFGDSNTHGQVPGRGPLDR YRREQRGG P.dejongeii RVM04532 (1) MKTI CFGDSNTWGYDPASMTAPFPRRHGPEVRWTG RSM05666_Sm (1) MKTVLCYGDSLTWGYDATGSG RHALEDRWPS RSM02162_Sm (1) MVEKRSVLCFGDSLTWGWIFVKESSPT-LRYPYEQRWTG At-Q8UAC0 (1) MKTVLAFGDSLTWGADPATGLR HPVEHR PD At-Q80FG4 (1) MVKSVLCFGDSLTWGSNAETGG RHSHDDLWPS M1-RMLO00301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAEGGR HALEDRWPS S261_M2aA12 (1) MKNILAFGDSLTWGFVAGQDA RHPFETRWPN M091_M4aEll (1) MKTILAYGDSLTYGANPIPGG-PR HAYEDRWPT Consensus (1) MKTVLCFGDSLTWGY P G RHA E RWP 51 100 ACT MSMEG (37) VLAQQLGADFEVIE---EGLSARUUNIDDPUDPRL-NGASYLPSCLAUHLP Q98MY5 Mesorhizobium loti (31) VLQASLGGGVQVIA—DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP

Smal993 Sinorhizobium meliloti (50) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE—EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHAP P.dejongeii RVM04532 (37) VLAKALGAGFRVIE—EGQNGRTTVHEDPLNICR-KGKDYLPACLESHKP RSM05666_Sm (32) VLQKALGSDAHVIA—EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAP RSM02162_Sm (39) AMAARLGDGYHIIE—EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP At-Q8ϋAC0 (32) VLEAELAGKAKVHP—EGLGGRTTCYDDHAGPACRNGARALEVALSCHMP At-Q8UFG4 (33) VLQKALGSDVHVIFTHEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHAP M1-RMLO00301 (45) VLQASLGGGVQVIA—DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP S261_M2aA12 (32) AAAGLGGKARVIE—EGQNGRTTVFDDAATFESRNGSVALPLLLISHQP M091_M4aEll (33) ALEQGLGGKARVIA—EGLGGRTTVHDDWFAADRNGARVLPTLLESHSP Consensus (51) VL A LGG VIE EGL GRTTAHDD A RNGAR LPT L SHAP 101 150 ACT MSMEG (84) LDLVIIMLGUNDUKAYFRRUPLDIA—LGMSVLVUQVLUSAGGVGUU PA GC821-2

Q98MY5 Mesorhizobium loti (79) IDLIVIMLGANDMKPWIHGNPVAAK—QGIQRLIDIVRGHDYPFDWPAP-

Smal993 Sinorhizobium meliloti (97) LDLVIIMLGTNDTKSYFHRTPYEIA—NGMGKLVGQVLTCAGGVGTPYPA Q92XZ1 Sinorhizobium meliloti (87) LDLIIIMLGTNDLKRRFNMPPSEVA—MGIGCLVHDIRELSPGRTGN P. ejongeii RVM04532 (84) LDLVILMLGTNDLKSTFNVPPGEIA—AGAGVLGRMILAGDAGPENR-PP RSM05666_Sm (80) LDLIVFMLGSNDMKPIIHGTAFGAV—KGIERLVNLVRRHDWPTETEEG- RSM02162_Sm (86) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA At-Q8UAC0 (80) LDLVIIMLGTNDIKPVHGGRAEAAVS—GMRRLAQIVETFIYKPREAVP- At-Q8UFG4 (83) LDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQVSDWEAP M1-RMLO00301 (93) iDLIVIMLGANDMKPWIHGNPVAAK—QGIQRLIDIVRGHDYPFDWPAP- S261_M2aA12 (80) LDLVIIMLGTNDIKFAARCRAFDAS—MGMERLIQIVRSANYMKGYKIP- M091_M4aEll (81) LDLIVIMLGTNDIKPHHGRTAGEAG—RGMARLVQIIRGHYAGRMQDEP- Consensus (101) LDLVIIMLGTNDMKP H P EAA GM RLV IVR YG P 151 200 ACT MSMEG (132) PKVLWSPPPLAPMPHPWFQLIFE— GGEQKUUELARVYSALASFMKVPF Q98MY5 Mesorhizobium loti (126) -QILIVSPPWSRTENADFREMFAG— GDEASKQLAPQYAALADEVGCGF

Smal993 Sinorhizobium meliloti (145) PKVLWAPPPLAPMPDPWFEGMFG— GGYEKSKELSGLYKALADFMKVEF Q92XZ1 Sinorhizobium meliloti (132) DPEIMIVAPPPMLEDLKEWESIFS--GAQEKSRKLALEFEIMADSLEAHF P. ejongeii RVM04532 (131) QLLmCPPKVRDLSAMPDLDAKIP--HGAARSAEFPRHYKAQAVALKCEY RSM05666_Sm (127) PEILIVSPPPLCETANSAFAAMFAG— GVEQSAMLAPLYRDLADELDCGF RSM02162_Sm (134) PKVLVVAPPPIAPMPDPWFEGMFG— GGYEKS1XELSGLYKALADFMKVEF At-Q8UAC0 (127) -KLLIVAPPPCVAGPGGEPAGGRD IEQSMRLAPLYRKLAAELGHHF At-Q8UFG4 (133) -DVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDCGF M1-RMLO00301 (140) -QILIVSPPWSRTENADFREMFAG— GDEASKQLAPQYAALADEVGCGF S261_M2aA12 ( 127 ) -EILIISPPSLVPTQDEWFNDLWG— HAIAESKLFAKHYKRVAEELKVHF M091_M4aEll ( 128 ) -QIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHF Consensus (151) ILIVSPPPL T DF AMFG G E SK LA YKALADELK F 201 241 ACT MSMEG (180) FDAGSVISUDGVDGIHFUEANNRDLGVALAEQVRSLL (SEQ ID NO: :662) Q98MY5 Mesorhizobium loti (173) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVMLEL— (SEQ ID NO: :663)

Smal993 Sinorhizobium meliloti (193) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF (SEQ ID NO: : 664) Q92XZ1 Sinorhizobium meliloti (180) FDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA (SEQ ID NO: :665) P.dejongeii RVM0 532 (179) FNΞQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG (SEQ ID NO: :666) RSM05666_Sm (175) FDGGSVARTTPIDGVHLDAENTRAVGRGLEPWRMMLGL— (SEQ ID NO: :667) RSM02162_Sm (182) AAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF (SEQ ID NO: :668) At-Q8UAC0 (172) FDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG (SEQ ID NO: :669) At-Q8UFG4 (182) FDAGSVARTTPVDGVHLDAENTRAIGRGLEPWRMMLGL— (SEQ ID NO: :670) M1-RMLO00301 (187) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVML (SEQ ID NO: :671) S261_M2aA12 (174) FDAGTVAVADKTDGGHLDAVNTKAIGVALVPWKSILAL— (SEQ ID NO: :672) M091_M4aEll (177) FDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL-- (SEQ ID NO: :673) Consensus (201) FDAGTVA TSPVDGIHLDAENTR IG ALA WR LLG (SEQ ID NO; :674) GC821-2

A guide tree (i.e., an approximation of a phylogenetic tree) of the CLUSTALW alignment of active clones/sequences is provided below.

GC821-2

Based on the results, the active clones were found to have an overall identity to M. smegmatis perhydrolase of 38.7 - 58.3%. Moraxella bovis AAK53448 was found to be an exception and the (translated) amino acid sequence is questionable.

Redundancy From the analyses above, it was evident that some redundancy exists in the alignment provided at the beginning of this Example that will have added undue weighting to the consensus sequence. Also, fiirther GDSL-GRTT sequences were added. Thus, in the revised alignment below, the following changes were made:

Removed: Natural isolate 14B Natural isolate 2D RSM02162_Sm Q98MY5 Mesorhizobium loti Added: BAB16197 (Arh Il) BAB16192 (Arh I) NP 00197751 (Mlo II) NP 00216984 (Bee) NP 522806 (Rso)

Non-redundant alignment: 1 50 20A (1) LPSGILCFGDSLTWGWIPVEEGVPTERFP-RDVRWTG 9B Natural Isolate ( 1) -GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPTQRFP-KRVRWTG M. parafortuitum COl (1) MAKRILCFGDSLTWGWIPVEEGVPTERFP-RDVRWTG MSAT (1) MAKRILCFGDSLTWGWVPVEDGAPTERFA-PDVRWTG GC821-2

Sm-RSM05666 (1) —MKTVLCYGDSLTWGYDATG- -SGRHALEDRWPS At-Q8ϋAC0 (1) —MKTVLAFGDSLTWGADPAT- -GLRHPVEHRWPD At-Q8UFG4 (1) -MVKSVLCFGDSLTWGSNAET- -GGRHSHDDLWPS M091_M4aEll (1) — KTI AYGDSLTYGANPIP- GGPRHAYEDRWPT M1-RML000301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAE GGRHALEDRWPS P.dejongeii RVM04532 (1) MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG Q92XZ1 Sinorhizobium meliloti (1) MEETVARTVLCFGDSNTHGQVPG—RGPLDRYR-REQRWGG S261_M2aA12 (1) MKNjILAFGDSLTWGFVAG QDARHPFETRWPN

Smal993 Sinorhizobium meliloti (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPTLRYP-YEQRWTG ZP_00197751 (1) MKTILCYGDSLTWGYDAVG PSRHAYEDRWPS ZP_00216984 (1) MTMTQKTVLCYGDSNTHGTRPMTHAGGLGRFA-REERWTG BAB16192 (1) -MICHKGGEEMRSVLCYGDSNTHGQIPG—GSPLDRYG-PNERWPG BAB16197 (1) MAESRSILCFGDSLTWGWIPVPESSPTLRYP-FEQRWTG NP_522806 (1) MQQILLYSDSLSWGIIPG TRRRLPFAARWAG Consensus (1) MKTILCFGDSLTWGWIPV P RR E RW G 51 100 20A (37) VLADLLGDRYEVIE EGLSARTTTADDPADPRLN-GSQYLPSCLASHL 9B Natural Isolate (49) VLADELGAGYEWE EGLSARTTTADDPTDPRLN-GSDYLPACLASHL M. parafortuitum COl (37) VLADLLGDRYEVIE EGLSARTTTAEDPADPRLN-GSQYLPSCLASHL MSAT (37) VLAQQLGADFEVIE EGLSARTTNΪDDPTDPRLN-GASYLPSCLATHL Sm-RSM05666 (32) VLQKALGSDAHVIA EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHA At-Q8UAC0 (32) VLEAELAGKAKVHP EGLGGRTTCYDDHAGPACRNGARALEVALSCHM At-Q8UFG4 (33) VLQKALGSDVHVIFT-HEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHA M091_M4aEll (33) ALEQGLGGKARVIA EGLGGRTTVHDDWFANADRNGARVLPTLLESHS M1-RML000301 (45) VLQASLGGGVQVIA DGLNGRTTAFDDHLAGADRNGARLLPTALTTHA P.dejongeii RVM04532 (37) VLAKALGAGFRVIE EGQNGRTTVHEDPLNICRK-GKDYLPACLESHK Q92XZ1 Sinorhizobium meliloti (39) VLQGLLGPNWQVIE EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHA S261_M2aA12 (32) ALAAGLGGKARVIE EGQNGRTTVFDDAATFESRNGSVALPLLLISHQ Smal993 Sinorhizobium meliloti (50) AMAARLGDGYHIIE EGLSARTTSLDDPNDARLN-GSTYLPMALASHL ZP_00197751 (32) VLQGRLGSSARVIA EGLCGRTTAFDDWVAGADRNGARILPTLLATHS ZP_00216984 (40) VLAQTLGASWRVIE EGLPARTTVHDDPIEGRHKNGLSYLRACVESHL BAB16192 (43) VLRRELGSQWYVIE EGLSGRTTVRDDPIEGTMKNGRTYLRPCLMSHA BAB16197 (39) AMAAALGDGYSIIE EGLSARTTSVEDPNDPRLN-GSAYLPMALASHL NP_522806 (32) VMEHALQAQGHAVRIVEDCLNGRTTVLDDPARPGRN-GLQGLAQRIEAHA Consensus (51) VIA LGA Y VIE EGL GRTT DDP D RNGA YLP L SH 101 150 20A (83) PLDLVILMLGINDTKANFGRTPFD—IATGMGVLATQVLTSAGG-VGTSY 9B Natural Isolate (95) PLDLVILMLGTNDTKANLNRTPVD—IASGMGVLATQVLTSAGG-VGTSY M. parafortuitum COl (83) PLDLVILMLGTNDTKANFGRTPFD—IATGMGVLATQVLTSAGG-VGTSY MSAT (83) PLDLVIIMLGTNDTKAYFRRTPLD—IALGMSVLVTQVLTSAGG-VGTTY Sm-RSM05666 (79) PLDLIVFMLGSNDMKPIIHGTAFG— AVKGIERLVNLVRRHDWPT— ETE At-Q8UAC0 (79) PLDLVIIMLGTNDIKPVHGGRAEA—AVSGMRRLAQIVETFIYK PRE At-Q8UFG4 (82) PLDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQV—SDW M091_M4aEll (80) PLDLIVIMLGTNDIKPHHGRTAGE—AGRGMARLVQIIRGHYAG RMQ M1-RMLO00301 ( 92 ) PIDLIVIMLGANDMKPWIHGNPVA—AKQGIQRLIDIVRGHDYP—FDW GC821-2

P.dejongeii RVM04532 (83) PLDLVILMLGTNDLKSTFNVPPGE- -IAAGAGVLGRMILAGDA GPEN Q92XZ1 Sinorhizobium meliloti (86) PLDLIIIMLGTNDLKRRFNMPPSE- ^■-VAMGIGCLVHDIRELSP GRTG S261_M2aA12 (79) PLDLVIIMLGTNDIKFAARCRAFD- -ASMGMERLIQIVRSANYM KGY

Smal993 Sinorhizobium meliloti (96) PLDLVIIMLGTNDTKSYFHRTPYE- -IANGMGKLVGQVLTCAGG-VGTPY ZP_00197751 (79) PLDLVIVMLGTNDMKSFVCGRAIG- -AKQGMERIVQIIRGQPYS FNY ZP_00216984 (87) PVDVWLMLGTNDLKTRFSVTPAD- -IATSVGVLLAKIAACGA GPSG BAB16192 (90) ILDLVIIMLGTNDLKARFGQPPSE- -VAMGIGCLVYDIRELAP GPGG BAB16197 (85) PLDLVIILLGTNDTKSYFRRTPYE- -IANGMGKLAGQVLTSAGG-IGTPY NP_522806 (81) PLALVILMLGTNDFQAIFRHTAQD- -AAQGVAQLVRAIRQAPIEP GM Consensus (101) PLDLVIIMLGTNDLKA F TP D IA GMGRLV VR G ^' G Y 151 200 20A 130) PAPQVLIVAPPPLGELPHPWFDL— FSGGREKTAELARVYSALASFMKV 9B Natural Isolate 142) PAPQVLIVAPPPLAEMPHPWFEL—VFDGGREKTAQLARVYSALASFMKV M. parafortuitum COl 130) PAPQVLIVAPPPLGELPHPWFDL—VFSGGREKTAELARVYSALASFMKV MSAT 130) PAPKVLWSPPPIAPMPHPWFQL—IFEGGEQKTTEIARVYSAI-^FMKV Sm-RSM05666 125) EGPEILIVSPPPLCETANSAFAAMFAGGVEQSAMLAP— YRDLADELDC At-Q8UAC0 12 ) AVPKLLIVAPPPCVAGP—GGEPAGGRDIEQSMRLAP— YRKLAAELGH At-Q8UFG4 130) EAPDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDC M091_M4aEll 125) DEPQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKV M1-RMLO00301 13 ) PAPQILIVSPPVVSRTENADFREMFAGGDEASKQLAP--QYAALADEVGC P.dejongeii RVM04532 128) RPPQLLLMCPPKVRDLSAMPDLDAKIPHGAAR-SAEFPRHYKAQAVALKC Q92XZ1 Sinorhizobium meliloti 131) NDPEIMIVAPPPMLEDLKEWES IFSGAQEKSRKIALEFEIMADSLEA S261_M2aA12 12 ) KIPEILIISPPSLVPTQDEWFNDLWGHAIAESKLFAK—HYKRVAEELKV

Smal993 Sinorhizobium meliloti 143) PAPKVLWAPPPLAPMPDPWFEG—MFGGGYEKSKELSGLYKALADFMKV ZP_00197751 12 ) KVPSILLVAPPPLCATENSDFAEIFEGGMAESQKLAP—LYAALAQQTGC ZP_^00216984 132) ASPKLVLMAPAPIVEVGFLGEI FAGGAAK-SRQLAKRYEQVASDAGA BAB16192 135) KPPEIMWAPPPMLDDIKEWEP IFSGAQEKSRRLALEFEIIADSLEV BAB16197 132) PAPKLLIVSPPPLAPMPDPWFEG--MFGGGYEKSLELAKQYKALANFLKV NP_522806 126) PVPPVLIWPPAITAPAGAMADK FADAQPKCAGLAQAYRATAQTLGC Consensus 151) AP ILIVAPPPL E WF IFGGA KS LA YKALA LKV 201 248 20A 178 ) PFFDAGSVISTDGVDGTHFTRGETI (SEQ ID NO: 675) 9B Natural Isolate 190) PFFDAGSVISTDGVDGTHFTRGETIDR (SEQ ID NO: 676) M. parafortuitum COl 178) PFFDAGSVISTDGVDGIHFTRGEQST (SEQ ID NO: 677) MSAT 178) PFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL (SEQ ID NO: 678) Sm-RSM05666 173) GFFDGGSVARTTPIDGVHLDAENTRAVGRGLEPWRMMLGL (SEQ ID NO: 679) At-Q8UAC0 170) HFFDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG (SEQ ID N0:680) At-Q80FG4 180) GFFDAGSVARTTPVDGVHLDAENTRAIGRGLEPWRMMLGL (SEQ ID NO:681) M091_M4aEll 175) HFFDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL (SEQ ID NO: 682) M1-RMLO00301 185) GFFDAGTVAQTTPLDGVHLDAENTRNIGKALTSWRVML (SEQ ID NO: 683) P. ejongeii RVM04532 177) EYFNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG (SEQ ID NO: 684) Q92XZ1 Sinorhizobium meliloti 178) HFFDAGTVCQCSPADGFHIDEDAHRLLGEAAQEVLAIGWPDA (SEQ ID NO: 685) S261_M2aA12 172) HFFDAGTVAVADKTDGGHLDAVNTKAIGVALVPWKSILAL (SEQ ID NO:686)

Smal993 Sinorhizobium meliloti 191) EFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF (SEQ ID NO: 687 ZP 00197751 172) AFFDAGTVARTTPLDGIHLDAENTRAIGAGLEPWRQALGL (SEQ ID NO: 688) GC821-2

ZP_00216984 (178) HFLDAGAIVEVSPVDGVHFAADQHRVLGQRVAALLQQIA (SEQ ID NO: 689) BAB16192 (182) HFFDAATVASCDPCDGFHINREAHEALGTALAREVEAIGWR (SEQ ID NO: 690) BAB16197 (180) DFLDAGEFVKTDGCDGIHFSAETNITLGHAIA1UVEAIFSQEAKNAAA (SEQ ID NO: 691) NP_522806 (173) HVFDANSVTPASRVDGIHLDADQHAQLGRAMAQWGTLLAQ (SEQ ID NO:692) Consensus (201) FFDAGSV TSPVDGIHLDAENTR LG ALA VR IL (SEQ ID NO:693)

The guide tree to the CLUSTALW alignment (which approximates to a phylogenetic tree) clearly indicates 3 groupings: 1 ) GDSL - ARTT group including Act 2) GDSL - GRTT group composed of members of the Rhizobiales and the metagenome; and 3) Intermediate group of mixed motifs. It is also contemplated that the results suggest some form of gene duplication and mutation events in the Rhizobiales and lateral gene transfer to Mycobacterium.

GC821-2

Using the non-redundant alignment a new Act consensus was constructed called "Act chimera".

1 KTILCFGDSL T GWIPVEDG APTERRAPEV RWTGVLAQQL GADYEVIEEG 51 LSGRTTNIDD PTDPRLRNGA SYLPSCLASH LPLDLVIIML GTNDLKAYFR 101 RTPLDIALGM GRLVTQVRTS AGGVGTTYPA PKILIVAPPP LAEMPHPWFQ 151 LIFGGAEQKS TELARVYKAL ASFLKVPFFD AGSVISTSPV DGIHLDAENT 201 RDLGVALAEQ VRSIL (SEQ ID NO: 694)

An alignment of Act-chimera with Ms Act (Chimera align) indicates 91.6% similarity and 86.0% identity, as indicated below. GC821-2

1 50 MSAT ( 1) MAKRILCFGDSLTWG VPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIE

Act-Chimera ( 1) --KTILCFGDSLT G IPVEDGAPTERRAPEVR TGVLAQQLGADYEVIE Consensus (1) K ILCFGDSLT G IPVEDGAPTER APDVR TGVLAQQLGADFEVIE 51 100 MSAT (51) EGLSARTTNIDDPTDPRLN-GASYLPSCLATHLPLDLVIIMLGTNDTKAY

Act-Chimera (49) EGLSGRTTNIDDPTDPRLRNGASYLPSCLASHLPLDLVIIMLGTNDLKAY Consensus (51) EGLSARTTNIDDPTDPRL GAS LPSCLASHLPLDLVIIMLGTND KAY 101 150 MSAT { 100) FRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPW

Act-Chimera ( 99) FRRTPLDIALGMGRLVTQVRTSAGGVGTTYPAPKILIVAPPPLAEMPHP Consensus (101) FRRTPLDIALGM LVTQV TSAGGVGTTYPAPKILIVAPPPLA MPHP 151 200 MSAT ( 150 ) FQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEA

Act-Chimera (149) FQLIFGGAEQKSTELARVYKALASFLKVPFFDAGSVISTSPVDGIHLDAE Consensus (151) FQLIF GAEQKSTELARVY ALASFLKVPFFDAGSVIST VDGIH 201 217 MSAT (200) NNRDLGVALAEQVRSLL (SEQ ID NO: 695)

Act-Chimera (199) NTRDLGVALAEQVRSIL (SEQ ID NO: 694) Consensus (201) N RDLGVALAEQVRSIL (SEQ ID NO: 696)

A BLASTP search with Act-chimera did not reveal any further sequences. The Act-chimera is "forced" on the Per sequence at the positions where no consensus exists. However, a basic 'unforced' consensus sequence did not provide any more information from a blastp search or from alignment analysis. Thus, comparison with the most distant homologues in the blastp 'hit' list was considered more useful in defining the important residues/positions in Act sequence space. This was a useful exercise, as these sequences were not used in the non-redundant alignment. For example, Rhodopirellula baltica (NP_865748; Psp; a Planctomycetes and quite different from either Mycobacterium or Rhizobiales), was compared as shown below. GC821-2

1 50 MSAT (1) MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRTGVLA QQLGADFE

NP_865746 (1) -MHSILIYGDSLS GIIPGTR RRFAFHQR PGVMEIELRQTGIDAR

Consensus (1) IL FGDSLS G IP RFA RW GVL Q G D 51 100 MSAT (48) VIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTK NP_865746 (46) VIEDCLNGRRTVLEDPIKPGRNGLDGLQQRIEINSPLSLWLFLGTNDFQ Consensus (51) VIED L AR T IDDP P NG L I PL LVII LGTND 101 150 MSAT (98) AYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLWSPPPLAPMPH NP_865746 (96) SVHEFHAEQSAQGLALLV—DAIRRSPFEPGMPTPKILLVAPPTVHH-PK Consensus (101) A A GLALLV P PKILLVAPP L P 151 200 MSAT (148) PWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFT NP_865746 (143) LDMAAKFQNAETKSTGLADAIRKVSTEHSCEFFDAATVTTTSWDGVHLD Consensus (151) F AE KST LA LAS FFDAASV ST VDGIH 201 222 MSAT (198) EANNRDLGVALAEQVRSLL (SEQ ID NO: 695)

NP_865746 (193) QEQHQALGTALASTIAEILADC (SEQ ID NO: 697)

Consensus (201) N LG ALA I IL (SEQ ID NO: 698)

The following is an alignment with Ralstonia eutropha (Reu):

1 50 MSAT (1) MAKRILCFGDSLT GVPVEDGAPTERFAPDVR TGVLA—

ZP_00166901 (1) MPLTAPSEVDPLQILVYADSLS GIVPGTR RRLPFPVR PGRLELG Consensus (1) IL FADSLSWG VP R VR G L 51 100 MSAT (40) —QQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLV

ZP_00166901 (47) LNADGGAPVRIIEDCLNGRRTV DDPFKPGRNGLQGLAQRIEIHSPVALV Consensus (51) GA IIED L AR T DDP P NG L I H PL LV 101 150 MSAT (88) IIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLW

ZP_00166901 (97) VLMLGNNDFQSMHPHNA HAAQGVGALV—HAIRTAPIEPGMPVPPILVV Consensus (101) IIMLG ND A A GM LV A I P P ILVV GC821-2

151 200 MSAT (138) SPPPLAPMPHP FQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVIS ZP_00166901 (145) VPPPIRT-PCGPLAPKFAGGEHKWAGLPEALRELCATVDCSLFDAGTVIQ Consensus (151) PPPI P F GGE K L L A M FDAGSVI 201 237 MSAT (188) TDGVDGIHFTEANNRDLGVALAEQVRSLL (SEQ ID NO: 695)

ZP_00166901 (194) SSAVDGVHLDADAHVALGDALQPVVRALLAESSGHPS (SEQ ID N0:699) Consensus (201) S AVDGIH LG AL VRALL (SEQ ID NO: 700)

Based on these results, the following conclusions were made. A BLASTp nr- database search with a perhydrolase consensus sequence revealed GDSL or GDSI lipases/esterases from a wide diversity of organisms. However, only 12 or 14 of these were reliable homologues of Per. Nearly all of these were derived from 1 small group of bacteria, namely the Rhizobiales (i.e., Gram-negative soil bacteria belonging the alpha- Proteobacteria). A few members of the beta-Proteobacteria were found, but no Mycobacterium sp. This provides an indication that the perhydrolase (Per) gene/protein is not widely distributed in nature. The Mycobacterium protein is characterized by the GDSL-ARTT motif, whereas most of the Rhizobiales are characterized by a GDSL-GRTT motif. There are also some mixed or intermediate motifs (e.g., GDSN-GRTT, GDSN-ARTT and SDSL-GRTT). This may indicate gene duplication and mutation event and lateral gene transfer. The consensus residues identified in these experiments were L6, W14, R27, W34, L38, R56,

D62, L74, L78, H81, P83, M90, K97, GllO, L114, L135, F180, and G205. Using the non-redundant alignment and comparison with distant homologues the follow sequence space can be defined starting at position 5 of the M. smegmatis perhydrolase and ending at position 195, with perhydrolase shown in residues in bold.

[I, V][L][X][F, Y][G^MSIL_^N][T, S][W, Y, H][G][X] ₂[P, A][X] ₁₄[R, L][W] [X] ₇[L][X] _S[V, I][I, V, H][X][E. D][G, C][L, Q][X][C_Tj A][R][T][T][X] ₂[D, E][D] GC821-2

[X] ₇[G][X] ₃[L][X] ₆[H][X][P, I][L, I, N][D, A][V, I][X] ₂[M, L][L][G][X][Ν][D]

[X] 36[P][X] ₆[P][P, A][X] 3i[A][X] ₁₉[D][G][X][H] (SEQ ID NO:701)

In sum, it is clear from the analyses above that the active clones/sequences with a

GDSxi - x₂RTT - Gx₃ND motif have all been found among the alp a-Proteobacteria -

Gram-negative bacteria associated with the soil rhizosphere. This is in sharp contrast to the prototype perhydrolase from M. smegmatis - a high GC content Gram-positive bacterium assigned to the class Actinobacteria. This division is illustrated in Figure 2, which provides a phylogenetic tree, showing the major branches of the bacteria and the origin of the active clones/sequences compared to M. smegmatis. EXAMPLE 14 Native Molecular Weight Estimation of Homologues of the Perhydrolase In this Example, experiments conducted to estimate the native molecular weights of M. smegmatis perhydrolase homologues are described.

Preparation of Samples for Purification (Size Determination) A single colony of the desired strains was inoculated in 50ml Terrific Broth and incubated overnight at 37°C with shaking at 200 rpm. The cells were pelleted by centrifugation for 10 minutes at 7000 rpm in a Sorvall SuperSpeed Centrifuge. The pellets were then resuspended in 10 ml 25mM Bis-Tris (pH 6.5) and lysed by passage through a French pressure cell twice. The lysates were then centrifuged at 15000 rpm in a

Sorvall SuperSpeed Centrifuge. The soluble fraction was heat treated at 55°C for 1 hour to precipitate cellular proteins. The samples were then centrifuged at 10000 rpm in a Sorvall SuperSpeed Centrifuge and the soluble fractions used for further purification or assay. GC821-2

Sizing Columns The supernatants (prepared as described above) were run on a Sephadex 200 sizing column in 20 mM phosphate (pH 8.0), with a flow rate of 0.5 ml/min. The column was calibrated prior to running the samples with MW standards (listed below) and purified M. smegmatis perhydrolase protein. The crude sample elution volumes were determined by collecting 0.5 ml fractions, and assaying the fractions for pNB activity. Molecular weights and elution volumes of the standards: Thyroglobulin MW 669 kDa : elution volume 16ml Aldolase MW 158 kDa: elution volume 24 ml Ovalbumin MW 43 kDa: elution volume 26 ml Ribonuclease MW 14 kDa: elution volume 32 ml Perhydrolase elution volume 24 ml

Results The following Table (Table 14- 1 ) provides the elution volume of some of the smegmatis perhydrolase homologues identified herein.

GC821-2

The data in the above Table and the assay results obtained for these homologues indicated that these enzymes have an amino acid sequence similar to the M. smegmatis perhydrolase. As with the M. smegmatis perhydrolase, these homologues exhibit perhydrolysis activity as multimers. As described herein, the perhydrolase is an octamer, while the homologues, although they elute in a similar volume, are contemplated to be dimers, trimers, tetramers, hexamers, and/ or octamers.

EXAMPLE 15 Crystal Structure of Perhydrolase In this Example, the crystallographic analysis of the perhydrolase is described. Perhydrolase crystals were obtained under two conditions: 2.0 M [NH ]₂SO , 2% PEG400, 0.1 M Tris pH 7.1 (giving triclinic, PI crystals) and 1.0 M ammonium dihydrogen phosphate, and 0.1M sodium citrate pH 5.6 (giving tetragonal, P4 crystals) Both crystal forms gave suitable diffraction beyond 2.0A resolution. Derivative protein for a MAD phase determination using selenium replacing sulfur containing methionine resulting in a protein molecule having four selenomethionines the N-terminal methionine is cleaved proteolytically. Ofthe two forms, triclininc Pl a= 83.77A b=90.07A c= 112.115A α =73.32° β = 77.30° γ = 88.07° and P4 a=b=98.18A c= 230.12A, the P4 crystal gave data that was possible to use for structure determination. Three wavelength MAD datasets were collected at wavelengths corresponding to the Se absorption edge, near the inflection point and a third, away from the absorption edge. GC821-2

Three hundred and thirty-three frames (0.3 degree oscillations per frame) for each wavelength with 1 sec exposure time were collected from a single tetragonal space group P4 crystal. The structure could be solved with either SOLVE or SHELX computer programs giving similar solutions for the 32 possible Se positions. The map was fitted using the program "O". It was possible to trace electron density for residues 3-216 in each of the eight independent molecules. The final structure of these eight molecules was refined using CNS. The current crystallographic R-factor is 21%. The coordinates are provided below.

CRYSTl 98. 184 98. 184 230.: L19 90.00 90.0Ci 90.00

SCALE1 0.010185 0.000000 0.000000 0.000000

SCALE2 0.000000 0.010185 0.000000 0.000000

SCALE3 0.000000 0.000000 0.004346 0.000000

ATOM 1 CB LYS 3 -8.167 -61.964 18.588 1. ,000 40. .95

ATOM 2 CG LYS 3 -8.685 -63.192 19.323 1. .000 22. .95

ATOM 3 CD LYS 3 -8.635 -64.400 18.399 1. .000 14. .97

ATOM 4 CE LYS 3 -7.963 -65.575 19.090 1. .000 19. ,83

ATOM 5 NZ LYS 3 -7.359 -66.511 18.099 1. .000 44. ,28

ATOM . 6 C LYS 3 -9.684 -60.377 17.426 1. ,000 13. ,89

ATOM 7 O LYS 3 -9.087 -59.356 17.767 1. .000 12. ,50

ATOM 8 N LYS 3 -8.000 -61.626 16.153 1. .000 15. ,57

ATOM 9 CA LYS 3 -8.919 -61.686 17.284 1. ,000 20. ,71

ATOM 10 N ARG 4 -10.987 -60.381 17.166 1. ,000 24, .56

ATOM 11 CA ARG 4 -11.695 -59.097 17.204 1. ,000 22. .65

ATOM 12 CB ARG 4 -12.299 -58.822 15.822 1. .000 21. .44

ATOM 13 CG ARG 4 -11.232 -58.465 14.792 1. .000 21. .56

ATOM 14 CD ARG 4 -11.845 -58.181 13.431 1, .000 29. .29

ATOM 15 NE ARG 4 -11.660 -56.790 13.020 1. ,000 32. .87

ATOM 16 CZ ARG 4 -12.643 -56.013 12.585 1. .000 30. .24

ATOM 17 NH1 ARG 4 -13.879 -56.487 12.494 1. .000 17. .82

ATOM 18 NH2 ARG 4 -12.399 -54.760 12.229 1. .000 44, .53

ATOM 19 C ARG 4 -12.735 -59.054 18.308 1. .000 14. .59

ATOM 20 O ARG 4 -13.604 -59.909 18.456 1, .000 18, .72

ATOM 21 N ILE 5 -12.639 -58.012 19.131 1. .000 13, .45

ATOM 22 CA ILE 5 -13.549 -57.882 20.263 1, .000 12 .08

ATOM 23 CB ILE 5 -12.747 -57.835 21.578 1, .000 15 .40

ATOM 24 CG2 ILE 5 -13.678 -57.677 22.765 1 .000 5 .80

ATOM 25 CGI ILE 5 -11.811 -59.034 21.741 1 .000 11 .66

ATOM 26 CDl ILE 5 -10.437 -58.632 22.232 1 .000 19 .35

ATOM 27 C ILE 5 -14.420 -56.640 20.142 1 .000 8 .96 GC821-2

ATOM 28 O ILE 5 -13.905 -55.529 20.021 1.000 13. 31

ATOM 29 N LEU 6 -15.736 -56.833 20.169 1.000 13.04

ATOM 30 CA LEU 6 -16.675 -55.728 20.059 1.000 8.54

ATOM 31 CB LEU 6 -17.879 -56.087 19.178 1.000 7.42

ATOM 32 CG LEU 6 -18.959 -54.996 19.120 1.000 14.12

ATOM 33 CDl LEU 6 -18.446 -53.783 18.359 1.000 12.19

ATOM 34 CD2 LEU 6 -20.245 -55.512 18. .494 1. 000000 27.94

ATOM 35 C LEU 6 -17.170 -55.293 21. .436 1. 000000 2.72

ATOM 36 O LEU 6 -17.719 -56.101 22. .179 1. 000000 13.36

ATOM 37 N CYS 7 -16.978 -54.020 21. .756 1. 000000 1.38

ATOM 38 CA CYS 7 -17.472 -53.469 23. .011 1. 000 3.17

ATOM 39 CB CYS 7 -16.411 -52.582 23, .667 1. ,000 7.01

ATOM 40 SG CYS 7 -14.867 -53.471 23.992 1.000 11.21

ATOM 41 C CYS 7 -18.755 -52.685 22.776 1.000 0.65

ATOM 42 O CYS 7 -18.756 -51.627 22.145 1.000 4.76

ATOM 43 N PHE 8 -19.859 -53.228 23.281 ,000 00

ATOM 44 CA PHE 8 -21.147 -52.568 23.053 ,000 14

ATOM 45 CB PHE 8 -22.115 -53.578 22.443 ,000 54

ATOM 46 CG PHE 8 -23.421 -53.000 21.937 ,000 3, .36

ATOM 47 CDl PHE 8 -23.456 -52.212 20.800 1.000 0, .89

ATOM 48 CD2 PHE 8 -24.602 -53.262 22.614 000 1, .39

ATOM 49 CE1 PHE 8 -24.644 -51.683 20.333 000 0, .00

ATOM 50 CE2 PHE 8 -25.793 -52.733 22.148 000 4 .42

ATOM 51 CZ PHE 8 -25.818 -51.944 21.012 000 2 .71

ATOM 52 C PHE 8 -21.677 -51.978 24.346 000 4, .46

ATOM 53 O PHE 8 -21.873 -52.672 25.348 000 6, .98

ATOM 54 N GLY 9 -21.923 -50.666 24.384 000 5, .61

ATOM 55 CA GLY 9 -22.396 -50.109 25.646 000 5, .44

ATOM 56 C GLY 9 -22.860 -48.673 25.522 1.000 5.66

ATOM 57 O GLY 9 -23.229 -48.222 24.440 1.000 14.54

ATOM 58 N ASP 10 -22.837 -47.964 26.641 000 3.89

ATOM 59 CA ASP 10 -23.322 -46.596 26.734 000 5.17

ATOM 60 CB ASP 10 -24.331 -46.467 27.880 000 99

ATOM 61 CG ASP 10 -23.807 -47.052 29.175 000 05

ATOM 62 OD1 ASP 10 -22.617 -46.829 29.494 000 17.93

ATOM 63 OD2 ASP 10 -24.564 -47.738 29.895 000 10.98

ATOM 64 C ASP 10 -22.154 -45.642 26.939 000 5.15

ATOM 65 O ASP 10 -21.022 -45.940 26.556 000 5.62

ATOM 66 N SER 11 -22.423 -44.497 27.554 000 9.02

ATOM 67 CA SER 11 -21.394 -43.493 27.802 000 43

ATOM 68 CB SER 11 -22.014 -42.331 28.585 1.000 25

ATOM 69 OG SER 11 -22.640 -42.813 29.763 000 93

ATOM 70 C SER 11 -20.199 -44.046 28.561 000 58

ATOM 71 O SER 11 -19.089 -43.508 28.501 000 16.71

ATOM 72 N LEU 12 -20.393 -45.133 29.308 000 6.56

ATOM 73 CA LEU 12 -19.264 -45.696 30.046 1.000 16.41

ATOM 74 CB LEU 12 -19.711 -46.759 31.042 1.000 17.05 GC821-2

ATOM 75 CG LEU 12 20 598 -46 336 32 210 1 000 18 22

ATOM 76 CDl LEU 12 20 866 -47 527 33 123 1 000 7 48

ATOM 77 CD2 LEU 12 19 973 -45 184 32. 988 1. 000 10 83

ATOM 78 C LEU 12 18 269 -46 285 29 048 1 000 14 99

ATOM 79 O LEU 12 17 065 -46. 307 29. 267 1. 000 6 10

ATOM 80 N THR 13 18 828 -46 764 27 940 1 000 14 77

ATOM 81 CA THR 13 18 014 -47 347 26. 876 1. 000 8 83

ATOM 82 CB THR 13 18 828 -48 381 26 080 1 000 6 87

ATOM 83 OG1 THR 13 19 109 -49 487 26 949 1 000 10 08

ATOM 84 CG2 THR 13 18 033 -48 940 24 914 1 000 16 85

ATOM 85 C THR 13 17 490 -46 245 25 970 1 000 4 56

ATOM 86 O THR 13' 16 315 -46 220 25 616 1 000 11 71

ATOM 87 N TRP 14 18 376 -45 317 25 612 1 000 5 57

ATOM 88 CA , TRP 14 17 992 -44 210 24 742 1 000 7 21

ATOM 89 CB TRP 14 19 208 -43 329 24 453 1 000 6 90

ATOM 90 CG TRP 14 18 917 -42 183 23 537 1 000 11 88

ATOM 91 CD2 TRP 14 18 731 -40 813 23 924 1 000 13 72

ATOM 92 CE2 TRP 14 18 483 -40 081 22 745 1 000 11 95

ATOM 93 CE3 TRP 14 18 752 -40 147 25 152 1 000 10 63

ATOM 94 CDl TRP 14 18 779 -42 222 22 181 1 000 8 28

ATOM 95 NE1 TRP 14 18 517 -40 963 21 694 1 000 7 16

ATOM 96 CZ2 TRP 14 18 255 -38 705 22 763 1 000 5 39

ATOM 97 CZ3 TRP 14 18 526 -38 783 25 168 1 000 12 55

ATOM 98 CH2 TRP 14 18 282 -38 084 23 981 1 000 12 81

ATOM 99 C TRP 14 16 880 -43 353 25 327 1 000 5 41

ATOM 100 O^' TRP 14 16 107 -42 745 24 582 1 000 4 90

ATOM 101 N GLY 15 16 794 -43 283 26 652 1 000 8 94

ATOM 102 CA GLY 15 15 794 -42 475 27 318 1 000 4 51

ATOM 103 C GLY 15 16 249 -41 098 27 755 1 000 10 98

ATOM 104 O GLY 15 15 480 -40 136 27 646 1 000 15 11

ATOM 105 N TRP 16 17 471 -40 952 28 255 1 000 23 34

ATOM 106 CA TRP 16 17 988 -39 691 28 792 1 000 15 10

ATOM 107 CB TRP 16 19 408 -39 890 29 327 1 000 6 11

ATOM 108 CG TRP 16 20 139 -38 694 29 846 1 000 1 78

ATOM 109 CD2 TRP 16 21 229 -38 008 29 213 1 000 8 98

ATOM 110 CE2 TRP 16 21 613 -36 942 30 051 1 000 7 76

ATOM 111 CE3 TRP 16 21 923 -38 186 28 009 1 000 15 66

ATOM 112 CDl TRP 16 19 927 -38 021 31 016 1 000 0 35

ATOM 113 NEl TRP 16 20 798 -36 973 31 154 1 000 8 35

ATOM 114 CZ2 TRP 16 22 649 -36 063 29 734 1 000 5 16

ATOM 115 CZ3 TRP 16 22 952 -37 .317 27 692 1 000 5 34

ATOM 116 CH2 TRP 16 23 306 -36 269 28 551 1 000 4 72

ATOM 117 C TRP 16 17 .059 -39 .154 29 881 1 000 7 .85

ATOM 118 O TRP 16 16 .846 -39 .815 30 .899 1 .000 3 .97

ATOM 119 N VAL 17 16 .533 -37 .952 29 685 1 .000 5 .45

ATOM 120 CA VAL 17 15 .750 -37 .256 30 .695 1 .000 12 .08

ATOM 121 CB VAL 17 14 .822 -36 .191 30 .082 1 .000 17 .55 GC821-2

ATOM 122 CGI VAL 17 14 084 -35 443 31.185 ,000 11.59

ATOM 123 CG2 VAL 17 13 841 -36 807 29.099 ,000 7.77

ATOM 124 C VAL 17 16 673 -36 565 31.696 ,000 13.86

ATOM 125 O VAL 17 17 390 -35 618 31.351 ,000 1.02

ATOM 126 N PRO 18 16 660 -37 034 32.936 .000 8.38

ATOM 127 CD PRO 18 15 770 -38 071 33.476 .000 8.64

ATOM 128 CA PRO 18 17 572 -36 501 33.948 .000 9.99

ATOM 129 CB PRO 18 17 201 -37 294 35.208 .000 12.31

ATOM 130 CG PRO 18 15 817 -37 789 34.954 ,000 7.46

ATOM 131 C PRO 18 17 327 -35 017 34.191 1.000 13.05

ATOM 132 O PRO 18 16 163 -34 619 34.306 1.000 18.63

ATOM 133 N VAL 19 18 381 -34 211 34.266 000 6.92

ATOM 134 CA VAL 19 18 214 -32 793 34.585 000 9.29

ATOM 135 CB VAL 19 18 482 -31 856 33.388 000 5.33

ATOM 136 CGI VAL 19 17 377 -31 995 32.354 000 6.78

ATOM 137 CG2 VAL 19 19 850 -32 150 32.796 000 3.72

ATOM 138 C VAL 19 19 151 -32 380 35.710 000 12.02

ATOM 139 O VAL 19 20 217 -32 962 35.913 000 14.52

ATOM 140 N GLU 20 18 771 -31 351 36.467 000 17.17

ATOM 141 CA GLU 20 19 662 -30 994 37.575 000 13.30

ATOM 142 CB GLU 20 18 918 -30 130 38.595 000 25.34

ATOM 143 CG GLU 20 18 276 -30 968 39.702 000 31.46

ATOM 144 CD GLU 20 16 871 -30 487 40.017 000 35.91

ATOM 145 OEl GLU 20 16 143 -30 157 39.055 000 40.11

ATOM 146 OE2 GLU 20 16 507 -30 431 41.210 000 45.47

ATOM 147 C GLU 20 20 913 -30 294 37.080 000 7.56

ATOM 148 O GLU 20 21 964 -30 361 37.723 000 11.30

ATOM 149 N ASP 21 20 852 -29 610 35.936 000 19.38

ATOM 150 CA ASP 21 22 099 -28 983 35.471 000 23.47

ATOM 151 CB ASP 21 21 815 -27 740 34.640 000 17.53

ATOM 152 CG ASP 21 21 114 -27 991 33.326 000 14.93

ATOM 153 OD1 ASP 21 20 984 -29 159 32.908 1.000 26.78

ATOM 154 OD2 ASP 21 20 685 -26 996 32.694 1.000 8.74

ATOM 155 C ASP 21 22 959 -29 988 34.707 1.000 19.54

ATOM 156 O ASP 21 23 988 -29 627 34.131 1.000 22.49

ATOM 157 N GLY 22 22 550 -31 250 34.697 1.000 13.19

ATOM 158 CA GLY 22 23 279 -32 377 34.166 1.000 15.71

ATOM 159 C GLY 22 23 507 -32 377 32.659 1.000 20.02

ATOM 160 O GLY 22 23 370 -33 431 32.036 1.000 23.32

ATOM 161 N ALA 23 23 846 -31 235 32.138 1.000 26.40

ATOM 162 CA ALA 23 24 265 -30 672 30.873 1.000 28.79

ATOM 163 CB ALA 23 24 483 -29 192 31.152 1.000 32.86

ATOM 164 C ALA 23 23 309 -30 988 29.745 1.000 22.68

ATOM 165 O ALA 23 22 922 -32 189 29.753 1.000 40.02

ATOM 166 N PRO 24 22 847 -30 255 28.748 1.000 12.97

ATOM 167 CD PRO 24 22 .892 -28 .855 28.309 1.000 15.92

ATOM 168 CA PRO 24 22 .051 -31 .028 27.767 1.000 5.31 GC821-2

ATOM 169 CB PRO 24 22 024 -30 134 26. 520 1.000 4.03

ATOM 170 CG PRO 24 22 002 -28 762 27 105 1.000 6.80

ATOM 171 C PRO 24 20 622 -31 273 28 222 1.000 14.45

ATOM 172 O PRO 24 20 034 -30 591 29 056 1.000 19.65

ATOM 173 N THR 25 20 062 -32 310 27 600 1.000 13.21

ATOM 174 CA THR 25 18 685 -32 690 27 894 1.000 11.82

ATOM 175 CB THR 25 18 691 -33 772 28 987 1.000 12.19

ATOM 176 OG1 THR 25 17 348 -34 104 29 355 1.000 19.38

ATOM 177 CG2 THR 25 19 372 -35 027 28 454 1.000 0.00

ATOM 178 C THR 25 18 009 -33 160 26 620 1.000 14.10

ATOM 179 O THR 25 18 555 -33 019 25 518 1.000 16.46

ATOM 180 N GLU 26 16 818 -33 724 26 762 1.000 12.30

ATOM 181 CA GLU 26 16 157 -34 314 25 598 1.000 13.24

ATOM 182 CB GLU 26 14 909 -33 518 25 225 1.000 15.75

ATOM 183 CG GLU 26 15 211 -32 066 24 873 1.000 25.45

ATOM 184 CD GLU 26 15 451 -31 152 26 056 1.000 27.41

ATOM 185 OEl GLU 26 14 687 -31 210 27 048 1.000 22.86

ATOM 186 OE2 GLU 26 16 416 -30 347 26 012 1.000 17.32

ATOM 187 C GLU 26 15 850 -35 775 25 891 1.000 8.80

ATOM ^• 188 0 GLU 26 16 279 -36 316 26 909 1.000 2.55

ATOM 189 N ARG 27 15 121 -36 421 25 001 1.000 13.28

ATOM 190 CA ARG 27 14 783 -37 838 25 124 1.000 12.71

ATOM 191 CB ARG 27 14 857 -38 447 23 726 1.000 6.07

ATOM 192 CG ARG 27 14 491 -39 908 23 585 1.000 4.38

ATOM 193 CD ARG 27 14 879 -40 387 22 186 1.000 11.29

ATOM 194 NE ARG 27 14 974 -41 840 22 110 1.000 13.10

ATOM 195 CZ ARG 27 15 191 -42 517 20 992 1.000 9.74

ATOM 196 NH1 ARG 27 15 337 -41 868 19 842 1.000 11.38

ATOM 197 NH2 ARG 27 15 262 -43 839 21 029 1.000 0.00

ATOM 198 C ARG 27 13 413 -38 031 25 746 1.000 8.79

ATOM 199 O ARG 27 12 534 -37 181 25 579 1.000 17.59

ATOM 200 N PHE 28 13 183 -39 133 26 461 1.000 12.29

ATOM 201 CA PHE 28 11 826 -39 379 26 955 1.000 9.91

ATOM 202 CB PHE 28 11 783 -40 575 27 900 1.000 10.13

ATOM 203 CG PHE 28 12 084 -40 263 29 355 1.000 11.54

ATOM 204 CDl PHE 28 11 250 -39 431 30 084 1.000 8.88

ATOM 205 CD2 PHE 28 13 194 -40 802 29 979 1.000 11.27

ATOM 206 CE1 PHE 28 11 535 -39 156 31 408 1.000 8.90

ATOM 207 CE2 PHE 28 13 486 -40 533 31 305 1.000 5.41

ATOM 208 CZ PHE 28 12 647 -39 703 32 020 1.000 0.61

ATOM 209 C PHE 28 10 901 -39 635 25 770 1.000 11.56

ATOM 210 O PHE 28 11 .370 -40 .112 24 736 1.000 13.14

ATOM 211 N ALA 29 -9 .612 -39 .349 25 896 1.000 13.02

ATOM 212 CA ALA 29 -8 .674 -39 .656 24 .818 1.000 13.91

ATOM 213 CB ALA 29 -7 .275 -39 .163 25 .151 1.000 6.49

ATOM 214 C ALA 29 -8 .662 -41 .157 24 .545 1.000 15.68

ATOM 215 O ALA 29 -8 .937 -41 .954 25 .446 1.000 31.74 GC821-2

ATOM 216 N PRO 30 -8.345 -41. ,537 23. ,314 1.000 11.44

ATOM 217 CD PRO 30 -7.982 -40. ,660 22. ,192 1.000 12.10

ATOM 218 CA PRO 30 -8.326 -42. ,955 22. ,936 1.000 18.85

ATOM 219 CB PRO 30 -7.822 -42. ,956 21. ,494 1.000 16.38

ATOM 220 CG PRO 30 -7.283 -41. .593 21. ,244 1.000 14.74

ATOM 221 C PRO 30 -7.386 -43. .767 23. ,826 1.000 13.40

ATOM 222 O PRO 30 -7.570 -44. ,969 23. ,979 1.000 8.18

ATOM 223 N ASP 31 -6.396 -43. ,115 24. ,412 1.000 22.50

ATOM 224 CA ASP 31 -5.426 -43. ,715 25. ,312 1.000 26.63

ATOM 225 CB ASP 31 -4.170 -42. ,841 25. .398 1.000 30.41

ATOM 226 CG ASP 31 -3.792 -42. ,143 24. ,108 1.000 39.21

ATOM 227 OD1 ASP 31 -2.577 -42. ,086 23, .802 1.000 39.00

ATOM 228 OD2 ASP 31 -4.673 -41. ,634 23. .375 1.000 37.50

ATOM 229 C ASP 31 -5.985 -43. .926 26, .721 1.000 17.49

ATOM 230 O ASP 31 -5.482 -44. .784 27. ,450 1.000 25.27

ATOM 231 N VAL 32 -6.989 -43. .150 27. .092 1.000 14.45

ATOM 232 CA VAL 32 -7.592 -43. .125 28, .421 1.000 12.64

ATOM 233 CB VAL 32 -7.966 -41. .683 28. .814 1.000 10.68

ATOM 234 CGI VAL 32 -8.580 -41. .609 30, .199 1.000 13.66

ATOM 235 CG2 VAL 32 -6.742 -40. .774 28. .752 1.000 20.51

ATOM 236 C VAL 32 -8.808 -44. .042 28. .507 1.000 9.73

ATOM 237 O VAL 32 -8.890 -44. .834 29. .452 1.000 2.23

ATOM 238 N ARG 33 -9.722 -43, .964 27, .553 1.000 10.63

ATOM 239 CA ARG 33 ^•10.888 -44. .824 27. .410 1.000 6.85

ATOM 240 CB ARG 33 ^■11.369 -44. .833 25, .961 1.000 16.41

ATOM 241 CG ARG 33 ^■12.281 -43. .727 25, .488 1.000 21.19

ATOM 242 CD ARG 33 ^■12.464 -43. .806 23, .974 1.000 26.66

ATOM 243 NE ARG 33 11.862 -42. .659 23. .309 1.000 30.35

ATOM 244 CZ ARG 33 ^•11.493 -42. .567 22, .044 1.000 31.60

ATOM 245 NH1 ARG 33 ^•11.658 -43. .585 21, .214 1.000 34.85

ATOM 246 NH2 ARG 33 ^■10.952 -41. .433 21, .610 1.000 52.70

ATOM 247 C ARG 33 ^■10.600 -46, .279 27, .775 1.000 9.71

ATOM 248 O ARG 33 -9.603 -46, .830 27, .300 1.000 16.85

ATOM 249 N TRP 34 ^■11.450 -46, .924 28, .577 1.000 10.64

ATOM 250 CA TRP 34 ^■11.166 -48, .311 28, .952 1.000 6.46

ATOM 251 CB TRP 34 ^■12.149 -48, .855 29 .979 1.000 12.45

ATOM 252 CG TRP 34 ^■13.561 -49, .106 29, .583 1.000 6.95

ATOM 253 CD2 TRP 34 ^•14.104 -50, .199 28 .835 1.000 9.27

ATOM 254 CE2 TRP 34 ^■15.493 -49, .986 28 .723 1.000 5.43

ATOM 255 CE3 TRP 34 ^•13.571 -51 .345 28 .240 1.000 14.72

ATOM 256 CDl TRP 34 ^■14.622 -48, .298 29 .888 1.000 4.49

ATOM 257 NE1 TRP 34 ^■15.786 -48 .820 29 .374 1.000 4.03

ATOM 258 CZ2 TRP 34 ^■16.337 -50 .864 28 .050 1.000 8.19

ATOM 259 CZ3 TRP 34 ^■14.405 -52 .216 27 .572 1.000 12.73

ATOM 260 CH2 TRP 34 -15.778 -51 .976 27 .479 1.000 8.32

ATOM 261 C TRP 34 ^■11.111 -49 .214 27 .723 1.000 7.27

ATOM 262 O TRP 34 -10.393 -50 .222 27 .767 1.000 11.53 GC821-2

ATOM 263 N THR 35 11.839 -48. ,887 26.659 1.000 1.15

ATOM 264 CA THR 35 11.730 -49. ,673 25.431 1.000 5.29

ATOM 265 CB THR 35 12.708 -49. ,239 24.331 1.000 3.10

ATOM 266 OG1 THR 35 12.629 -47. ,820 24.163 1.000 15.85

ATOM 267 CG2 THR 35 14.146 -49. .549 24.726 1.000 5.16

ATOM 268 C THR 35 10.307 -49. ,555 24.882 1.000 14.32

ATOM 269 O THR 35 -9.738 -50. ,494 24.333 1.000 12.77

ATOM 270 N GLY 36 -9.756 -48. ,361 25.060 1.000 15.72

ATOM 271 CA GLY 36 -8.392 -48. ,056 24.689 1.000 15.87

ATOM 272 C GLY 36 -7.407 -48. .785 25.583 1.000 14.86

ATOM 273 O GLY 36 -6.374 -49. ,252 25.101 1.000 22.97

ATOM 274 N VAL 37 -7.686 -48. .905 26.884 1.000 12.48

ATOM 275 CA VAL 37 -6.696 -49. .577 27.728 1.000 11.76

ATOM 276 CB VAL 37 -6.921 -49. ,365 29.229 1.000 10.95

ATOM 277 CGI VAL 37 -6.092 -50. .382 30.009 1.000 0.00

ATOM 278 CG2 VAL 37 -6.577 -47. .940 29.630 1.000 10.31

ATOM 279 C VAL 37 -6.707 -51. .081 27.471 1.000 16.75

ATOM 280 O VAL 37 -5.669 -51. .735 27.494 1.000 14.29

ATOM 281 N LEU 38 -7.911 -51. .586 27.238 1.000 14.60

ATOM 282 CA LEU 38 -8.094 -52. .999 26.917 1.000 11.25

ATOM 283 CB LEU 38 -9.573 -53. .266 26.660 1.000 12.92

ATOM 284 CG LEU 38 -9.975 -54. .663 26.198 1.000 15.77

ATOM 285 CDl LEU 38 -9.747 -55. .691 27.293 1.000 0.00

ATOM 286 CD2 LEU 38 11.425 -54. .677 25.733 1.000 24.28

ATOM 287 C LEU 38 -7.224 -53. .347 25.720 1.000 7.67

ATOM 288 O LEU 38 -6.408 -54. .262 25.740 1.000 13.04

ATOM 289 N ALA 39 -7.404 -52. .568 24.659 1.000 9.64

ATOM 290 CA^' ALA 39 -6.603 -52. ,667 23.451 1.000 3.53

ATOM 291 CB ALA 39 -6.894 -51. .487 22.530 1.000 6.32

ATOM 292 C ALA 39 -5.112 -52. .704 23.761 1.000 9.32

ATOM 293 O ALA 39 -4.411 -53. ,632 23.367 1.000 28.59

ATOM 294 N GLN 40 -4.653 -51. .665 24.456 1.000 21.51

ATOM 295 CA GLN 40 -3.251 -51. .553 24.833 1.000 18.93

ATOM 296 CB GLN 40 -2.974 -50, .365 25.744 1.000 28.00

ATOM 297 CG GLN 40 -3.597 -49. .034 25.378 1.000 37.51

ATOM 298 CD GLN 0 -3.070 -47. .877 26.214 1.000 40.85

ATOM 299 OEl GLN 40 -1.998 -47, .335 25.933 1.000 61.34

ATOM 300 NE2 GLN 40 -3.809 -47, .475 27.248 1.000 9.83

ATOM 301 C GLN 40 -2.822 -52, .851 25.525 1.000 10.96

ATOM 302 O GLN 40 -1.856 -53, .475 25.106 1.000 18.66

ATOM 303 N GLN 41 -3.563 -53, .239 26.552 1.000 15.02

ATOM 304 CA GLN 41 -3.253 -54, .423 27.337 1.000 22.27

ATOM 305 CB GLN 41 -4.258 -54, .582 28.484 1.000 16.69

ATOM 306 CG GLN 41 -4.064 -53 .605 29.624 1.000 14.55

ATOM 307 CD GLN 41 -2.788 -53, .852 30.406 1.000 16.86

ATOM 308 OEl GLN 41 -2.759 -54 .650 31.344 1.000 13.75

ATOM 309 NE2 GLN 41 -1.731 -53 .158 30.008 1.000 21.79 GC821-2

ATOM 310 C GLN 41 -3.261 -55, .694 26, .493 1.000 28. .40

ATOM 311 O GLN 41 -2.442 -56. .589 26, .703 1.000 26. ,71

ATOM 312 N LEU 42 -4.190 -55, .776 25, .546 1.000 28. .62

ATOM 313 CA LEU 42 -4.373 -57. .007 24. ,780 1.000 26. ,50

ATOM 314 CB LEU 42 -5.707 -56, .920 24, .012 1.000 19. .31

ATOM 315 CG LEU 42 -6.934 -57. .122 24. .914 1.000 16. ,32

ATOM 316 CDl LEU 42 -8.226 -57, .077 24, .119 1.000 10. .94

ATOM 317 CD2 LEU 42 -6.810 -58, .438 25. .673 1.000 15. ,03

ATOM 318 C LEU 42 -3.217 -57, .312 23, .846 1.000 23. .29

ATOM 319 O LEU 42 -2.770 -58. .457 23, .728 1.000 20. ,82

ATOM 320 N GLY 43 -2.693 -56, .312 23, .141 1.000 22, .18

ATOM 321 CA GLY 43 -1.605 -56. .590 22, .215 1.000 18. .95

ATOM 322 C GLY 43 -2.086 -56, .793 20, .791 1.000 23, .97

ATOM 323 O GLY 43 -3.284 -56, .838 20, .514 1.000 27, .50

ATOM 324 N ALA 44 -1.136 -56, .927 19, .879 1.000 22, .72

ATOM 325 CA ALA 44 -1.317 -57, .012 18, .448 1.000 24, ,25

ATOM 326 CB ALA 44 0.048 -56, .939 17, .755 1.000 13, .44

ATOM 327 C ALA 44 -2.034 -58, .272 17, .990 1.000 23. .83

ATOM • 328 O ALA 44 -2.146 -58, .520 16, .787 1.000 17. .77

ATOM 329 N ASP 45 -2.524 -59. .086 18, .917 1.000 21. ,59

ATOM 330 CA ASP 45 -3.230 -60, ,298 18, .495 1.000 17. .80

ATOM ^■ 331 CB ASP 45 -2.705 -61. .491 19. .296 1.000 18. .22

ATOM 332 CG ASP 45 -1.201 -61. .625 19, .113 1.000 24, .69

ATOM 333 OD1 ASP 45 -0.710 -61. ,174 18, .053 1.000 34. .10

ATOM 334 OD2 ASP 45 -0.517 -62. .159 20, .007 1.000 33, .14

ATOM 335 C ASP 45 -4.732 -60. ,107 18. .647 1.000 11. ,82

ATOM 336 O ASP 45 -5.535 -60. .992 18, .364 1.000 23. ,89

ATOM 337 N PHE 46 -5.097 -58. ,914 19. .097 1.000 9. ,27

ATOM 338 CA PHE 46 -6.485 -58. .519 19. .253 1.000 12. ,25

ATOM 339 CB PHE 46 -6.909 -58. ,479 20. ,722 1.000 14. ,52

ATOM 340 CG PHE 46 -6.474 -59. .693 21. .529 1.000 11. ,99

ATOM 341 CDl PHE 46 -5.160 -59. ,814 21. ,956 1.000 12. ,17

ATOM 342 CD2 PHE 46 -7.383 -60. .690 21. .846 1.000 8. ,34

ATOM 343 CE1 PHE 46 -4.760 -60. ,917 22. .683 1.000 13. ,46

ATOM 344 CE2 PHE 46 -6.990 -61. .794 22. .575 1.000 6. ,30

ATOM 345 CZ PHE 46 -5.680 -61. ,904 22. ,998 1.000 8. ,44

ATOM 346 C PHE 46 -6.725 -57. .149 18, .615 1.000 13. ,30

ATOM 347 O PHE 46 -5.816 -56. ,366 18. ,366 1.000 27. ,22

ATOM 348 N GLU 47 -7.992 -56, .883 18, .349 1.000 12. ,78

ATOM 349 CA GLU 47 -8.469 -55, .616 17, ,833 1.000 9. ,15

ATOM 350 CB GLU 47 -8.667 -55, .644 16, .325 1.000 11, .20

ATOM 351 CG GLU 47 -8.791 -54, .276 15, .670 1.000 21, .84

ATOM 352 CD GLU 47 -9.726 -54, .293 14, .474 1.000 25, .88

ATOM 353 OEl GLU 47 -9.575 -55, .205 13, .632 1.000 30, .74

ATOM 354 OE2 GLU 47 ^•10.602 -53, .408 14, .388 1.000 7, .59

ATOM 355 C GLU 47 -9.781 -55, .280 18, .550 1.000 11. .37

ATOM 356 O GLU 47 ^•10.722 -56, .071 18 .545 1.000 11, .73 GC821-2

ATOM 357 N VAL 48 -9.775 -54 . 103 19. 160 1.000 10.53

ATOM 358 CA VAL 48 -10. 954 -53. 604 19.843 1.000 8. 11

ATOM 359 CB VAL 48 -10.595 -52.826 21.115 1.000 9.71

ATOM 360 CGI VAL 48 -11. 842 -52.251 21.773 000 15.31

ATOM 361 CG2 VAL 48 -9.849 -53.732 22.085 000 7.41

ATOM 362 C VAL 48 -11.745 -52.714 18.882 000 12.72

ATOM 363 O VAL 48 -11.147 -51.879 18.203 000 10.16

ATOM 364 N ILE 49 -13.046 -52. 943 18.862 000 13.04

ATOM 365 CA ILE 49 -14.031 -52. 170 18. 122 1. 000 14 . 10

ATOM 366 CB ILE 49 -14 . 879 -53.068 17.203 1. 000 16.77

ATOM 367 CG2 ILE 49 -15.735 -52.214 16.285 000 1.57

ATOM 368 CGI ILE 49 -14.049 -54.081 16.415 000 18.10

ATOM 369 CDl ILE 49 -14.687 -54.559 15.133 000 14.33

ATOM 370 C ILE 49 -14.930 -51.406 19.091 000 9. ,02

ATOM 371 O ILE 49 -15.531 -52.013 19.983 000 15.82

ATOM 372 N GLU 50 -15.000 -50.085 18.932 000 5.34

ATOM 373 CA GLU 50 -15.730 -49 ,277 19.911 1.000 12.03

ATOM 374 CB GLU 50 -14.967 -47 ,984 20.222 1.000 10.36

ATOM 375 CG GLU 50 13 623 -48 203 20 889 1 000 7 32

ATOM 376 CD GLU 50 12 768 -46 966 21 056 1 000 7 06

ATOM 377 OEl GLU 50 12 744 -46 077 20 177 1 000 5 78

ATOM 378 OE2 GLU 50 12 079 -46 870 22 101 1 000 25 19

ATOM 379 C GLU 50 17 145 -48 962 19 446 1 000 6 79

ATOM 380 0 GLU 50 17 358 -48 318 18 423 1 000 8 80

ATOM 381 N GLU 51 18 118 -49 429 20 225 1 000 9 34

ATOM 382 CA GLU 51 19 524 -49 179 19 924 1 000 16 23

ATOM 383 CB GLU 51 20 173 -50 400 19 270 1 000 15 22

ATOM 384 CG GLU 51 19 757 -50 596 17 820 1 000 18 39

ATOM 385 CD GLU 51 20 348 -49 531 16 917 1 000 17 99

ATOM 386 OEl GLU 51 21 352 -48 912 17 332 1 000 26 29

ATOM 387 OE2 GLU 51 19 820 -49 309 15 809 1 000 15 93

ATOM 388 C GLU 51 20 295 -48 788 21 184 1 000 10 51

ATOM 389 O GLU 51 21 202 -49 495 21 623 1 000 7 29

ATOM 390 N GLY 52 19 906 -47 655 21 751 1 000 5 90

ATOM 391 CA GLY 52 20 533 -47 140 22 961 1 000 3 93

ATOM 392 C GLY 52 21 329 -45 887 22 635 1 000 6 21

ATOM 393 O GLY 52 20 785 -44 950 22 057 1 000 16 40

ATOM 394 N LEU 53 22 607 -45 890 22 989 1 000 11 68

ATOM 395 CA LEU 53 23 498 -44 764 22 710 1 000 7 60

ATOM 396 CB LEU 53 24 627 -45 195 21 792 1 000 4 45

ATOM 397 CG LEU 53 25 576 -44 164 21 185 1 000 3 84

ATOM 398 CDl LEU 53 26 721 -43 872 22 141 1 000 15 09

ATOM 399 CD2 LEU 53 24 856 -42 874 20 817 1 .000 3 .41

ATOM 400 C LEU 53 24 035 -44 204 24 023 1 .000 5 .05

ATOM 401 O LEU 53 24 664 -44 920 24 801 1 .000 5 .74

ATOM 402 N SER 54 23 .771 -42 .918 24 .251 1 .000 9 .85

ATOM 403 CA SER 54 -24.192 -42.296 25.502 1.000 10.24 GC821-2

ATOM 404 CB SER 54 -23.797 -40.819 25.524 1.000 7.63

ATOM 405 OG SER 54 -22.395 -40.683 25.640 1.000 4.65

ATOM 406 C SER 54 -25.695 -42.448 25.691 1.000 7.74

ATOM 407 O SER 54 -26.438 -42.326 24.717 1.000 10.39

ATOM 408 N ALA^' 55 -26.127 -42.713 26.920 1.000 0.00

ATOM 409 CA ALA 55 -27.554 -42.749 27.218 1.000 00..0000

ATOM 410 CB ALA 55 -28.209 -41.474 26, .713 1. ,000 0.00

ATOM 411 C ALA 55 -28.235 -43.982 26, .640 1. ,000 6.11

ATOM 412 O ALA 55 -29.442 -44.179 26, .816 1. ,000 2.57

ATOM 413 N ARG 56 -27.474 -44.843 25, .971 1. .000 8.50

ATOM 414 CA ARG 56 -27.997 -46.084 25, .433 1. ,000 5.94

ATOM 415 CB ARG 56 -26.919 -46.868 24, .672 1. ,000 0.00

ATOM 416 CG .ARG 56 -27.420 -48.244 24. .247 1. ,000 2.73

ATOM 417 CD ARG 56 -26.467 -48.951 23, ,307 1. ,000 0.00

ATOM 418 NE ARG 56 -26.552 -48.440 21, .935 1. .000 6.44

ATOM 419 CZ ARG 56 -25.465 -48.325 21.170 1.000 11.18

ATOM 420 NH1 ARG 56 -24.283 -48.678 21.666 1.000 0.00

ATOM 421 NH2 ARG 56 -25.549 -47.861 19.928 1.000 1.13

ATOM 422 C ARG 56 -28.539 -47.009 26.526 1.000 12.43

ATOM 423 O ARG 56 -27.886 -47.179 27.556 1.000 10.16

ATOM 424 N THR 57 -29.697 -47.592 26.262 1.000 9.24

ATOM 425 CA THR 57 -30.376 -48.548 27.120 1.000 9.36

ATOM 426 CB THR 57 -31.855 -48.161 27.315 1.000 4.78

ATOM 427 OG1 THR 57 -32.608 -48.509 26.146 1.000 3.70

ATOM 428 CG2 THR 57 -31.992 -46.656 27.484 0Q0 0.00

ATOM 429 C THR 57 -30.284 -49.953 26.532 000 10.18

ATOM 430 O THR 57 -29.873 -50.099 25.378 000 12.60

ATOM 431 N THR 58 -30.648 -50.987 27.286 000 5.87

ATOM 432 CA THR 58 -30.574 -52.349 26.769 000 65

ATOM 433 CB THR 58 -30.850 -53.410 27.853 000 35

ATOM 434 OG1 THR 58 -32.151 -53.196 28.413 000 12.48

ATOM 435 CG2 THR 58 -29.859 -53.311 29. 002 1 . 000 11. 47

ATOM 436 C THR 58 -31.556 -52.569 25, .624 1, .000 1, .31

ATOM 437 O THR 58 -31.162 -52.902 24, .506 1, .000 7. .78

ATOM 438 N ASN 59 -32.856 -52 404 25. ,867 1. .000 4. .91

ATOM 439 CA ASN 59 -33.810 -52.604 24, .772 1, .000 11, .25

ATOM 440 CB ASN 59 -34.150 -54.090 24, .624 1, .000000 99.. .1199

ATOM 441 CG ASN 59 -35.186 -54.548 25. .629 1. .000000 99.. .5500

ATOM 442 OD1 ASN 59 -35.293 -54.000 26. .725 1. .000000 1133.. .3366

ATOM 443 ND2 ASN 59 -35.965 -55.556 25.263 1.000000 4.31

ATOM 444 C ASN 59 -35.070 -51.775 24.960 000000 8.67

ATOM 445 O ASN 59 -36.172 -52.160 24.574 000 12.75

ATOM 446 N ILE 60 -34.938 -50.587 25.548 000 10.46

ATOM 447 CA ILE 60 -36.128 -49.752 25.722 000 10.70

ATOM 448 CB ILE 60 -36.572 -49.721 27.198 1.000 11.36

ATOM 449 CG2 ILE 60 -35.465 -49.223 28.112 1.000 0.00

ATOM 450 CGI ILE 60 -37.872 -48.940 27.417 1.000 8.05 GC821-2

ATOM 451 CDl ILE 60 -38.291 -48.800 28. .860 1.000 27. .90

ATOM 452 C ILE 60 -35.879 -48.350 25. ,177 1.000 16. .37

ATOM 453 O ILE 60 -34.813 -47.773 25. .374 1.000 28. .53

ATOM 454 N ASP 61 -36.861 -47.811 24. ,470 1.000 18. .37

ATOM 455 CA ASP 61 -36.838 -46.520 23, .821 1.000 12, .62

ATOM 456 CB ASP 61 -38.110 -46.353 22. .977 1.000 12, .58

ATOM 457 CG ASP 61 -38.111 -47.199 21. .725 1.000 12, .09

ATOM 458 OD1 ASP 61 -37.044 -47.723 21. .349 1.000 16, .37

ATOM 459 OD2 ASP 61 -39.197 -47.332 21, .122 1.000 23. .20

ATOM 460 C ASP 61 -36.796 -45.350 24, .794 1.000 11, .54

ATOM 461 O ASP 61 -37.626 -45.279 25, .702 1.000 8, .66

ATOM 462 N ASP 62 -35.860 -44.428 24, .603 1.000 8, .03

ATOM 463 CA ASP 62 -35.844 -43.228 25, .431 1.000 14, .39

ATOM 464 CB ASP 62 -34.430 -42.656 25, .565 1.000 13, .94

ATOM 465 CG ASP 62 -34.384 -41.598 26. .656 1.000 18, .06

ATOM 466 OD1 ASP 62 -33.609 -41.768 27, .622 1.000 13, .05

ATOM 467 OD2 ASP 62 -35.129 -40.604 26, .536 1.000 20. .19

ATOM 468 C ASP 62 -36.759 -42.162 24, .844 1.000 13, .14

ATOM 469 O ASP 62 -36.506 -41.698 23. .731 1.000 14, ,36

ATOM 470 N PRO 63 -37.800 -41.751 25, .553 1.000 8, .49

ATOM 471 CD PRO 63 -38.102 -42.088 26. .951 1.000 4, .73

ATOM 472 CA PRO 63 -38.805 -40.853 24, .972 1.000 16, .60

ATOM 473 CB PRO 63 -39.802 -40.646 26, .123 1.000 11, .61

ATOM 474 CG PRO 63 -39.020 -40.960 27, .352 1.000 88,. .0044

ATOM 475 C PRO 63 -38.251 -39.504 2244,. .553311 1. ,000000 1199,..7700

ATOM 476 O PRO 63 -38.924 -38.738 2233..,883355 1. ,000000 1100...2266

ATOM 477 N THR 64 -37.024 -39.180 24.922 ,000 22.29

ATOM 478 CA THR 64 -36.429 -37.908 24.534 ,000 19.30

ATOM 479 CB THR 64 -35.852 -37.191 25.769 ,000 20.62

ATOM 480 OG1 THR 64 -34.550 -37.713 26.045 ,000 30.42

ATOM 481 CG2 THR 64 -36.718 -37.467 26.992 .000 7.89

ATOM 482 C THR 64 -35.329 -38.087 23.497 ,000 19.22

ATOM 483 O THR 64 -34.609 -37.132 23.183 .000 11.15

ATOM 484 N ASP 65 -35.189 -39.301 22.965 .000 15.61

ATOM 485 CA ASP 65 -34.139 -39.542 21.967 .000 18.78

ATOM 486 CB ASP 65 -32.777 -39.286 22.605 .000 20.50

ATOM 487 CG ASP 65 -31.613 -39.348 21.638 .000 17.33

ATOM 488 OD1 ASP 65 -31.767 -39.935 20.550 .000 19.33

ATOM 489 OD2 ASP 65 -30.538 -38.810 21.983 .000 15.26

ATOM 490 C ASP 65 -34.241 -40.945 21.382 .000 14.84

ATOM 491 O ASP 65 -33.982 -41.936 22.060 .000 8.38

ATOM 492 N PRO 66 -34.638 -41.026 20.115 .000 15.75

ATOM 493 CD PRO 66 -34.896 -39.870 19.235 .000 23.61

ATOM 494 CA PRO 66 -34.882 -42.301 19.441 ,000 9.14

ATOM 495 CB PRO 66 -35.693 -41.871 18.206 .000 14.38

ATOM 496 CG PRO 66 -35.210 -40.494 17.902 000 16.45

ATOM 497 C PRO 66 -33.621 -43.029 18.995 1.000 8.15 GC821-2

ATOM 498 O PRO 66 33 695 -44 041 18 283 1 000 12 38

ATOM 499 N ARG 67 32 446 -42 557 19 404 1 000 11 98

ATOM 500 CA ARG 67 31 209 -43 225 19 020 1 000 7 77

ATOM 501 CB ARG 67 30 081 -42 211 18 831 1 000 8 16

ATOM 502 CG ARG 67 30 162 -41 308 17 614 1 000 7 27

ATOM 503 CD ARG 67 29 078 -40 228 17 713 1 000 11 05

ATOM 504 NE ARG 67 29 378 -39 266 18 769 1 000 11 17

ATOM 505 CZ ARG 67 28 768 -38 115 19 001 1 000 13 35

ATOM 506 NH1 ARG 67 27 756 -37 708 18 245 1 000 3 80

ATOM 507 NH2 ARG 67 29 168 -37 347 20 010 1 000 9 93

ATOM 508 C ARG 67 30 728 -44 239 20 048 1 000 8 92

ATOM 509 O ARG 67 29 714 -44 887 19 774 1 000 13 65

ATOM 510 N LEU 68 31 389 -44 365 21 191 1 000 9 14

ATOM 511 CA LEU 68 30 805 -45 057 22 335 1 000 13 92

ATOM 512 CB LEU 68 31 052 -44 223 23 608 1 000 7 80

ATOM 513 CG LEU 68 30 899 -42 707 23 481 1 000 8 78

ATOM 514 CDl LEU 68 31 285 -41 987 24 770 1 000 13 12

ATOM 515 CD2 LEU 68 29 477 -42 333 23 090 1 000 3 77

ATOM 516 C LEU 68 31 299 -46 478 22 571 1 000 16 19

ATOM 517 O LEU 68 30 895 -47 092 23 574 1 000 5 21

ATOM 518 N ASN 69 32 139 -47 056 21 716 1 000 7 75

ATOM 519 CA ASN 69 32 520 -48 457 21 927 1 000 6 53

ATOM 520 CB ASN 69 33 807 -48 842 21 198 1 000 6 25

ATOM 521 CG ASN 69 34 377 -50 172 21 658 1 000 11 70

ATOM 522 OD1 ASN 69 33 732 -51 219 21 664 1 000 2 64

ATOM 523 ND2 ASN 69 35 646 -50 164 22 057 1 000 10 84

ATOM 524 C ASN 69 31 406 -49 404 21 480 1 000 8 62

ATOM 525 O ASN 69 31 204 -49 617 20 287 1 000 14 61

ATOM . 526 N GLY 70 30 697 -49 972 22 452 1 000 8 79

ATOM 527 CA GLY 70 29 582 -50 854 22 212 1 000 1 64

ATOM 528 C GLY 70 29 911 -52 031 21 316 1 000 6 17

ATOM 529 O GLY 70 29 189 -52 293 20 355 1 000 12 06

ATOM 530 N ALA 71 30 982 -52 744 21 622 1 000 1 39

ATOM 531 CA ALA 71 31 442 -53 885 20 843 1 000 5 92

ATOM 532 CB ALA 71 32 688 -54 457 21 529 1 000 3 81

ATOM 533 C ALA 71 31 766 -53 565 19 392 1 000 4 67

ATOM 534 O ALA 71 31 565 -54 391 18 490 1 000 0 00

ATOM 535 N SER 72 32 295 -52 371 19 121 1 000 3 88

ATOM 536 CA SER 72 32 687 -52 033 17 752 1 000 6 33

ATOM 537 CB SER 72 33 678 -50 870 17 759 1 000 4 05

ATOM 538 OG SER 72 33 023 -49 637 18 004 1 000 25 62

ATOM 539 C SER 72 31 468 -51 730 16 884 1 000 7 90

ATOM 540 O SER 72 31 568 -51 720 15 .658 1 000 12 06

ATOM 541 N TYR 73 30 315 -51 505 17 498 1 000 8 51

ATOM 542 CA TYR 73 29 070 -51 210 16 .789 1 .000 8 .77

ATOM 543 CB TYR 73 28 .394 -50 .029 17 .478 1 .000 10 .31

ATOM 544 CG TYR 73 27 124 -49 .453 16 .913 1 .000 11 .92 GC821-2

ATOM 545 CDl TYR 73 -27.113 -48.329 16.090 1.000 8.49

ATOM 546 CE1 TYR 73 -25.931 -47.812 15.586 1.000 1.47

ATOM 547 CD2 TYR 73 25 888 -50.018 17 201 1.000 10.36

ATOM 548 CE2 TYR 73 24 704 -49.512 16 703 1.000 9.07

ATOM 549 CZ TYR 73 24 727 -48.398 15 890 1.000 5.36

ATOM 550 OH TYR 73 23 544 -47.902 15 391 1.000 10.80

ATOM 551 C TYR 73 28 148 -52.419 16 730 1.000 13.31

ATOM 552 O TYR 73 27 404 -52.630 15 764 1.000 10.40

ATOM 553 N LEU 74 28 172 -53.261 17 759 1.000 8.99

ATOM 554 CA LEU 74 27 204 -54.342 17 901 1.000 7.76

ATOM 555 CB LEU 74 27 554 -55.155 19 155 1.000 9.47

ATOM 556 CG LEU 74 26 402 -55.532 20 080 1.000 10.36

ATOM 557 CDl LEU 74 26 786 -56.729 20 939 1.000 25.33

ATOM 558 CD2 LEU 74 25 137 -55.819 19 288 1.000 13.92

ATOM 559 C LEU 74 27 088 -55.253 16 687 1.000 5.72

ATOM 560 O LEU 74 25 980 -55.383 16 141 1.000 7.01

ATOM 561 N PRO 75 28 141 -55.907 16 219 1.000 6.99

ATOM 562 CD PRO 75 29 553 -55.794 16 615 1.000 1.55

ATOM 563 CA PRO 75 27 965 -56.896 15 140 1.000 7.57

ATOM 564 CB PRO 75 29 384 -57.401 14 855 1.000 5.01

ATOM 565 CG PRO 75 30 158 -57.063 16 086 1.000 6.27

ATOM 566 C PRO 75 27 364 -56.285 13 882 1.000 4.16

ATOM 567 0 PRO 75 26 651 -56.971 13 158 1.000 4.35

ATOM 568 N SER 76 27 640 -55.014 13 615 1.000 6.22

ATOM 569 CA SER 76 27 050 -54.322 12 473 1.000 0.00

ATOM 570 CB SER 76 27 758 -52.978 12 261 1.000 0.00

ATOM 571 OG SER 76 29 120 -53.249 11 920 1.000 0.00

ATOM 572 C SER 76 25 554 -54.127 12 674 1.000 0.69

ATOM 573 O SER 76 24 767 -54.280 11 740 1.000 4.06

ATOM 574 N CYS 77 25 202 -53.802 13 911 1.000 2.82

ATOM 575 CA CYS 77 23 851 -53.599 14 384 1.000 2.99

ATOM 576 CB CYS 77 23 878 -53.202 15 868 1.000 0.00

ATOM 577 SG CYS 77 22 325 -52.508 16 451 1.000 8.78

ATOM 578 C CYS 77 22 962 -54.831 14 225 1.000 13.77

ATOM 579 O CYS 77 21 828 -54.700 13 755 1.000 12.12

ATOM 580 N LEU 78 23 455 -55.996 14 621 1.000 15.71

ATOM 581 CA LEU 78 22 751 -57.268 14 538 1.000 10.13

ATOM 582 CB LEU 78 23 617 -58.387 15 129 1.000 2.73

ATOM 583 CG LEU 78 23 777 -58.354 16 651 1.000 7.98

ATOM 584 CDl LEU 78 24 866 -59.319 17 085 1.000 3.36

ATOM 585 CD2 LEU 78 22 451 -58.676 17 330 1.000 8.53

ATOM 586 C LEU 78 22 385 -57.650 13 106 1.000 9.88

ATOM 587 O LEU 78 -21.222 -57.855 12.761 1.000 12.55

ATOM 588 N ALA 79 -23.407 -57.748 12.271 1.000 11.93

ATOM 589 CA ALA 79 -23.297 -58.022 10.848 1.000 2.98

ATOM 590 CB ALA 79 -24.699 -58.042 10.255 1.000 0.32

ATOM 591 C ALA 79 -22.393 -57.026 10.127 1.000 7.73 GC821-2

ATOM 592 O ALA 79 21 724 -57 408 9.163 1.000 13.15

ATOM 593 N THR 80 22 337 -55 774 10.560 1.000 10.93

ATOM 594 CA THR 80 21 427 -54 757 10.044 1.000 6.56

ATOM 595 CB THR 80 21 703 -53 373 10.669 1.000 9.10

ATOM 596 OG1 THR 80 23 013 -52 897 10.320 1.000 4.47

ATOM 597 CG2 THR 80 20 722 -52 328 10.148 1.000 8.02

ATOM 598 C THR 80 19 970 -55 117 10.317 1.000 10.87

ATOM 599 O THR 80 19 103 -55 052 9.450 1.000 12.66

ATOM 600 N HIS 81 19 659 -55 512 11.548 1.000 13.90

ATOM 601 CA HIS 81 18 282 -55 720 11.978 1.000 13.04

ATOM 602 CB HIS 81 18 119 -55 195 13.418 1.000 15.15

ATOM 603 CG HIS 81 18 279 -53 704 13.502 1.000 10.10

ATOM 604 CD2 HIS 81 19 202 -52 927 14.111 1.000 6.25

ATOM 605 ND1 HIS 81 17 404 -52 833 12.889 1.000 7.20

ATOM 606 CE1 HIS 81 17 775 -51 589 13.117 1.000 7.73

ATOM 607 NE2 HIS 81 18 867 -51 616 13.863 1.000 6.24

ATOM 608 C HIS 81 17 827 -57 166 11.896 1.000 9.61

ATOM 609 O HIS 81 16 674 -57 460 12.216 1.000 10.35

ATOM 610 N LEU 82 18 689 -58 081 11.470 1.000 4.74

ATOM 611 CA LEU 82 18 257 -59 461 11.247 1.000 6.06

ATOM 612 CB LEU 82 19 399 -60 263 10.631 1.000 6.90

ATOM 613 CG LEU 82 20 535 -60 716 11.541 1.000 6.83

ATOM 614 CDl LEU 82 21 388 -61 774 10.851 1.000 11.79

ATOM 615 CD2 LEU 82 19 987 -61 246 12.856 1.000 23.45

ATOM 616 C LEU 82 17 042 -59 500 10.337 1.000 6.51

ATOM 617 O LEU 82 16 972 -58 722 9.375 1.000 1.45

ATOM 618 N PRO 83 16 056 -60 360 10.556 1.000 7.15

ATOM 619 CD PRO 83 14 823 -60 374 9.731 1.000 0.00

ATOM 620 CA PRO 83 16 043 -61 394 11.583 1.000 5.44

ATOM 621 CB PRO 83 14 941 -62 341 11.067 1.000 9.33

ATOM 622 CG PRO 83 13 968 -61 405 10.415 1.000 7.09

ATOM 623 C PRO 83 15 638 -60 922 12.973 1.000 10.31

ATOM 624 O PRO 83 14 716 -60 125 13.110 1.000 16.21

ATOM 625 N LEU 84 16 319 -61 434 13.994 1.000 14.34

ATOM 626 CA LEU 84 16 009 -61 132 15.382 1.000 10.66

ATOM 627 CB LEU 84 17 165 -60 373 16.049 1.000 7.23

ATOM 628 CG LEU 84 17 485 -59 010 15.434 1.000 2.01

ATOM 629 CDl LEU 84 18 843 -58 518 15.902 1.000 8.19

ATOM 630 CD2 LEU 84 16 382 -58 019 15.766 1.000 5.93

ATOM 631 C LEU 84 15 734 -62 386 16.203 1.000 7.34

ATOM 632 O LEU 84 16 299 -63 447 15.945 1.000 8.40

ATOM 633 N ASP 85 14 879 -62 247 17.208 1.000 8.68

ATOM 634 CA ASP 85 14 607 -63 332 18.146 1.000 10.21

ATOM 635 CB ASP 85 13 093 -63 433 18.382 1.000 15.96

ATOM 636 CG ASP 85 12 338 -63 789 17.117 1.000 11.01

ATOM 637 OD1 ASP 85 12 .343 -64 .975 16.727 1.000 9.49

ATOM 638 OD2 ASP 85 -11.739 -62.878 16.518 1.000 28.18 GC821-2

ATOM 639 C ASP 85 -15.313 -63.142 19.477 11..000000 00..00

ATOM 640 O ASP 85 -15.778 -64.067 20.137 11..000000 55..48

ATOM 641 N LEU 86 -15.414 -61.907 19.958 1.000 77..62

ATOM 642 CA LEU 86 -16.080 -61.695 21.243 1.000 88..84

ATOM 643 CB LEU 86 -15.085 -61.690 22.403 1..000000 1122..15

ATOM 644 CG LEU 86 -15.655 -61.580 23.822 1.000 13.

ATOM 645 CDl LEU 86 -16.562 -62.757 24.151 1.000 7.

ATOM 646 CD2 LEU 86 -14.535 -61.477 24.850 1.000 10.

ATOM 647 C LEU 86 -16.841 -60.374 21.221 1.000 66..69

ATOM 648 O LEU 86 -16.327 -59.409 20.649 1.000 88..05

ATOM 649 N VAL 87 -18.013 -60.361 21.842 1.000 4. ,26

ATOM 650 CA VAL 87 -18.752 -59.127 22.049 1.000 2..21

ATOM 651 CB VAL 87 -20.150 -59.126 21.413 1.000 88..44

ATOM 652 CGI VAL 87 -20.848 -57.808 21.722 1.000 22..51

ATOM 653 CG2 VAL 87 -20.104 -59.352 19.911 1.000 00..00

ATOM 654 C VAL 87 -18.893 -58.869 23.551 1.000 77..05

ATOM 655 O VAL 87 -19.472 -59.660 24.289 1.000 55..76

ATOM 656 N ILE 88 -18.351 -57.746 24.010 1.000 77..24

ATOM 657 CA ILE 88 -18.499 -57.336 25.400 1.000 66..18

ATOM 658 CB ILE 88 -17.233 -56.652 25.938 1.000 6.54

ATOM 659 CG2 ILE 88 -17.458 -56.098 27.333 1.000 11.40

ATOM 660 CGI ILE 88 -16.001 -57.559 25.902 1.000 21

ATOM 661 CDl ILE 88 -14.734 -56.856 26.339 1.000 20

ATOM 662 C ILE ' 88 -19.693 -56.394 25.506 .000 68

ATOM 663 O ILE 88 -19.817 -55.458 24.716 000 10.14

ATOM 664 N ILE 89 -20.574 -56.672 26.457 000 7.74

ATOM 665 CA ILE 89 -21.765 -55.857 26.645 ,000 12.20

ATOM 666 CB ILE 89 -23.052 -56.635 26.306 000 12.51

ATOM 667 CG2 ILE 89 -24.253 -55.703 26.339 000 11.52

ATOM 668 CGI ILE 89 -22.981 -57.390 24.979 000 6.47

ATOM 669 CDl ILE 89 -24.250 -58.111 24.597 000 8.71

ATOM 670 C ILE 89 -21.861 -55.340 28.078 000 11.05

ATOM 671 O ILE 89 -22.169 -56.106 28.989 000 3 02

ATOM 672 N MET 90 -21.590 -54.049 28.236 000 7 01

ATOM 673 CA MET 90 -21.808 -53.359 29.492 000 11 48

ATOM 674 CB MET 90 -20.535 -52.721 30.043 000 9 27

ATOM 675 CG MET 90 -20.756 -52.097 31.415 000 10.33

ATOM 676 XD MET 90 -19.202 -51.706 32.246 ,000 17.92

ATOM 677 CE MET 90 -18.544 -50.475 31.124 ,000 12.70

ATOM 678 C MET 90 -22.872 -52.262 29.325 ,000 12.90

ATOM 679 O MET 90 -22.524 -51.143 28.954 000 0.00

ATOM 680 N LEU 91 -24.108 -52.639 29.604 ,000 8.70

ATOM 681 CA LEU 91 -25.292 -51 802 29.511 000 10.58

ATOM 682 CB LEU 91 -26.114 52.105 28.254 ,000 9.42

ATOM 683 CG LEU 91 -25.573 51.564 26.932 ,000 4, .10

ATOM 684 CDl LEU 91 -26.427 -52.046 25.772 1.000 0.00

ATOM 685 CD2 LEU 91 -25.506 -50.044 26.961 1.000 2.02 GC821-2

ATOM 686 C LEU 91 -26. 169 -52 . 031 30. 734 1 . 000 2 .21

ATOM 687 O LEU 91 25 989 -53 066 31. 388 1 000 10 59

ATOM 688 N GLY 92 27 087 -51 117 31. 025 1 000 4 69

ATOM 689 CA GLY 92 27 963 -51 321 32 172 1 000 7 16

ATOM 690 C GLY 92 28 189 -50 092 33 027 1 000 0 00

ATOM 691 O GLY 92 29 266 -49 924 33 603 1 000 8 09

ATOM 692 N THR 93 27 204 -49 219 33 133 1 000 0 16

ATOM 693 CA THR 93 27 241 -48 005 33 929 1 000 9 42

ATOM 694 CB THR 93 25 927 -47 205 33 768 1 000 17 05

ATOM 695 OG1 THR 93 24 811 -48 063 34 024 1 000 26 81

ATOM 696 CG2 THR 93 25 847 -46 068 34 778 1 000 0 34

ATOM 697 C THR 93 28 386 -47 075 33 551 1 000 9 26

ATOM 698 O THR 93 29 037 -46 491 34 419 1 000 14 18

ATOM 699 N ASN 94 28 614 -46 927 32 250 1 000 0 69

ATOM 700 CA ASN 94 29 609 -45 981 31 755 1 000 5 12

ATOM 701 CB ASN 94 29 333 -45 677 30 274 1 000 9 42

ATOM 702 CG ASN 94 27 990 -44 983 30 120 1 000 10 74

ATOM 703 OD1 ASN 94 27 679 -44 062 30 873 1 000 21 66

ATOM 704 ND2 ASN 94 27 175 -45 417 29 174 1 000 18 23

ATOM 705 C ASN 94 31 029 -46 481 31 986 1 000 5 80

ATOM 706 O ASN 94 31 889 -45 654 32 317 1 000 4 04

ATOM 707 N ASP 95 31 282 -47 777 31 863 1 000 4 02

ATOM 708 CA ASP 95 32 568 -48 411 32 137 1 000 7 86

ATOM 709 CB ASP 95 32 522 -49 913 31 880 1 000 5 49

ATOM 710 CG ASP 95 32 090 -50 392 30 521 1 000 10 09

ATOM 711 OD1 ASP 95 30 998 -50 021 30 040 1 000 16 22

ATOM 712 OD2 ASP 95 32 843 -51 184 29 907 1 000 15 98

ATOM 713 C ASP 95 33 020 -48 208 33 591 1 000 9 17

ATOM 714 O ASP 95 34 188 -48 361 33 958 1 000 0 43

ATOM 715 N THR 96 32 051 -47 882 34 421 1 000 11 45

ATOM 716 CA THR 96 32 122 -47 529 35 823 1 000 16 75

ATOM 717 CB THR 96 30 697 -47 638 36 412 1 000 24 78

ATOM 718 OG1 THR 96 30 607 -48 784 37 274 1 000 17 62

ATOM 719 CG2 THR 96 30 350 -46 409 37 229 1 000 12 12

ATOM 720 C THR 96 32 697 -46 132 35 997 1 000 12 12

ATOM 721 O THR 96 33 047 -45 678 37 088 1 000 10 94

ATOM 722 N LYS 97 32 820 -45 406 34 883 1 000 12 18

ATOM 723 CA LYS 97 33 387 -44 060 34 954 1 000 14 27

ATOM 724 CB LYS 97 33 247 -43 336 33 620 1 000 13 25

ATOM 725 CG LYS 97 31 996 -42 477 33 500 1 000 11 50

ATOM 726 CD LYS 97 31 819 -41 935 32 086 1 000 3 08

ATOM 727 CE LYS 97 30 344 -41 856 31 717 1 000 0 00

ATOM 728 NZ LYS 97 30 131 -41 152 30 416 1 000 0 00

ATOM 729 C LYS 97 34 848 -44 112 35 403 1 000 12 44

ATOM 730 O LYS 97 35 636 -44 914 34 911 1 000 8 04

ATOM 731 N ALA 98 35 179 -43 .246 36 .355 1 .000 11 .97

ATOM 732 CA ALA 98 36 .454 -43 .218 37 .047 1 .000 4 .97 ATOM 733 CB ALA 98 -36.522 -41.982 37.943 .000 3.,36

ATOM 734 C ALA 98 -37.641 -43.246 36.100 .000 12. .00

ATOM 735 O ALA 98 -38.651 -43.905 36.355 .000 22. .61

ATOM 736 N TYR 99 -37.535 -42.518 34.988 .000 12. ,39

ATOM 737 CA TYR 99 -38.695 -42.403 34.107 .000000 7. .,2255

ATOM 738 CB TYR 99 -38.521 -41.297 33.087 .000000 9..,1111

ATOM 739 CG TYR 99 -37.300 -41.251 32.217 .000 15. ,58

ATOM 740 CDl TYR 99 -37.261 -41.912 30.995 .000 13. ,09

ATOM 741 CE1 TYR 99 -36.144 -41.874 30.186 .000 9. ,06

ATOM 742 CD2 TYR 99 -36.173 -40.533 32.598 .000 14. .48

ATOM 743 CE2 TYR 99 -35.051 -40.482 31.796 .000 15, .13

ATOM 744 CZ TYR 99 -35.044 -41.154 30.591 .000 11. .74

ATOM 745 OH TYR 99 -33.925 -41.102 •29.794 .000 6, .20

ATOM 746 C TYR 99 -38.990 -43.726 33.413 .000. 11, .25

ATOM 747 O TYR 99 -40.121 -43.927 32.963 .000 12. ,89

ATOM 748 N PHE 100 -37.993 -44.606 33.351 .000000 4.,,6633

ATOM 749 CA PHE 100 -38.237 -45.908 32.731 .000000 1...0011

ATOM 750 CB PHE 100 -36.903 -46.556 32.348 000 3.41

ATOM 751 CG PHE 100 -36.316 -45.980 31.070 000 11.77

ATOM 752 CDl PHE 100 -35.018 -45.506 31.032 000 ^" 7.50

ATOM 753 CD2 PHE 100 -37.080 -45.919 29.917 000 16.94

ATOM 754 CE1 PHE 100 -34.489 -44.981 29,868 000 7.31

ATOM 755 CE2 PHE 100 -36.557 -45.398 28.748 000 12.92

ATOM 756 CZ PHE 100 -35.260 -44.925 28.722 .000 7.58

ATOM 757 c PHE 100 -39.051 -46.829 33.628 000 6.94

ATOM 758 0 PHE 100 -39.711 -47.750 33.131 000 9.31

ATOM 759 N ARG 101 -39.032 -46.629 34.943 000 12.10

ATOM 760 CA ARG 101 -39.783 -47.468 35.869 000 12.96

ATOM 761 CB ARG 101 -41.294 -47.296 35.695 000 16.21

ATOM 762 CG ARG 101 -41.890 -45.959 36.087 1.000 19.51

ATOM 763 CD ARG 101 -43.376 -45.918 35.740 1.000 25.82

ATOM 764 NE ARG 101 -43.818 -44.553 35.466 000 31.88

ATOM 765 CZ ARG 101 -43.797 -43.583 36.373 000 33.97

ATOM 766 NH1 ARG 101 -43.355 -43.839 37.599 000 43.49

ATOM 767 NH2 ARG 101 -44.206 -42.361 36.067 000 44.85

ATOM 768 C ARG 101 -39.472 -48.955 35.704 000 12.20

ATOM 769 O ARG 101 -40.376 -49.782 35. ,878 1. ,000 12. ,48

ATOM 770 N ARG 102 -38.238 -49.319 35. ,378 1. ,000 8. ,86

ATOM 771 CA ARG 102 -37.887 -50.733 35. ,264 1. ,000 11. ,00

ATOM 772 CB ARG 102 -36.899 -50.962 3344. ,115 1. ,000 6. ,96

ATOM 773 CG ARG 102 -37.497 -50.805 3322,. .772200 1, ,000 9. ,64

ATOM 774 CD ARG 102 -36.518 -51.198 31. .624 1, .000 8, .07

ATOM 775 NE ARG 102 -37.140 -51.842 30. .474 1, .000 4, .64

ATOM 776 CZ ARG 102 -36.540 -52.606 2299,. .557711 1. .000 7. .34

ATOM 777 NH1 ARG 102 -35.240 -52.877 2299,. .662288 1, .000 1, .45

ATOM 778 NH2 ARG 102 -37.232 -53.131 28 .567 1 .000 6 .11

ATOM 779 C ARG 102 -37.320 -51.275 36.577 1.000 11.09 GC821-2

ATOM 780 O ARG 102 -36.734 -50.567 37, .394 1.000 10. .02

ATOM 781 N THR 103 -37.497 -52.573 36, .785 1.000 11. ,01

ATOM 782 CA THR 103 -36.898 -53.307 37, .893 1.000 12. .65

ATOM 783 CB THR 103 -37.844 -54.376 38. .462 1.000 7. .64

ATOM 784 OG1 THR 103 -38.083 -55.384 37. .468 1.000 11. .29

ATOM 785 CG2 THR 103 -39.199 -53.771 38. .790 1.000 15. .33

ATOM 786 C THR 103 -35.618 -53.966 37, .390 1.000 10. .55

ATOM 787 O THR 103 -35.409 -53.986 36, .173 1.000 9. .17

ATOM 788 N PRO 104 -34.765 -54.474 38. .264 1.000 10. .17

ATOM 789 CD PRO 104 -34.799 -54.363 39, .731 1.000 14. .03

ATOM 790 CA PRO 104 -33.598 -55.230 3377...880033 1.000 66.. .8811

ATOM 791 CB PRO 104 -32.968 -55.748 39.094 1.000 5 . 25

ATOM 792 CG PRO 104 -33.402 -54.759 40.129 1.000 8 . 07

ATOM 793 C PRO 104 -34.010 -56.400 36.911 1.000 5 . 89

ATOM 794 O PRO 104 -33.251 -56.728 35.998 1.000 5 . 49

ATOM 795 N LEU 105 -35.164 -56.994 37.173 1.000 2 . 55

ATOM 796 CA LEU 105 -35.690 -58.071 36.341 1.000 10. 27

ATOM 797 CB LEU 105 -36.989 -58.642 36.890 1 , 000 11 . 51

ATOM 798 CG LEU 105 -37.304 -60.122 36.695 1.000 16.39

ATOM 799 CDl LEU 105 -38.804 -60.319 36. 480 1.000 4 . 05

ATOM 800 CD2 LEU 105 -36.533 -60.744 35. 542 1. 000 15. 49

ATOM 801 C LEU 105 -35.923 -57.566 34 . 915 1.000 14 . 30

ATOM 802 O LEU 105 -35.415 -58.168 33. 969 1. 000 14 .22

ATOM 803 N ASP 106 -36.686 -56.484 34 . 791 1.000 11 . 11

ATOM 804 CA ASP 106 -36.922 -55.878 33.482 . 000000 8.08

ATOM 805 CB ASP 106 -37.636 -54.538 33.621 000 14.02

ATOM 806 CG" ASP 106 -39.046 -54.638 34.152 000 13.88

ATOM 807 OD1 ASP 106 -39.726 -55.653 33.875 000 19.94

ATOM 808 OD2 ASP 106 -39.479 -53.686 34.843 000000 4.29

ATOM 809 C ASP 106 -35.607 -55.668 32.734 000000 7.79

ATOM 810 O ASP 106 -35.504 -55.987 31.554 000 10.52

ATOM 811 N ILE 107 -34.614 -55.131 33.438 000 5.00

ATOM 812 CA ILE 107 -33.321 -54.814 32.845 1.000 6.63

ATOM 813 CB ILE 107 -32.444 -54.016 33.828 1.000 14.49

ATOM 814 CG2 ILE 107 -31.125 -53.622 33.184 1.000 7.24

ATOM 815 CGI ILE 107 -33.146 -52.790 34.415 1.000 16.93

ATOM 816 CDl ILE 107 -32.174 -51.779 34.992 1.000 19.38

ATOM 817 C ILE 107 -32.564 -56.059 32.405 000 .12

ATOM 818 O ILE 107 -31.877 -56.024 31.381 000 .80

ATOM 819 N ALA 108 -32.691 -57.148 33.157 000 .34

ATOM 820 CA ALA 108 -32.021 -58.398 32.812 000 .25

ATOM 821 CB ALA 108 -32.089 -59.399 33.956 000 2.49

ATOM 822 C ALA 108 -32.637 -59.018 31.568 000 2.89

ATOM 823 O ALA 108 -31.952 -59.619 30.738 000 11.68

ATOM 824 N LEU 109 -33.956 -58.864 31.449 000 0.00

ATOM 825 CA LEU 109 -34.609 -59.401 30.251 000 6.18

ATOM 826 CB LEU 109 -36.125 -59.391 30.435 1.000 12.37 GC821-2

ATOM 827 CG LEU 109 36 674 -60. 463 31 386 1 000 15 66

ATOM 828 CDl LEU 109 37 985 -60 004 32 001 1 000 27 44

ATOM 829 CD2 LEU 109 36 854 -61. 794 30 672 1 000 3 14

ATOM 830 C LEU 109 34 171 -58 620 29 022 1 000 10 30

ATOM 831 O LEU 109 34 035 -59 139 27 915 1 000 18 00

ATOM 832 N GLY 110 33 918 -57 323 29 193 1 000 11 78

ATOM 833 CA GLY 110 33 426 -56. 535 28 069 1 000 8 26

ATOM 834 C GLY 110 32 028 -56 976 27 666 1 000 7 06

ATOM 835 O GLY 110 31 757 -57 155 26 482 1 000 18 68

ATOM 836 N MET 111 31 149 -57 149 28 651 1 000 5 04

ATOM 837 CA MET 111 29 812 -57 661 28 414 1 000 4 52

ATOM 838 CB MET 111 28 962 -57 717 29 683 1 000 1 61

ATOM 839 CG MET 111 27 663 -58 503 29 542 1 000 0 00

ATOM 840 XD MET 111 26 456 -57 694 28 453 1 000 16 83

ATOM 841 CE MET 111 25 895 -56 355 29 497 1 000 5 08

ATOM 842 C MET 111 29 915 -59 066 27 821 1 000 6 40

ATOM 843 O MET 111 29 098 -59 476 27 005 1 000 8 66

ATOM 844 N SER 112 30 937 -59 795 28 270 1 000 9 55

ATOM 845 CA SER 112 31 140 -61 133 27 731 1 000 8 05

ATOM 846 CB SER 112 32 322 -61 821 28 405 1 000 10 37

ATOM 847 OG SER 112 33 488 -61 744 27 609 1 000 8 11

ATOM 848 C SER 112 31 341 -61 034 26 217 1 000 6 07

ATOM 849 O SER 112 30 761 -61 823 25 471 1 000 9 26

ATOM 850 N VAL 113 32 142 -60 065 25 803 1 000 4 80

ATOM 851 CA VAL 113 32 424 -59 788 .24 401 1 000 9 22

ATOM 852 CB VAL 113 33 414 -58 615 24 266 1 000 9 35

ATOM 853 CGI VAL 113 33 350 -57 979 22 886 1 000 0 53

ATOM 854 CG2 VAL 113 34 830 -59 090 24 567 1 000 15 43

ATOM 855 C ^' VAL 113 31 149 -59 490 23 616 1 000 18 19

ATOM 856 0 VAL 113 31 027 -59 900 22 456 1 000 17 08

ATOM 857 N LEU 114 30 199 -58 791 24 235 1 000 16 22

ATOM 858 CA LEU 114 28 948 -58 431 23 570 1 000 9 05

ATOM 859 CB LEU 114 28 220 -57 329 24 341 1 000 4 93

ATOM 860 CG LEU 114 28 938 -55 983 24 427 1 000 6 23

ATOM 861 CDl LEU 114 28 122 -54 973 25 221 1 000 8 47

ATOM 862 CD2 LEU 114 29 228 -55 450 23 032 1 000 0 00

ATOM 863 C LEU 114 28 018 -59 628 23 407 1 000 5 15

ATOM 864 O LEU 114 27 310 -59 762 22 410 1 000 8 05

ATOM 865 N VAL 115 28 028 -60 503 24 403 1 000 5 78

ATOM 866 CA VAL 115 27 223 -61 717 24 373 1 000 8 93

ATOM 867 CB VAL 115 27 202 -62 383 25 762 1 000 8 05

ATOM 868 CGI VAL 115 26 501 -63 729 25 720 1 000 0 00

ATOM 869 CG2 VAL 115 26 543 -61 .439 26 .759 1 .000 0 .00

ATOM 870 C VAL 115 27 .763 -62 .685 23 .330 1 .000 9 .50

ATOM 871 O VAL 115 27 007 -63 .390 22 .662 1 .000 9 .58

ATOM 872 N THR 116 29 .087 -62 .715 23 .179 1 .000 8 .15

ATOM 873 CA THR 116 29 .688 -63 .617 22 .199 1 .000 8 .38 GC821-2

ATOM 874 CB THR 116 -31.222 -63.622 22.327 1.000 12.50

ATOM 875 OG1 THR 116 -31.575 -64.207 23.585 1.000 13.40

ATOM 876 CG2 THR 116 -31.848 -64.479 21.233 .000 10.82

ATOM 877 C THR 116 -29.316 -63.241 20.771 ,000 56

ATOM 878 0 THR 116 -29.011 -64.127 19.966 ,000 27

ATOM 879 N GLN 117 -29.345 -61.945 20.473 ,000 8.17

ATOM 880 CA GLN 117 -28.956 -61.430 19.160 ,000 '9.93

ATOM 881 CB GLN 117 -29.166 -59.920 19.080 1.000 66

ATOM 882 CG GLN 117 -30.592 -59.440 19.279 .000 21

ATOM 883 CD GLN 117 -30.699 -57.933 19.390 .000 09

ATOM 884 OEl GLN 117 -29.801 -57.260 19.896 ,000 12.85

ATOM 885 NE2 GLN 117 -31.811 -57.376 18.914 ,000 7.39

ATOM 886 C GLN 117 -27.499 -61.761 18.847 ,000 11.60

ATOM 887 O GLN 117 -27.105 -62.023 17.706 ,000 9.03

ATOM 888 N VAL 118 -26.652 -61.751 19.879 ,000 11.77

ATOM 889 CA VAL 118 -25.258 -62.146 19.659 ,000 8.34

ATOM 890 CB VAL 118 -24.340 -61.768 20.831 ,000 0.49

ATOM 891 CGI VAL 118 -22.892 -62.118 20.499 ,000 21.94

ATOM 892 CG2 VAL 118 -24.452 -60.291 21.169 ,000 3.31

ATOM 893 C VAL 118 -25.166 -63.652 19.417 ,000 10.48

ATOM 894 O VAL 118 -24.354 -64.107 18.607 ,000 10.54

ATOM 895 N LEU 119 -25.993 -64.431 20.112 ,000000 7.97

ATOM 896 CA LEU 119 -25.916 -65.885 19.993 ,000000 8.73

ATOM 897 CB LEU 119 -26.679 -66.572 21.135 ,000000 8.06

ATOM 898 CG LEU 119 -25.981 -66.556 22.498 ,000 21.06

ATOM 899 CDl LEU 119 -26.800 -67.296 23.548 .000 5.53

ATOM 900 CD2 LEU 119 -24.580 -67.150 22.403 .000 21.96

ATOM 901 C LEU 119 -26.446 -66.362 18.649 000 5.78

ATOM 902 O LEU 119 -26.022 -67.409 18.153 .000 14.06

ATOM 903 N THR 120 -27.364 -65.608 18.053 ,000 8.82

ATOM 904 CA THR 120 -27.964 -65.985 16.780 ,000000 0.00

ATOM 905 CB THR 120 -29.497 -65.798 16.815 .000000 6.15

ATOM 906 OG1 THR 120 -29.805 -64.405 16.969 000 10.14

ATOM 907 CG2 THR 120 -30.121 -66.535 17.994 000 0.76

ATOM 908 C THR 120 -27.419 -65.198 15.594 000 10.30

ATOM 909 O THR' 120 -28.061 -65.190 14.537 000 13.46

ATOM 910 N SER 121 -26.272 -64.533 15.700 000 11.26

ATOM 911 CA SER 121 -25.774 -63.675 14.636 000 7 70

ATOM 912 CB SER 121 -25.000 -62.487 15.240 1.000 5 36

ATOM 913 OG SER 121 -23.826 -62.954 15.886 000 3 70

ATOM 914 C SER 121 -24.852 -64.353 13.629 000 7.89

ATOM 915 O SER 121 -24.360 -63.660 12.730 000 13.24

ATOM 916 N ALA 122 -24.603 -65.645 13.755 000 11.50

ATOM 917 CA ALA 122 -23.748 -66.370 12.820 000 12.48

ATOM 918 CB ALA 122 -23.820 -67.868 13.098 000 3 73

ATOM 919 C ALA 122 -24.124 -66.083 11.370 000 7 92

ATOM 920 O ALA 122 -25.311 -66.050 11.042 1.000 8 42 GC821-2

ATOM 921 N GLY 123 -23. 125 -65.859 10.529 1.000 7.14

ATOM 922 CA GLY 123 23. 316 -65. 625 9. 115 1. 000 3. 98

ATOM 923 C GLY 123 23. 643 -64 196 8. 735 1. 000 12 34

ATOM 924 O GLY 123 23. 445 -63 822 7. 571 1. 000 1. 55

ATOM 925 N GLY 124 24. 132 -63 404 9 683 1. 000 19 09

ATOM 926 CA GLY 124 24. 506 -62 016 9. 471 1. 000 13 26

ATOM 927 C GLY 124 25. 277 -61 809 8. 186 1. 000 10 25

ATOM 928 O GLY 124 26 403 -62 278 8 018 1 000 10 97

ATOM 929 N VAL 125 24 684 -61 110 7 217 1. 000 12 50

ATOM 930 CA VAL 125 25 365 -60 956 5 930 1 000 9 40

ATOM 931 CB VAL 125 25 557 -59 477 5 559 1 000 14 11

ATOM 932 CGI VAL 125 26 156 -59 326 4 168 1 000 13 51

ATOM 933 CG2 VAL 125 26 455 -58 786 6 578 1 000 22 31

ATOM 934 C VAL 125 24 588 -61 675 4 833 1 000 6 71

ATOM 935 0 VAL 125 23 580 -61 151 4 368 1 000 4 54

ATOM 936 N GLY 126 25 047 -62 850 4 427 1 000 14 20

ATOM 937 CA GLY 126 24 466 -63 654 3 377 1 000 9 15

ATOM 938 C GLY 126 23 012 -64 018 3 580 1 000 10 06

ATOM ^' 939 O GLY 126 22 225 -64 068 2 629 1 000 4 29

ATOM 940 N THR 127 22 595 -64 295 4 811 1 000 6 29

ATOM 941 CA THR 127 21 214 -64 701 5 050 1 000 3 83

ATOM 942 CB THR 127 20 470 -63 707 5 957 1 000 8 35

ATOM 943 OG1 THR 127 20 719 -64 001 7 339 1 000 16 55

ATOM 944 CG2 THR 127 20 987 -62 295 5 716 1 000 11 34

ATOM 945 C THR 127 21 143 -66 099 5 663 1 000 1 10

ATOM 946 O THR 127 22 159 -66 699 6 001 1 000 4 52

ATOM 947 N THR 128 19 921 -66 590 5 790 1 000 9 21

ATOM 948 CA THR 128 19 546 -67 893 6 299 1 000 8 72

ATOM 949 CB THR 128 18 451 -68 505 5 397 1 000 10 99

ATOM 950 OG1 THR 128 17 447 -67 497 5 236 1 000 7 85

ATOM 951 CG2 THR 128 18 976 -68 853 4 015 1 000 3 45

ATOM 952 C THR 128 18 995 -67 821 7 718 1 000 13 03

ATOM 953 O THR 128 18 450 -68 788 8 255 1 000 8 50

ATOM 954 N TYR 129 19 127 -66 646 8 315 1 000 10 .20

ATOM 955 CA TYR 129 18 542 -66 357 9 615 1 000 7 58

ATOM 956 CB TYR 129 18 323 -64 853 9 722 1 000 8 22

ATOM 957 CG TYR 129 17 246 -64 280 8 835 1 000 11 97

ATOM 958 CDl TYR 129 17 514 -63 176 8 031 1 000 8 62

ATOM 959 CE1 TYR 129 16 547 -62 636 7 211 1 000 7 23

ATOM 960 CD2 TYR 129 15 970 -64 827 8 799 1 000 12 .10

ATOM 961 CE2 TYR 129 14 991 -64 .290 7 .982 1 .000 16 .92

ATOM 962 CZ TYR 129 15 288 -63 .196 7 .193 1 .000 16 .10

ATOM 963 OH TYR 129 14 315 -62 .655 6 .383 1 .000 11 .56

ATOM 964 C TYR 129 19 416 -66 .840 10 .765 1 .000 9 .63

ATOM 965 O TYR 129 20 .644 -66 .723 10 .714 1 .000 13 .75

ATOM 966 N PRO 130 18 .789 -67 .380 11 .804 1 .000 8 .51

ATOM 967 CD PRO 130 ^■17 .336 -67 .523 12 .004 1 .000 10 .11 GC821-2

ATOM 968 CA PRO 130 -19.549 -67.914 12.938 1.000 5.53

ATOM 969 CB PRO 130 -18.522 -68.804 13.647 1.000 8.51

ATOM 970 CG PRO 130 -17.227 -68.097 13.397 .000 11.17

ATOM 971 C PRO 130 -19.983 -66.791 13.872 000 77

ATOM 972 O PRO 130 -19.500 -65.667 13.730 000 72

ATOM 973 N ALA 131 -20.873 -67.117 14.799 000 61

ATOM 974 CA ALA 131 -21.305 -66.205 15.844 000 73

ATOM 975 CB ALA 131 -22.537 -66.747 16.554 000 00

ATOM 976 C ALA 131 -20.174 -65.984 16.842 000 8.30

ATOM 977 O ALA 131 -19.502 -66.942 17.223 000 12.18

ATOM 978 N PRO 132 -19.937 -64.752 17.273 ,000 14.28

ATOM 979 CD PRO 132 -20.610 -63.516 16.842 000 11.04

ATOM 980 CA PRO 132 -18.901 -64.505 18, .284 1. ,000 12, .37

ATOM 981 CB PRO 132 -18.696 -62.992 18. ,181 1. ,000 14, .35

ATOM 982 CG PRO 132 -20.032 -62.472 17, .753 1. .000 12, .70

ATOM 983 C PRO 132 -19.395 -64.884 19, .675 1. .000 12, .80

ATOM 984 O PRO 132 -20.608 -65.027 19. .856 1. ,000 21, .24

ATOM 985 N LYS 133 -18.497 -65.051 20. .641 1. .000 14, .17

ATOM 986 CA LYS 133 -18.903 -65.337 22, .017 1. .000 14, .31

ATOM 987 CB LYS 133 -17.760 -65.881 22. .869 1. .000 14, .22

ATOM 988 CG LYS 133 -17.050 -67.101 22, .317 1. .000 13, .51

ATOM 989 CD LYS 133 -15.746 -67.358 23, .057 1, .000 18, .76

ATOM 990 CE LYS 133 -15.463 -68.849 23, .174 1. .000 21, .23

ATOM 991 NZ LYS 133 -15.154 -69.237 24, .580 1, .000 37, .08

ATOM 992 C LYS 133 -19.441 -64.066 22, .667 1. .000 10. .23

ATOM 993 O LYS 133 -19.319 -62.982 22, .091 1. .000 4, .45

ATOM 994 N VAL 134 -20.032 -64.194 23, .853 1, .000 4. .74

ATOM 995 CA VAL 134 -20.562 -63.000 24. ,507 1. .000 10, .55

ATOM 996 CB VAL 134 -22.106 -62.964 24, .490 1. .000 11. .86

ATOM 997 CGI VAL 134 -22.586 -61.523 24, .423 1, .000 0, .00

ATOM 998 CG2 VAL 134 -22.659 -63.778 23, .334 1. .000 29. .88

ATOM 999 C VAL 134 -20.129 -62.885 25, .963 1, .000 12, .01

ATOM 1000 O VAL 134 -20.215 -63.837 26, .736 1, .000 27, .94

ATOM 1001 N LEU 135 -19.676 -61.703 26, ,357 1, .000 12, .21

ATOM 1002. CA LEU 135 -19.364 -61.443 27, .757 1, .000 14 .41

ATOM 1003 CB LEU 135 -17.975 -60.835 27, .898 1, .000 17, .37

ATOM 1004 CG LEU 135 -17.123 -61.223 29, .105 1, .000 1 L88. .5577

ATOM 1005 CDl LEU 135 -15.993 -60.213 29.264 1.000 4.42

ATOM 1006 CD2 LEU 135 -17.932 -61.341 30.387 1.000 6.01

ATOM 1007 C LEU 135 -20.397 -60.497 28 . 360 1 . 000 17 . 03

ATOM 1008 O LEU 135 -20.485 -59.326 27 . 984 1.000 14 . 19

ATOM 1009 N VAL 136 -21.196 -60.988 29.303 ,000 19.10

ATOM 1010 CA VAL 136 -22.167 -60.110 29.954 .000 14.45

ATOM 1011 CB VAL 136 -23.344 -60.925 30.511 1.000 13.65

ATOM 1012 CGI VAL 136 -24.272 -60.045 31.335 1.000 8.06

ATOM 1013 CG2 VAL 136 -24.080 -61.596 29.362 1.000 0.00

ATOM 1014 C VAL 136 -21.498 -59.327 31.073 1.000 10.63 GC821-2

ATOM 1015 O VAL 136 20 929 -59 948 31 971 1.000 ^•7.12

ATOM 1016 N VAL 137 21 556 -57 997 31 027 1.000 7.93

ATOM 1017 CA VAL 137 20 882 -57 215 32 056 1.000 6.63

ATOM 1018 CB VAL 137 19 699 -56 397 31 497 1.000 6.08

ATOM 1019 CGI VAL 137 19 115 -55 512 32 595 1.000 6.59

ATOM 1020 CG2 VAL 137 18 609 -57 291 30 936 1.000 10.34

ATOM 1021 C VAL 137 21 828 -56 255 32 775 1.000 6.02

ATOM 1022 O VAL 137 22 319 -55 273 32 219 1.000 11.10

ATOM 1023 N SER 138 22 061 -56 558 34 040 1.000 6.05

ATOM 1024 CA SER 138 22 800 -55 715 34 972 1.000 9.77

ATOM 1025 CB SER 138 23 139 -56 523 36 223 1.000 16.98

ATOM 1026 OG SER 138 23 850 -55 804 37 202 1.000 19.18

ATOM 1027 C SER 138 21 944 -54 496 35 276 1.000 8.41

ATOM 1028 O SER 138 20 779 -54 646 35 652 1.000 13.52

ATOM 1029 N PRO 139 22 459 -53 287 35 096 1.000 12.22

ATOM 1030 CD PRO 139 23 803 -52 952 34 599 1.000 11.54

ATOM 1031 CA PRO 139 21 657 -52 087 35 389 1.000 6.14

ATOM 1032 CB PRO 139 22 422 -51 015 34 608 1.000 7.78

ATOM 1033 CG PRO 139 23 848 -51 455 34 731 1.000 3.74

ATOM 1034 C PRO 139 21 620 -51 775 36 875 1.000 3.92

ATOM 1035 O PRO 139 22 460 -52 217 37 664 1.000 10.47

ATOM 1036 N PRO 140 20 636 -51 014 37 347 1.000 8.52

ATOM 1037 CD PRO 140 19 524 -50 412 36 611 1.000 3.33

ATOM 1038 CA PRO 140 20 591 -50 724 38 788 1.000 13.50

ATOM 1039 CB PRO 140 19 251 -50 012 38 971 1.000 12.27

ATOM 1040 CG PRO 140 18 843 -49 543 37 623 1.000 6.73

ATOM 1041 C PRO 140 21 748 -49 832 39 228 1.000 15.77

ATOM 1042 0 PRO 140 22 321 -49 073 38 445 1.000 21.96

ATOM 1043 N PRO 141 22 103 -49 939 40 505 1.000 4.93

ATOM 1044 CD PRO 141 21 487 -50 799 41 528 1.000 0.26

ATOM 1045 CA PRO 141 23 230 -49 172 41 036 1.000 3.17

ATOM 1046 CB PRO 141 23 254 -49 560 42 521 1.000 4.18

ATOM 1047 CG PRO 141 22 591 -50 897 42 556 1.000 0.00

ATOM 1048 C PRO 141 23 014 -47 671 40 890 1.000 10.32

ATOM 1049 O PRO 141 21 876 -47 203 40 900 1.000 17.58

ATOM 1050 N LEU 142 24 120 -46 942 40 760 1.000 9.20

ATOM 1051 CA LEU 142 24 079 -45 490 40 729 1.000 7.44

ATOM 1052 CB LEU 142 25 421 -44 900 40 288 1.000 7.55

ATOM 1053 CG LEU 142 25 775 -45 119 38 812 1.000 13.23

ATOM 1054 CDl LEU 142 27 262 -44 901 38 566 1.000 0.00

ATOM 1055 CD2 LEU 142 24 932 -44 218 37 921 1.000 1.85

ATOM 1056 C LEU 142 23 711 -44 945 42 109 1.000 13.38

ATOM 1057 O LEU 142 23 .764 -45 680 43 099 1.000 20.55

ATOM 1058 N ALA 143 23 363 -43 670 42 126 1.000 15.81

ATOM 1059 CA ALA 143 22 .960 -42 .941 43 .322 1.000 13.69

ATOM 1060 CB ALA 143 21 .461 -42 .676 43 .239 1.000 3.16

ATOM 1061 C ALA 143 23 .762 -41 .656 43 .475 1.000 16.69 GC821-2

ATOM 1062 0 ALA 143 24 500 -41 280 42 552 1 000 10 61

ATOM 1063 N PRO 144 23 668 -40 968 44 609 1 000 19 19

ATOM 1064 CD PRO 144 22 997 -41 377 45 852 1 000 16 93

ATOM 1065 CA PRO 144 24 315 -39 659 44 745 1 000 19 29

ATOM 1066 CB PRO 144 23 730 -39 076 46 031 1 000 17 13

ATOM 1067 CG PRO 144 22 904 -40 130 46 664 1 000 12 97

ATOM 1068 C PRO 144 24 009 -38 723 43 578 1 000 17 14

ATOM 1069 O PRO 144 22 902 -38 626 43 048 1 000 12 89

ATOM 1070 N MET 145 25 049 -38 002 43 161 1 000 18 09

ATOM 1071 CA MET 145 24 925 -37 064 42 052 1 000 14 70

ATOM 1072 .CB MET 145 25 912 -37 398 40 942 1 000 21 06

ATOM 1073 CG MET 145 25 711 -38 740 40 263 1 000 24 88

ATOM 1074 XD MET 145 27 259 -39 577 39 860 1 000 18 47

ATOM 1075 CE MET 145 27 956 -39 804 41 495 1 000 34 91

ATOM 1076 C MET 145 25 155 -35 645 42 559 1 000 11 49

ATOM 1077 O MET 145 26 205 -35 342 43 116 1 000 18 46

ATOM 1078 N PRO 146 24 182 -34 763 42 367 1 000 6 41

ATOM 1079 CD PRO 146 22 909 -34 993 41 683 1 000 8 62

ATOM 1080 CA PRO 146 24 325 -33 388 42 851 1 000 10 88

ATOM .1081 CB PRO 146 22 916 -32 814 42 759 1 000 10 59

ATOM 1082 CG PRO 146 22 064 -33 819 42 072 1 000 12 17

ATOM 1083 C PRO 146 25 292 -32 588 41 972 1 000 13 13

ATOM 1084 O PRO 146 25 999 -31 712 42 484 1 000 17 39

ATOM 1085 N HIS 147 25 311 -32 901 40 677 1 000 10 50

ATOM 1086 CA HIS 147 26 203 -32 215 39 758 1 000 9 69

ATOM 1087 CB HIS 147 25 865 -32 480 38 279 1 000 14 24

ATOM 1088 CG HIS 147 26 441 -31 373 37 431 1 000 6 69

ATOM 1089 CD2 HIS 147 25 875 -30 297 36 850 1 000 5 99

ATOM 1090 ND1 HIS 147 27 780 -31 296 37 134 1 000 11 40

ATOM 1091 CE1 HIS 147 28 018 -30 226 36 391 1 000 11 68

ATOM 1092 NE2 HIS 147 26 871 -29 600 .36 201 1 000 12 68

ATOM 1093 C HIS 147 27 658 -32 596 40 ^"013 1 000 5 47

ATOM 1094 O HIS 147 28 052 -33 761 39 960 1 000 11 15

ATOM 1095 N PRO 148 28 463 -31 575 40 291 1 000 12 88

ATOM 1096 CD PRO 148 28 098 -30 148 40 322 1 000 12 98

ATOM 1097 CA PRO 148 29 877 -31 806 40 602 1 000 13 30

ATOM 1098 CB PRO 148 30 440 -30 401 40 811 1 000 14 82

ATOM 1099 CG PRO 148 29 426 -29 455 40 267 1 000 16 64

ATOM 1100 C PRO 148 30 600 -32 508 39 456 1 000 15 39

ATOM 1101 O PRO 148 31 525 -33 290 39 689 1 000 15 71

ATOM 1102 N TRP 149 30 218 -32 263 38 201 1 000 21 29

ATOM 1103 CA TRP 149 30 909 -32 947 37 109 1 000 15 64

ATOM 1104 CB TRP 149 30 571 -32 328 35 750 1 000 17 31

ATOM 1105 CG TRP 149 31 296 -33 043 34 639 1 000 10 06

ATOM 1106 CD2 TRP 149 32 715 -33 086 34 444 1 000 4 30

ATOM 1107 CE2 TRP 149 32 952 -33 862 33 295 1 .000 8 55

ATOM 1108 CE3 TRP 149 33 805 -32 541 35 129 1 .000 4 24 GC821-2

ATOM 1109 CDl TRP 149 -30.748 -33.774 33.629 1.000 11.09

ATOM 1110 NE1 TRP 149 -31.736 -34.272 32.813 1.000 5.61

ATOM llll CZ2 TRP 149 -34.240 -34.107 32.815 1.000 12.36

ATOM 1112 CZ3 TRP 149 -35,076 -32.785 34.654 1.000 13.41

ATOM 1113 CH2 TRP 149 -35.286 -33.563 33.505 1.000 14.13

ATOM 1114 C TRP 149 -30.566 -34.432 37.101 1.000 12.85

ATOM 1115 O TRP 149 -31.447 -35.290 37.033 1.000 7.92

ATOM 1116 N PHE 150 -29.270 -34.728 37.186 000 11.11

ATOM 1117 CA PHE 150 -28.841 -36.125 37.305 000 11.76

ATOM 1118 CB PHE 150 -27.321 -36.192 37.483 000 8.65

ATOM 1119 CG PHE 150 -26.581 -36.170 36.150 000 13.44

ATOM 1120 CDl PHE 150 -25.315 -35.623 36.047 000 14.41

ATOM 1121 CD2 PHE 150 -27.167 -36.697 35.014 000 12.01

ATOM 1122 CE1 PHE 150 -24.650 -35.604 34.838 000 14.96

ATOM 1123 CE2 PHE 150 -26.511 -36.684 33.797 000 13.41

ATOM 1124 CZ PHE 150 -25.246 -36.136 33.711 000 18.95

ATOM 1125 C PHE 150 -29.555 -36.813 38.459 000 10.90

ATOM 1126 O PHE 150 -30.059 -37.930 38.354 000 7.95

ATOM 1127 N GLN 151 -29.606 -36.120 39.598 000 12.36

ATOM 1128 CA GLN 151 -30.294 -36.665 40.759 000 19.45

ATOM 1129 CB GLN 151 -30.306 -35.680 41.932 000 12.11

ATOM 1130 CG GLN 151 -28.947 -35.446 42.561 000 16.34

ATOM 1131 CD GLN 151 -29.048 -34.481 43.734 000 22.05

ATOM 1132 OEl GLN 151 -29.693 -34.803 44.729 000 39.76

ATOM 1133 NE2 GLN 151 -28.423 -33.317 43.598 000 16.49

ATOM 1134 C GLN 151 -31.745 -37.027 40.441 1.000 20.77

ATOM 1135 O GLN 151 -32.232 -38.044 40.936 1.000 19.36

ATOM 1136 N LEU 152 -32.397 -36.183 39.644 1.000 11.67

ATOM 1137 CA LEU 152 -33.818 -36.360 39.365 1.000 13.95

ATOM 1138 CB LEU 152 -34.438 -35.101 38.764 1.000 14.14

ATOM 1139 CG LEU 152 -34.837 -33.957 39.688 1.000 12.09

ATOM 1140 CDl LEU 152 -34.781 -32.631 38.935 1.000 11.66

ATOM 1141 CD2 LEU 152 -36.225 -34.162 40.274 1.000 12.14

ATOM 1142 C LEU 152 -34.053 -37.544 38.428 1.000 13.07

ATOM 1143 O LEU 152 -34.913 -38.372 38.729 1.000 13.96

ATOM 1144 N ILE 153 -33.310 -37.613 37.326 1.000 13.21

ATOM 1145 CA ILE 153 -33.519 -38.661 36.334 1.000 12.12

ATOM 1146 CB ILE 153 -32.814 -38.377 34.991 1.000 9.74

ATOM 1147 CG2 ILE 153 -33.360 -37.106 34.355 1.000 0.00

ATOM 1148 CGI ILE 153 -31.284 -38.333 35.061 000 8.16

ATOM 1149 CDl ILE 153 -30.635 -38.332 33.684 000 0.00

ATOM 1150 C ILE 153 -33.054 -40.024 36.836 000 9.56

ATOM 1151 O ILE 153 -33.540 -41.043 36.342 000 4.79

ATOM 1152 N PHE 154 -32.138 -40.069 37.797 000 12.41

ATOM 1153 CA PHE 154 -31.645 -41.349 38.301 000 8.75

ATOM 1154 CB PHE 154 -30.113 -41.372 38.348 1.000

ATOM 1155 CG PHE 154 -29.456 -41.758 37.031 1.000 8.38 GC821-2

ATOM 1156 CDl PHE 154 28 597 -40 887 36 384 1. 000 9. 10

ATOM 1157 CD2 PHE 154 29 703 -42 990 36 458 1. 000 0. 00

ATOM 1158 CE1 PHE 154 28 000 -41 232 35 188 1 000 9 85

ATOM 1159 CE2 PHE 154 29 119 -43 344 35 260 1 000 5 02

ATOM 1160 CZ PHE 154 28 258 -42 468 34 624 1. 000 8. 39

ATOM 1161 C PHE 154 32 199 -41. 648 39 690 1. 000 11. 55

ATOM 1162 O PHE 154 31 683 -42 515 40 400 1. 000 10. 77

ATOM 1163 N GLU 155 33 246 -40 936 40 093 1. 000 15. 11

ATOM 1164 CA GLU 155 33 898 -41 221 41 367 1. 000 19 95

ATOM 1165 CB GLU 155 35 134 -40 343 41 542 1 000 26 08

ATOM 1166 CG GLU 155 35 558 -40 107 42 980 1 000 33 00

ATOM 1167 CD GLU 155 36 339 -41 267 43 568 1 000 44 51

ATOM 1168 OEl GLU 155 37 432 -41 585 43 051 1 000 49 47

ATOM 1169 OE2 GLU 155 35 862 -41 867 44 558 1 000 61 39

ATOM 1170 C GLU 155 34 270 -42 702 41 449 1 000 18 82

ATOM 1171 O GLU 155 34 978 -43 212 40 582 1 000 14 49

ATOM 1172 N GLY 156 33 779 -43 376 42 481 1 000 12 58

ATOM 1173 CA GLY 156 33 993 -44 787 42 696 1 000 6 50

ATOM 1174 C GLY 156 33 061 -45 684 41 914 1 000 12 22

ATOM 1175 O GLY 156 33 205 -46 914 41 914 1 000 27 90

ATOM 1176 N GLY 157 32 082 -45 107 41 224 1 000 9 19

ATOM 1177 CA GLY 157 31 216 -45 877 40 358 1 000 8 21

ATOM 1178 C GLY 157 30 007 -46 514 40 991 1 000 8 61

ATOM 1179 O GLY 157 29 563 -47 579 40 549 1 000 17 22

ATOM 1180 N GLU 158 29 442 -45 887 42 018 1 000 7 58

ATOM 1181 CA GLU 158 28 299 -46 453 42 721 1 000 7 50

ATOM • 1182 CB GLU 158 27 807 -45 505 43 814 1 000 9 84

ATOM 1183 CG GLU 158 26 756 -46 097 44 739 1 000 11 00

ATOM 1184 CD GLU 158 26 031 -45 053 45 564 1 000 24 40

ATOM 1185 OEl GLU 158 26 158 -43 845 45 267 1 000 33 57

ATOM 1186 OE2 GLU 158 25 325 -45 439 46 523 1 000 39 11

ATOM 1187 C GLU 158 28 696 -47 807 43 302 1 000 13 34

ATOM 1188 O GLU 158 27 956 -48 787 43 225 1 000 29 78

ATOM 1189 N GLN 159 29 895 -47 840 43 875 1 000 10 17

ATOM 1190 CA GLN 159 30 481 -49 058 44 406 1 000 15 50

ATOM 1191 CB GLN 159 31 856 -48 764 45 017 1 000 19 57

ATOM 1192 CG GLN 159 32 548 -49 952 45 647 1 000 24 93

ATOM 1193 CD GLN 159 31 737 -50 676 46 704 1 000 30 24

ATOM 1194 OEl GLN 159 31 940 -50 499 47 909 1 000 40 80

ATOM 1195 NE2 GLN 159 30 800 -51 510 46 265 1 000 20 75

ATOM 1196 C GLN 159 30 605 -50 132 43 336 1 000 17 89

ATOM 1197 O GLN 159 30 218 -51 285 43 .544 1 .000 21 .71

ATOM 1198 N LYS 160 31 .154 -49 .791 42 .168 1 .000 15 .99

ATOM 1199 CA LYS 160 31 361 -50 855 41 .176 1 .000 6 .75

ATOM 1200 CB LYS 160 32 .314 -50 369 40 .090 1 .000 10 .24

ATOM 1201 CG LYS 160 33 .666 -49 .907 40 .607 1 .000 6 .13

ATOM 1202 CD LYS 160 34 .386 -49 .041 39 .581 1 .000 11 .21 GC821-2

ATOM 1203 CE LYS 160 35 897 -49 190 39 702 1 000 9 55

ATOM 1204 NZ LYS 160 36 616 -48 235 38 811 1 000 20 37

ATOM 1205 C LYS 160 30 029 -51 305 40 591 1 000 14 32

ATOM 1206 O LYS 160 29 842 -52 475 40 257 1 000 14 42

ATOM 1207 N THR 161 29 082 -50 375 40 465 1 000 10 29

ATOM 1208 CA THR 161 27 771 -50 734 39 933 1 000 13 43

ATOM 1209 CB THR 161 26 878 -49 508 39 672 1 000 10 03

ATOM 1210 OG1 THR 161 27 070 -48 557 40 730 1 000 30 01

ATOM 1211 CG2 THR 161 27 263 -48 788 38 389 1 000 13 57

ATOM 1212 C THR 161 27 057 -51 683 40 896 1 000 12 06

ATOM 1213 O THR 161 26 160 -52 415 40 481 1 000 6 51

ATOM 1214 N THR 162 27 457 -51 664 42 165 1 000 8 39

ATOM 1215 CA THR 162 26 894 -52 551 43 177 1 000 9 75

ATOM 1216 CB THR 162 27 286 -52 130 44 604 1 000 12 96

ATOM 1217 OG1 THR 162 26 705 -50 863 44 941 1 000 11 98

ATOM 1218 CG2 THR 162 26 735 -53 132 45 605 1 000 20 35

ATOM 1219 C THR 162 27 349 -53 991 42 956 1 000 10 87

ATOM 1220 0 THR 162 26 764 -54 942 43 471 1 000 12 87

ATOM 1221 N GLU 163 28 410 -54 170 42 174 1 000 16 58

ATOM 1222 CA GLU 163 28 949 -55 496 41 905 1 000 20 69

ATOM 1223 CB GLU 163 30 486 -55 450 41 861 1 000 21 36

ATOM 1224 CG GLU 163 31 136 -54 918 43 122 1 000 19 81

ATOM 1225 CD GLU 163 30 918 -55 799 44 332 1 000 20 57

ATOM 1226 OEl GLU 163 30 336 -56 894 44 181 1 000 13 38

ATOM 1227 OE2 GLU 163 31 340 -55 394 45 441 1 000 37 36

ATOM 1228 C GLU 163 28 455 -56 101 40 596 1 000 12 31

ATOM 1229 O GLU 163 28 614 -57 306 40 384 1 000 8 17

ATOM 1230 N LEU 164 27 880 -55 296 39 710 1 000 14 12

ATOM 1231 CA LEU 164 27 561 -55 746 38 356 1 000 8 92

ATOM 1232 CB LEU 164 26 960 -54 602 37 541 1 000 5 54

ATOM 1233 CG LEU 164 27 903 -53 857 36 593 1 000 10 39

ATOM 1234 CDl LEU 164 29 295 -53 740 37 197 1 000 23 43

ATOM 1235 CD2 LEU 164 27 352 -52 485 36 240 1 000 2 48

ATOM 1236 C LEU 164 26 621 -56 943 38 361 1 000 6 54

ATOM 1237 O LEU 164 26 847 -57 925 37 653 1 000 4 26

ATOM 1238 N ALA 165 25 562 -56 865 39 159 1 000 7 24

ATOM 1239 CA ALA 165 24 609 -57 965 39 239 1 000 11 41

ATOM 1240 CB ALA 165 23 542 -57 659 40 276 1 000 11 40

ATOM 1241 C ALA 165 25 312 -59 284 39 551 1 000 16 26

ATOM 1242 O ALA 165 24 980 -60 302 38 947 1 000 18 13

ATOM 1243 N ARG 166 26 266 -59 245 40 .469 1 .000 20 04

ATOM 1244 CA ARG 166 27 014 -60 397 40 947 1 000 10 10

ATOM 1245 CB ARG 166 27 875 -59 .992 42 .145 1 .000 15 .40

ATOM 1246 CG ARG 166 28 .600 -61 .127 42 .843 1 .000 15 .67

ATOM 1247 CD ARG 166 29 .286 -60 .640 44 .115 1 .000 20 .34

ATOM 1248 NE ARG 166 30 .097 -59 .453 43 .851 1 .000 31 .99

ATOM 1249 CZ ARG 166 31 .261 -59 .505 43 .202 1 .000 37 .46 GC821-2

ATOM 1250 NH1 ARG 166 31 718 -60 673 42 770 1 000 41 26

ATOM 1251 NH2 ARG 166 31 974 -58 410 42 979 1 000 44 85

ATOM 1252 C ARG 166 27 899 -60 991 39 862 1 000 10 33

ATOM 1253 O ARG 166 27 .862 -62 186 39 569 1 000 11 28

ATOM 1254 N VAL 167 28 724 -60 143 39 253 1 000 10 14

ATOM 1255 CA VAL 167 29 647 -60 637 38 231 1 000 8 08

ATOM 1256 CB VAL 167 30 800 -59 642 38 007 1 000 12 63

ATOM 1257 CGI VAL 167 31 873 -60 262 37 129 1 000 23 15

ATOM 1258 CG2 VAL 167 31 423 -59 212 39 331 1 000 16 49

ATOM 125,9 C VAL 167 28 941 -60 943 36 916 1 000 8 93

ATOM 1260 0 VAL 167 29 342 -61 889 36 230 1 000 11 00

ATOM 1261 N TYR 168 27 906 -60 209 36 507 1 000 6 53

ATOM 1262 CA TYR 168 27 225 -60 549 35 262 1 000 5 82

ATOM 1263 CB TYR 168 26 220 -59 494 34 815 1 000 12 35

ATOM 1264 CG TYR 168 26 746 -58. 249 34 148 1 000 10 53

ATOM 1265 CDl TYR 168 25 898 -57 415 33 429 1 000 4 25

ATOM 1266 CE1 TYR 168 26 377 -56 273 32 816 1 000 3 59

ATOM 1267 CD2 TYR 168 28 085 -57 889 34 230 1 000 9 22

ATOM 1268 CE2 TYR 168 28 565 -56 750 33 624 1 000 11 67

ATOM 1269 CZ TYR 168 27 708 -55 940 32 912 1 000 8 76

ATOM 1270 OH TYR 168 28 194 -54 801 32 308 1 000 13 56

ATOM 1271 C TYR 168 26 466 -61 863 35 444 1 000 9 45

ATOM 1272 O TYR 168 26 398 -62 696 34 544 1 000 5 20

ATOM 1273 N SER 169 25 896 -61 972 36 648 1 000 5 94

ATOM 1274 CA SER 169 25 145 -63 174 36 999 1 000 11 65

ATOM 1275 CB SER 169 24 663 -63 109 38 445 1 000 12 52

ATOM 1276 OG SER 169 23 611 -64 024 38 688 1 000 13 86

ATOM 1277 C SER 169 26 034 -64 389 36 740 1 000 14 93

ATOM 1278 O SER 169 25 709 -65 240 35 912 1 000 25 35

ATOM 1279 N ALA 170 27 161 -64 434 37 448 1 000 9 54

ATOM 1280 CA ALA 170 28 154 -65 483 37 259 1 000 7 33

ATOM 1281 CB ALA 170 29 397 -65 155 38 069 1 000 3 12

ATOM 1282 C ALA 170 28 495 -65 659 35 785 1 000 12 27

ATOM 1283 O ALA 170 28 526 -66 772 35 262 1 000 20 56

ATOM 1284 N LEU 171 28 753 -64 558 35 081 1 000 15 11

ATOM 1285 CA LEU 171 29 115 -64 661 33 665 1 000 17 04

ATOM 1286 CB LEU 171 29 329 -63 272 33 076 1 000 13 64

ATOM 1287 CG LEU 171 29 846 -63 164 31 645 1 000 21 08

ATOM 1288 CDl LEU 171 28 692 -63 043 30 658 1 000 45 18

ATOM 1289 CD2 LEU 171 30 734 -64 340 31 270 1 000 17 34

ATOM 1290 C LEU 171 28 052 -65 404 32 868 1 000 18 57

ATOM 1291 O LEU 171 28 328 -66 409 32 219 1 000 17 64

ATOM 1292 N ALA 172 26 825 -64 890 32 920 1 .000 22 .46

ATOM 1293 CA ALA 172 25 735 -65 489 32 157 1 .000 17 47

ATOM 1294 CB ALA 172 24 454 -64 699 32 .377 1 .000 10 .29

ATOM 1295 C ALA 172 25 .549 -66 .953 32 .536 1 .000 13 .15

ATOM 1296 O ALA 172 25 192 -67 .797 31 .713 1 .000 17 .25 GC821-2

ATOM 1297 N SER 173 25 802 -67 242 33 809 1 000 11 55

ATOM 1298 CA SER 173 25 653 -68 595 34 337 1 000 15 80

ATOM 1299 CB SER 173 25 837 -68 578 35 856 1 000 15 14

ATOM 1300 OG SER .173 26 298 -69 837 36 293 1 000 15 66

ATOM 1301 C SER 173 26 640 -69 565 33 691 1 000 10- 39

ATOM 1302 O SER 173 26 263 -70 667 33 284 1 000 5 06

ATOM 1303 N PHE 174 27 882 -69 119 33 601 1 000 6 57

ATOM 1304 CA PHE 174 28 970 -69 778 32 908 1 000 4 04

ATOM 1305 CB PHE 174 30 288 -69 024 33 .114 1 000 4 43

ATOM 1306 CG PHE 174 31 524 -69 765 32 .626 1 000 3 57

ATOM 1307 CDl PHE 174 32 219 -70 606 33 475 1 000 0 40

ATOM 1308 CD2 PHE 174 31 988 -69 615 31 331 1 000 11 71

ATOM 1309 CE1 PHE 174 33 343 -71 281 33 .051 1 000 1 63

ATOM 1310 CE2 PHE 174 33 114 -70 285 30 886 1 000 10 57

ATOM 1311 CZ PHE 174 33 795 -71 119 31 756 1 000 10 59

ATOM 1312 c PHE 174 28 701 -69 872 31 408 1 000 8 80

ATOM 1313 O PHE 174 28 846 -70 949 30 834 1 000 0 14

ATOM 1314 N MET 175 28 328 -68 751 30 793 1 000 7 91

ATOM. 1315 CA .MET 175 28 058 -68 739 29 356 1 000 5 97

ATOM 1316 CB MET 175 28 103 -67 321 28 780 1 000 0 00

ATOM 1317 CG MET 175 29 492 -66 712 28 751 1 000 7 42

ATOM 1318 XD MET 175 29 573 -65 056 28 023 1 000 16 37

ATOM 1319 CE MET 175 30 064 -65 488 26 348 1 000 21 02

ATOM 1320 C MET. 175 26 715 -69 399 29 045 1 000 6 31

ATOM 1321 O MET 175 26 332 -69 479 27 880 1 000 8 17

ATOM 1322 N LYS 176 26 020 -69 872 30 070 1 000 8 77

ATOM 1323 CA LYS 176 24 762 -70 598 29 939 1 000 10 68

ATOM 1324 CB LYS 176 24 970 -71 945 29 239 1 000 10 45

ATOM 1325 CG LYS 176 25 907 -72 900 29 971 1 000 3 74

ATOM 1326 CD LYS 176 25 133 -73 755 30 964 1 000 5 05

ATOM 1327 CE LYS 176 26 084 -74 568 31 833 1 000 6 09

ATOM 1328 NZ LYS 176 26 739 -73 721 32 861 1 000 24 38

ATOM 1329 C LYS 176 23 733 -69 760 29 190 1 000 12 34

ATOM 1330 O LYS 176 23 084 -70 178 28 231 1 000 24 85

ATOM 1331 N VAL 177 23 601 -68 520 29 648 1 000 12 09

ATOM 1332 CA VAL 177 22 709 -67 581 28 953 1 000 12 10

ATOM 1333 CB VAL 177 23 569 -66 629 28 106 1 000 9 74

ATOM 1334 CGI VAL 177 23 831 -65 319 28 835 1 000 18 59

ATOM 1335 CG2 VAL 177 22 921 -66 372 26 753 1 000 20 30

ATOM 1336 C VAL 177 21 848 -66 876 29 982 1 000 13 62

ATOM 1337 O VAL 177 22 292 -66 730 31 126 1 000 20 25

ATOM 1338 N PRO 178 20 635 -66 454 29 637 1 000 10 56

ATOM 1339 CD PRO 178 20 019 -66 530 28 312 1 000 2 11

ATOM 1340 CA PRO 178 19 760 -65 842 30 642 1 000 10 32

ATOM 1341 CB PRO 178 18 433 -65 656 29 913 1 000 6 70

ATOM 1342 CG PRO 178 18 623 -66 026 28 499 1 000 0 81

ATOM 1343 C PRO 178 20 .281 -64 483 31 .119 1 .000 20 .65 GC821-2

ATOM 1344 O PRO 178 20 796 -63 674 30 351 1 000 22 70

ATOM 1345 N PHE 179 20 124 -64 253 32 412 1 000 22 55

ATOM 1346 CA PHE 179 20 474 -63 025 33 107 1 000 19 13

ATOM 1347 CB PHE 179 21 518 -63 283 34 194 1 000 8 91

ATOM 1348 CG PHE 179 21 661 -62 215 35 268 1 000 8 12

ATOM 1349 CDl PHE 179 22 433 -61 087 35 044 1 000 10 36

ATOM 1350 CD2 PHE 179 21 031 -62 337 36 499 1 000 2 04

ATOM 1351 CE1 PHE 179 22 590 -60 103 36 004 1 000 2 43

ATOM 1352 CE2 PHE 179 21 183 -61 367 37 470 1 000 0 76

ATOM 1353 CZ PHE 179 21 963 -60 248 37 228 1 000 2 96

ATOM 1354 C PHE 179 19 231 -62 400 33 736 1 000 13 74

ATOM 1355 O PHE 179 18 309 -63 110 34 128 1 000 15 60

ATOM 1356 N PHE 180 19 214 -61 080 33 838 1 000 14 28

ATOM 1357 CA PHE 180 18 178 -60 371 34 573 1 000 13 03

ATOM 1358 CB PHE 180 17 004 -59 952 33 686 1 000 17 94

ATOM 1359 CG PHE 180 15 933 -59 164 34 433 1 000 21 76

ATOM 1360 CDl PHE 180 14 960 -59 807 35 176 1 000 21 38

ATOM 1361 CD2 PHE 180 15 904 -57 780 34 391 1 000 19 62

ATOM 1362 CE1 PHE 180 13 979 -59 108 35 859 1 000 15 07

ATOM 1363 CE2 PHE 180 14 941 -57 064 35 075 1 000 21 73

ATOM 1364 CZ PHE 180 13 979 -57 727 35 816 1 000 21 65

ATOM 1365 C PHE 180 18 822 -59 164 35 256 1 000 12 16

ATOM 1366 O PHE 180 19 594 -58 423 34 648 1 000 11 01

ATOM 1367 N ASP 181 18 504 -58 988 36 536 1 000 7 72

ATOM 1368 CA ASP 181 19 062 -57 864 37 286 1 000 10 61

ATOM 1369 CB ASP 181 19 521 -58 346 38 659 1 000 5 77

ATOM 1370 CG ASP 181 19 986 -57 225 39 559 1 000 4 11

ATOM 1371 OD1 ASP 181 20 116 -56 076 39 092 1 000 8 61

ATOM 1372 OD2 ASP 181 20 217 -57 508 40 750 1 000 11 49

ATOM 1373 C ASP 181 18 037 -56 743 37 378 1 000 15 44

ATOM 1374 O ASP 181 17 023 -56 872 38 060 1 000 16 84

ATOM 1375 N ALA 182 18 293 -55 639 36 672 1 000 18 65

ATOM 1376 CA ALA 182 17 359 -54 517 36 678 1 000 18 00

ATOM 1377 CB ALA 182 17 778 -53 459 35 668 1 000 7 66

ATOM 1378 C ALA 182 17 240 -53 911 38 075 1 000 18 92

ATOM 1379 O ALA 182 16 198 -53 340 38 400 1 000 8 61

ATOM 1380 N GLY 183 18 296 -54 044 38 872 1 000 15 67

ATOM 1381 CA GLY 183 18 374 -53 516 40 219 1 000 13 53

ATOM 1382 C GLY 183 17 444 -54 230 41 176 1 000 14 96

ATOM 1383 O GLY 183 17 268 -53 846 42 330 1 000 25 31

ATOM 1384 N SER 184 16 830 -55 306 40 696 1 000 16 38

ATOM 1385 CA SER 184 15 940 -56 105 41 525 1 000 12 32

ATOM 1386 CB SER 184 16 009 -57 574 41 116 1 000 14 55

ATOM 1387 OG SER 184 15 237 -57 867 39 967 1 .000 12 36

ATOM 1388 C SER 184 14 516 -55 .572 41 .439 1 .000 13 .33

ATOM 1389 O SER 184 13 .644 -55 .986 42 .204 1 .000 12 .05

ATOM 1390 N VAL 185 14 276 -54 .640 40 .515 1 .000 9 .89 GC821-2

ATOM 1391 CA VAL 185 12. 902 -54. 156 40 358 1 000 14 54

ATOM 1392 CB VAL 185 12 320 -54 649 39 021 1 000 16 34

ATOM 1393 CGI VAL 185 12. 034 -56 141 39 100 1 000 13 09

ATOM 1394 CG2 VAL 185 13 274 -54 346 37 877 1 000 20 34

ATOM 1395 C VAL 185 12 802 -52 642 40 445 1 000 20 13

ATOM 1396 O VAL 185 11 718 -52 101 40 682 1 000 11 67

ATOM 1397 N ILE 186 13 912 -51 929 40 260 1 000 19 83

ATOM 1398 CA ILE 186 13. 905 -50 479 40 381 1 000 13 97

ATOM 1399 CB ILE 186 13 716 -49 752 39 031 1 000 8 30

ATOM 1400 CG2 ILE 186 12 362 -50 070 38 428 1 000 12 39

ATOM 1401 CGI ILE 186 14 830 -50 005 38 014 1 000 10 45

ATOM 1402 CDl ILE 186 14 956 -48 929 36 957 1 000 3 60

ATOM 1403 C ILE 186 15 209 -49 957 40 979 1 000 13 38

ATOM 1404 O ILE 186 16 256 -50 583 40 857 1 000 12 90

ATOM 1405 N SER 187 15 120 -48 788 41 596 1 000 11 99

ATOM 1406 CA SER 187 16 287 -48 046 42 052 1 000 9 16

ATOM 1407 CB SER 187 16 110 -47 594 43 498 1 000 10 88

ATOM 1408 OG SER 187 14 889 -46 879 43 658 1 000 16 58

ATOM 1409 C SER 187 16 517 -46 839 41 145 1 000 11 87

ATOM 1410 O SER 187 15 567 -46 304 40 563 1 000 16 73

ATOM 1411 N THR 188 17 767 -46 410 41 015 1 000 15 17

ATOM 1412 CA THR 188 18 077 -45 244 40 189 1 000 13 51

ATOM 1413 CB THR 188 19 571 -45 151 39 848 1 000 12 88

ATOM 1414 OG1 THR 188 19 969 -46 308 39 101 1 000 16 33

ATOM 1415 CG2 THR 188 19 843 -43 943 38 961 1 000 8 08

ATOM 1416 C THR 188 17 639 -43 978 40 916 1 000 14 09

ATOM 1417 0 THR 188 18 293 -43 535 41 860 1 000 10 72

ATOM 1418 N ASP 189 16 518 -43 414 40 474 1 000 15 51

ATOM 1419 CA ASP 189 15 911 -42 313 41 210 1 000 11 58

ATOM , 1420 CB ASP 1.89 14 407 -42 594 41 362 1 000 12 86

ATOM 1421 CG ASP 189 14 158 -43 791 42 261 1 000 4 55

ATOM 1422 OD1 ASP 189 14 915 -43 960 43 239 1 000 13 27

ATOM 1423 OD2 ASP 189 13 208 -44 549 41 989 1 000 6 91

ATOM 1424 C ASP 189 16 120 -40 949 40 567 1 000 15 34

ATOM 1425 O ASP 189 15 910 -39 948 41 263 1 000 18 48

ATOM 1426 N GLY 190 16 510 -40 918 39 303 1 000 19 39

ATOM 1427 CA GLY 190 16 710 -39 718 38 515 1 000 15 08

ATOM 1428 C GLY 190 17 385 -38 613 39 303 1 000 18 57

ATOM 1429 O GLY 190 18 263 -38 908 40 119 1 000 20 64

ATOM 1430 N VAL 191 16 952 -37 381 39 057 1 000 13 86

ATOM 1431 CA VAL 191 17 428 -36 226 39 806 1 000 10 59

ATOM 1432 CB VAL 191 16 825 -34 905 39 286 1 000 17 05

ATOM 1433 CGI VAL 191 15 324 -34 875 39 559 1 000 30 84

ATOM 1434 CG2 VAL 191 17 092 -34 .701 37 .803 1 .000 8 .10

ATOM 1435 C VAL 191 18 950 -36 129 39 774 1 000 10 47

ATOM 1436 O VAL 191 19 .542 -35 .686 40 .761 1 .000 13 .60

ATOM 1437 N ASP 192 19 .571 -36 .534 38 .668 1 .000 1 .46 GC821-2

ATOM 1438 CA ASP 192 21 018 -36 447 38 540 1 000 0 70

ATOM 1439 CB ASP 192 21 387 -36 356 37 056 1 000 2 10

ATOM 1440 CG ASP 192 20 918 -37 566 36 268 1 000 9 82

ATOM 1441 OD1 ASP 192 20 296 -38 478 36 857 1 000 8 20

ATOM 1442 OD2 ASP 192 21 182 -37 597 35 047 1 000 6 78

ATOM 1443 C ASP 192 21 754 -37 622 39 173 1 000 7 73

ATOM 1444 O ASP 192 22 988 -37 674 39 136 1 000 7 10

ATOM 1445 N GLY 193 21 027 -38 572 39 753 1 000 15 10

ATOM 1446 CA GLY 193 21 631 -39 747 40 351 1 000 17 83

ATOM 1447 C GLY 193 22 153 -40 758 39 352 1 000 18 93

ATOM 1448 O GLY 193 22 820 -41 732 39 718 1 000 10 12

ATOM 1449 N ILE 194 21 867 -40 565 38 062 1 000 11 77

ATOM 1450 CA ILE 194 22 330 -41 546 37 081 1 000 7 87

ATOM 1451 CB ILE 194 23 401 -40 945 36 154 1 000 9 95

ATOM 1452 CG2 ILE 194 23 790 -41 927 35 063 1 000 0 00

ATOM 1453 CGI ILE 194 24 643 -40 441 36 896 1 000 9 90

ATOM 1454 CDl ILE 194 25 248 -39 237 36 206 1 000 8 85

ATOM 1455 C ILE 194 21 191 -42 068 36 225 1 000 2 97

ATOM 1456 0 ILE 194 21 086 -43 251 35 924 1 000 6 72

ATOM 1457 N HIS 195 20 277 -41 195 35 792 1 000 6 33

ATOM 1458 CA HIS 195 19 256 -41 719 34 884 1 000 10 76

ATOM 1459 CB HIS 195 19 089 -40 790 33 673 1 000 11 36

ATOM 1460 CG HIS 195 20 402 -40 647 32 958 1 000 11 50

ATOM 1461 CD2 HIS 195 20 981 -41 395 31 989 1 000 5 43

ATOM 1462 ND1 HIS 195 21 283 -39 633 33 253 1 000 7 30

ATOM 1463 CE1 HIS 195 22 351 -39 753 32 485 1 000 9 11

ATOM 1464 NE2 HIS 195 22 192 -40 814 31 711 1 000 8 18

ATOM 1465 C HIS 195 17 918 -41 941 35 577 1 000 8 63

ATOM 1466 O HIS 195 17 762 -41 602 36 743 1 000 13 71

ATOM 1467 N PHE 196 17 010 -42 529 34 812 1 000 6 37

ATOM 1468 CA PHE 196 15 725 -43 017 35 249 1 000 9 06

ATOM 1469 CB PHE 196 15 233 -44 136 34 320 1 000 5 38

ATOM 1470 CG PHE 196 16 048 -45 412 34 451 1 000 10 20

ATOM 1471 CDl PHE 196 15 822 -46 481 33 602 1 000 8 01

ATOM 1472 CD2 PHE 196 17 027 -45 509 35 427 1 000 6 21

ATOM 1473 CE1 PHE 196 16 571 -47 637 33 722 1 000 11 17

ATOM 1474 CE2 PHE 196 17 779 -46 662 35 546 1 000 14 06

ATOM 1475 CZ PHE 196 17 549 -47 727 34 694 1 000 13 03

ATOM 1476 C PHE 196 14 663 -41 925 35 273 1 000 12 92

ATOM 1477 O PHE 196 14 757 -40 983 34 494 1 000 15 16

ATOM 1478 N THR 197 13 694 -42 112 36 158 1 000 13 17

ATOM 1479 CA THR 197 12 477 -41 318 36 183 1 000 17 95

ATOM 1480 CB THR 197 11 886 -41 168 37 593 1 000 20 94

ATOM 1481 OG1 THR 197 11 650 -42 458 38 .173 1 000 20 .14

ATOM 1482 CG2 THR 197 12 882 -40 454 38 .499 1 .000 31 .55

ATOM 1483 C THR 197 11 443 -41 .978 35 .269 1 .000 10 .26

ATOM 1484 O THR 197 11 713 -43 037 34 .705 1 .000 14 .53 GC821-2

ATOM 1485 N GLU 198 10. ,283 -41. ,362 35. ,133 1. ,000 9. ,05

ATOM 1486 CA GLU 198 -9. ,192 -41. ,943 34. ,362 1. ,000 12. ,89

ATOM 1487 CB GLU 198 -8. ,023 -40. ,960 34. ,314 1. ,000 20. ,40

ATOM 1488 CG GLU 198 -6. ,903 -41. ,349 33. ,362 1. ,000 32. .30

ATOM 1489 CD GLU 198 -5. ,764 -40. .346 33. ,328 1. ,000 35. ,77

ATOM 1490 OEl GLU 198 -5. ,127 -40. ,141 34. ,385 1. ,000 42. ,59

ATOM 1491 OE2 GLU 198 -5. ,498 -39. ,761 32. ,256 1. .000 25. .40

ATOM 1492 C GLU 198 -8. ,779 -43. ,279 34. ,970 1. ,000 16. ,23

ATOM 1493 O GLU 198 -8. ,636 -44. ,296 34. ,292 1. ,000 14. .85

ATOM 1494 N ALA 199 -8. ,596 -43. ,284 36. ,291 1. ,000 11. ,36

ATOM 1495 CA ALA 199 -8. ,233 -44. ,489 37. ,022 1. .000 5, .99

ATOM 1496 CB ALA 199 -8. .047 -44. .154 38. ,499 1. .000 2, .34

ATOM 1497 C ALA 199 -9. ,273 -45. ,594 36. ,873 1. .000 7. .89

ATOM 1498 O ALA 199 -8. .922 -46. .767 36. ,748 1, .000 16. .70

ATOM 1499 N ASN 200 ^■10. .548 -45, .210 36. .897 1, .000 13, .48

ATOM 1500 CA ASN 200 ^•11. .644 -46. .155 36. ,715 1. .000 11. .59

ATOM 1501 CB ASN 200 ^■13. .007 -45, .474 36. .805 1. .000 4. .12

ATOM 1502 CG ASN 200 ^■13. .492 -45. .192 38. ,209 1, ,000 11. .67

ATOM 1503 OD1 ASN 200 •13. ,045 -45. .767 39. ,200 1. .000 6. ,19

ATOM 1504 ND2 ASN 200 ^•14. .455 -44. .276 38. ,330 1. .000 13. .74

ATOM 1505 C ASN 200 ^■11. .505 -46, .869 35. .366 1. .000 8. .88

ATOM 1506 O ASN 200 ^•11. .667 -48. ,084 35. ,305 1. ,000 9. .08

ATOM 1507 N ASN 201 ^•11. .208 -46. .111 34. ,315 1. ,000 14. .48

ATOM 1508 CA ASN 201 ^■11, .074 -46, .639 32. ,963 1. .000 14, .27

ATOM 1509 CB ASN 201 ^■10. ,903 -45. ,495 31. ,960 1. .000 16. .17

ATOM 1510 CG ASN 201 ^■12. .221 -44. .853 31. ,570 1. .000 14. .25

ATOM 1511 OD1 ASN 201 ^■13, .050 -45. .436 30. ,871 1, .000 13. .77

ATOM 1512 ND2 ASN 201 ^■12. ,441 -43. ,624 32. .021 1. ,000 16. ,01

ATOM 1513 C ASN 201 -9. .908 -47. ,620 32. ,870 1. .000 12. ,95

ATOM 1514 O ASN 201 ^•10, .050 -48. .720 32. ,334 1. .000 11. ,02

ATOM 1515 N ARG 202 -8. .775 -47. ,207 33. ,412 1. .000 15. ,80

ATOM 1516 CA ARG ' 202 -7. .571 -48. ,020 33. ,532 1, .000 14, .85

ATOM 1517 CB ARG 202 -6, .491 -47, .250 34. ,294 1. .000 17. .85

ATOM 1518 CG ARG 202 -5. .109 -47. .874 34. ,325 1, .000 17. .66

ATOM 1519 CD ARG 202 -4, .141 -47, .026 35. .143 1, .000 19, .69

ATOM 1520 NE ARG 202 -3. .646 -45, .881 34, .388 1, .000 30, .64

ATOM 1521 CZ ARG 202 -2, .410 -45, ,407 34. ,412 1, .000 36. .54

ATOM 1522 NH1 ARG 202 -1. .470 -45. .972 35, .164 1, .000 35, .38

ATOM 1523 NH2 ARG 202 -2, .093 -44, .353 33. .669 1, .000 23, .31

ATOM 1524 C ARG 202 -7, .862 -49. .344 34. .229 1, .000 6, .52

ATOM 1525 O ARG 202 -7, .636 -50, .401 33. .644 1 .000 9 .98

ATOM 1526 N ASP 203 -8. .365 -49. .285 35. ,464 1. .000 3. .83

ATOM 1527 CA ASP 203 -8, .597 -50, .500 36, .237 1 .000 12 .72

ATOM 1528 CB ASP 203 -9 .148 -50 .181 37, .631 1 .000 9 .96

ATOM 1529 CG ASP 203 -8, .170 -49, .370 38, .458 1 .000 16 .04

ATOM 1530 OD1 ASP 203 -6 .980 -49 .324 38 .086 1 .000 18 .66

ATOM 1531 OD2 ASP 203 -8 .584 -48 .772 39 .474 1 .000 22 .09 GC821-2

ATOM 1532 C ASP 203 -9.548 -51.455 35.524 1.000 18.07

ATOM 1533 O ASP 203 -9.383 -52.674 35.579 1.000 12.38

ATOM 1534 N LEU 204 -10.550 -50.890 34.859 1.000 23,73

ATOM 1535 CA LEU 204 -11.541 -51.706 34.169 1.000 21.34

ATOM 1536 CB LEU 204 -12.745 -50.872 33.727 1.000 26.39

ATOM 1537 CG LEU 204 -14.123 -51.510 33.908 1.000 26.92

ATOM 1538 CDl LEU 204 -15.079 -51.066 32.809 1.000 10.26

ATOM 1539 CD2 LEU 204 -14.019 -53.027 33.942 ,000 35.07

ATOM 1540 C LEU 204 -10.938 -52.392 32.948 ,000 10.84

ATOM 1541 0 LEU 204 -11.212 -53.567 32.707 .000 16.23

ATOM 1542 N GLY 205 -10.143 -51.649 32.189 ,000 8.26

ATOM 1543 CA GLY 205 -9.534 -52.173 30.984 ,000 6.27

ATOM 1544 C GLY 205 -8.472 -53.215 31.265 ,000 8.34

ATOM 1545 O GLY 205 -8.228 -54.094 30.436 ,000 9.21

ATOM 1546 N VAL 206 -7.829 -53.130 32.425 ,000 8.74

ATOM 1547 CA VAL 206 -6.833 -54.135 32.796 ,000 9.33

ATOM 1548 CB VAL 206 -5.942 -53.653 33.957 ,000 16.14

ATOM 1549 CGI VAL 206 -5.020 -54.754 34.457 ,000 6.58

ATOM 1550 CG2 VAL 206 -5.124 -52.445 33.514 ,000 6.33

ATOM 1551 C VAL 206 -7.526 -55.447 33.154 ,000 5.34

ATOM 1552 O VAL 206 -7.118 -56.498 32.664 ,000 5.68

ATOM 1553 N ALA 207 -8.564 -55.384 33.982 ,000 4.56

ATOM 1554 CA ALA 207 -9.349 -56.547 34.369 ,000 8.39

ATOM 1555 CB ALA 207 -10.323 -56.180 35.490 ,000 0.79

ATOM 1556 C • ALA 207 -10.144 -57.160 33.219 ,000 10.03

ATOM 1557 O ALA 207 -10.485 -58.346 33.261 ,000 13.69

ATOM 1558 N LEU 208 -10.471 -56.382 32.193 ,000 14.72

ATOM 1559 CA LEU 208 -11.278 -56.888 31.082 000 11.49

ATOM 1560 CB LEU 208 -12.065 -55.755 30.422 000 12.04

ATOM 1561 CG LEU 208 -13.325 -55.317 31.175 ,000 10.97

ATOM 1562 CDl LEU 208 -13.985 -54.127 30.497 000 18.17

ATOM 1563 CD2 LEU 208 -14.302 -56.477 31.290 000 17.03

ATOM 1564 C LEU 208 -10.391 -57.604 30.067 ,000 6.10

ATOM 1565 O LEU 208 -10.857 -58.502 29.369 000 15.12

ATOM 1566 N ALA 209 -9.132 -57.191 30.019 000 10.78

ATOM 1567 CA ALA 209 -8.103 -57.815 29.203 .000 16.00

ATOM 1568 CB ALA 209 -6.827 -56.992 29.220 ,000 18.55

ATOM 1569 C ALA 209 ,829 -59.238 29.694 000 19.15

ATOM 1570 O ALA 209 ,639 -60.143 28.882 1.000 13.89

ATOM 1571 N GLU 210 -7.822 -59.396 31.015 000 9.97

ATOM 1572 CA GLU 210 -7.645 -60.692 31.653 000 11.15

ATOM 1573 CB GLU 210 -7.535 -60.520 33.168 000 ^•21.07

ATOM 1574 CG GLU 210 -6.097 -60.365 33.647 000 39.63

ATOM 1575 CD GLU 210 -5 ,696 -58.921 33.860 000 47.94

ATOM 1576 OEl GLU 210 -5 ,958 -58.391 34.960 000 64.71

ATOM 1577 OE2 GLU 210 -5 ,097 -58.319 32.949 000 43.70

ATOM 1578 C GLU 210 .791 -61.634 31.308 000 10.80 GC821-2

ATOM 1579 O GLU 210 -8 589 -62 787 30 927 1 000 10 93

ATOM 1580 N GLN 211 10 007 -61 120 31 441 1 000 10 29

ATOM 1581 CA GLN 211 11 190 -61 871 31 035 1 000 17 12

ATOM 1582 CB GLN 211 12 443 -61 052 31 363 1 000 15 73

ATOM 1583 CG GLN 211 12 542 -60 709 32 844 1 000 19 97

ATOM 1584 CD GLN 211 12 936 -61 923 33 671 1 000 20 12

ATOM 1585 OEl GLN 211 13 886 -62 628 33 331 1 000 17 44

ATOM 1586 NE2 GLN 211 12 218 -62 166 34 759 1 000 12 84

ATOM 1587 C GLN 211 11 146 -62 237 29 556 1 000 19 66

ATOM 1588 O GLN 211 11 399 -63 384 29 170 1 000 12 73

ATOM 1589 N VAL 212 10 822 -61 287 28 679 1 000 17 48

ATOM 1590 CA VAL 212 10 785 -61 612 27 249 1 000 19 02

ATOM 1591 CB VAL 212 10 426 -60 369 26 415 1 000 14 47

ATOM 1592 CGI VAL 212 10 189 -60 744 24 958 1 000 15 00

ATOM 1593 CG2 VAL 212 11 527 -59 320 26 523 1 000 8 88

ATOM 1594 C VAL 212 -9 816 -62 745 26 936 1 000 23 29

ATOM 1595 O VAL 212 10 192 -63 735 26 294 1 000 25 62

ATOM 1596 N ARG 213 -8 557 -62 645 27 361 1 000 21 16

ATOM 1597 CA ARG 213 -7 617 -63 740 27 126 1 000 22 08

ATOM 1598 CB ARG 213 -6 251 -63 462 27 752 1 000 19 45

ATOM 1599 CG ARG 213 -5 577 -62 178 27 300 1 000 20 41

ATOM 1600 CD ARG 213 -4 621 -61 690 28 380 1 000 26 40

ATOM 1601 NE ARG 213 -3 847 -60 527 27 952 1 000 29 86

ATOM 1602 CZ ARG 213 -3 556 -59 504 28 745 1 000 26 00

ATOM 1603 NH1 ARG 213 -3 968 -59 485 30 007 1 000 15 34

ATOM 1604 NH2 ARG 213 -2 847 -58 491 28 268 1 000 17 74

ATOM 1605 C ARG 213 -8 157 -65 052 27 695 1 000 21 76

ATOM 1606 O ARG 213 -7 893 -66 138 27 182 1 000 28. 34

ATOM 1607 N SER 214 -8 924 -64 952 28 780 1 000 15 76

ATOM 1608 CA SER 214 -9 486 -66 151 29 389 1 000 15 09

ATOM 1609 CB SER 214 10 043 -65 824 30 781 1 000 19 35

ATOM 1610 OG SER 214 11 053 -66 745 31 144 1 000 46 77

ATOM 1611 C SER 214 10 561 -66 790 28 529 1 000 15 48

ATOM 1612 O SER 214 10 692 -68 016 28 535 1 000 24 87

ATOM 1613 N LEU 215 11 355 -66 030 27 772 1 000 21 40

ATOM 1614 CA LEU 215 12 367 -66 673 26 938 1 000 21 52

ATOM 1615 CB LEU 215 13 .655 -65 855 26 860 1 000 22 40

ATOM 1616 CG LEU 215 14 176 -65 153 28 103 1 000 20 48

ATOM 1617 CDl LEU 215 15 071 -63 990 27 697 1 000 27 15

ATOM 1618 CD2 LEU 215 14 931 -66 118 29 006 1 000 13 10

ATOM 1619 C LEU 215 11 884 -66 920 25 510 1 000 20 60

ATOM 1620 O LEU 215 12 536 -67 682 24 789 1 000 31 41

ATOM 1621 N LEU 216 10 790 -66 303 25 077 1 000 21 43

ATOM 1622 CA LEU 216 10 291 -66 503 23 718 1 000 19 55

ATOM 1623 CB LEU 216 10 114 -65 148 23 021 1 .000 19 47

ATOM 1624 CG LEU 216 11 385 -64 305 22 870 1 000 16 11

ATOM 1625 CDl LEU 216 11 095 -63 042 22 076 1 .000 17 .60 GC821-2

ATOM 1626 CD2 LEU 216 -12. 495 -65. 108 22 . 211 1. 000 4 . 00

ATOM 1627 C LEU 216 -8 . 983 -67 .283 23. 688 1. 000 24 .37

ATOM 1628 OT1 LEU 216 -8 . 472 -67 . 525 22 . 571 1. 000 29.22

ATOM 1629 OT2 LEU 216 -8 . 463 -67 . 655 24 . 758 1 .000 19.02

In addition to the above-described determinations, a carbamate-inhibited perhydrolase crystal was also produced and analyzed. In these experiments, a N- hexylcarbamate derivative of wild type perhydrolase was used. Wild-type perhydrolase (14.5 mg in 1 mL, 67mM NaPO4 pH 7 buffer) was titrated at room temperature with 1.25 μL aliquots of 400 mM p-nitrophenyl-N-hexylcarbamate dissolved in DMSO. Perhydrolase. activity was measured with ?-nitrophenylbutyrate assay (See, Example 2), as a function of time after each addition of the inhibitor. Several additions over several hours were required for complete inhibition of the enzyme. After inhibition was complete, the buffer of the inhibited enzyme solution was exchanged for 10 mM HEPES pH 8.3. This solution was stored at - 80'C until used for crystallization screening experiments were conducted as described above. The inhibitor ^-nitrophenyl-N- hexylcarbamate was prepared by methods known in the art (See e.g., Hosie et al, J. Biol. Chem., 262:260-264 [1987]). Briefly, the carbamate-inhibited perhydrolase was crystallized by vapor diffusion using the hanging drop method known in the art. A ml solution of inhibited perhydrolase (15 mg/ml in 10 mM HEPES, pH 8.2), was mixed with 4 μL of a reservoir solution (30% PEG-4,000 with 0.2 M lithium sulfate and 0.1 M Tris, pH 8.5) on a plastic coverslip, then inverted and sealed for a well of 6x4 Linbro plate containing 0.5 ml of the reservoir solution and allowed to equilibrate. Crystals formed within a few days. The crystals were flash frozen in liquid nitrogen and analyzed as described above. While the native octamer was determined in space group P4 with unit cell dimensions: a= 98.184 b= 98.184 and c= 230.119 α=90.00 β=90.00 γ=90.00, this crystal diffracted GC821-2

to about 2.0 A. The carbamate-inhibited crystal grew in the space group PI with unit cell dimensions a=67.754, b=80.096, and c=85.974 c =104.10° , β=l 12.10°, and γ=97.40° and these crystals diffract to a resolution exceeding 1.0 A. The carbamate was bound in a manner to exploit the interactions between the keto oxygen of the carbamate and residues forming the oxyanion hole, the amide N atoms of Ser 11 and Ala 55 and Asn 94 ND2. The hydrophobic side chain extends along the hydrophobic surface of the binding site out into the surface opening between pairs of dimers in the octamer structure. The carbamate moiety direction highlights the pivotal role of the S54V mutation. The hydrophobic moiety passes adjacent to the side chain of ser 54. Mutating the serine side to valine increased the hydrophobicity, and also served as a gatekeeper to prevent hydrophilic nucleophiles (e.g., water) for competing with desired deacylating nucleophiles. The t residues surrounding the carbamate moiety on the same and neighboring molecules forming the extended entry are expected to influence the selection of the optimal de-acylating nucleophile. In addition, residues with surface-accessible side chain atoms were identified using the program "AreaMol," within the CCP4 program package. Table 15-1 lists these residues. In this Table, the residue number, residue name, number of surface-accessible side chain atoms having at least 10.0 square atoms of accessible surface area, and maximum surface area (square angstroms) for any side chain atom within that residue (or CA for GLY residues) in the octameric structure of perhydrolase are provided.

GC821-2

GC821-2

EXAMPLE 16 Stain Removal In this Example, experiments conducted to assess the stain removal abilities of perhydrolase are described. Individual wells of 24 well culture plates were used to mimic conditions found in ordinary washing machines. Each well was filled with commercially available detergent (e.g., Ariel [Procter & Gamble], WOB [AATCC], and WFK [WFK]), and pre-stained cloth discs cut to fit inside of each well were added.. Temperature and agitation were accomplished by attaching the plate to the inside of a common laboratory incubator/shaker. To measure bleaching effectiveness of the perhydrolase, fabric stained with tea ( EMPA # 167, available commercially from Test Fabrics) was used. A single cloth disc was placed in each well, and 1 ml of detergent liquid, containing enzyme, ester substrate, and peroxide was added. After agitation at 100 - 300 rpm @ 20 - 60°C, the fabric discs were removed, rinsed with tap water, and allowed to dry overnight. The reflectance of each individual cloth disc was measured, and plotted as an "L" value. These results are provided in Figure 21, which shows that the addition of the perhydrolase of the present invention to the detergent consistently provides a greater degree of bleaching than the detergents alone. In this Figure, "E" indicates the results for each of GC821-2

the detergents tested in combination with the perhydrolase of the present invention.

EXAMPLE 17 Cotton Bleaching In this Example, experiments to assess the use of the perhydrolase of the present invention for bleaching of cotton fabrics are described. In these experiments, six cotton swatches per canister were treated at 55°C for 60 minutes in a Launder-O-meter. The substrates used in these experiments were: 3 (3"x3") 428U and 3 (3"x3") 400U per experiments. Two different types of 100% unbleached cotton fabrics from Testfabrics were tested (style 428U (desized but not bleached army carded cotton sateen); and style 400U (desized but not bleached cotton print cloth). The liquor ratio was about 26 to 1 (~7.7 g fabric/~ 200 ml volume liquor). The perhydrolase enzyme was tested at 12.7 mgP/ml, with ethyl acetate (3 % (v/v)), hydrogen peroxide (

1500 ppm), and Triton X-100 (0.001%), in a sodium phosphate buffer (100 mM) for pH 7 and pH 8; as well as in a sodium carbonate (100 mM) buffer, for pH 9 and pH 10. Bleaching effects were quantified with total color difference by taking 4 CIE L*a*b* values per each swatch before and after the treatments using a Chroma Meter CR-200 (Minolta) , and total color difference of the swatches after the treatments were calculated according to the following:

Total color difference (ΔE)

GC821-2

(where ΔL, Δa, Δb, are differences in CIE L*, CDE a*, and CIE b* values respectively before and after the treatments).

Higher ΔE values indicate greater bleaching effects. The results (See, Figure 22) indicated that the perhydrolase showed significantly improved bleaching effects on both types of 100% cotton fabrics at pH 7 and pH 8 under the conditions tested. It was also observed that high amounts of motes (e.g., pigmented spots) disappeared on the enzyme treated substrates.

EXAMPLE 18 Linen Bleaching In this Example, experiments conducted to assess the linen bleaching capability of the perhydrolase of the present invention are described. The same methods and conditions as describe above for cotton testing (in Example 17) were used to test linen swatches. As indicated above, experiments were conduction in a Launder-O-meter using a linen fabric (linen suiting, Style L-53; Testfabrics). In these experiments, 3 (4"x4") linen swatches were treated with 12.7 mgP/ml of the perhydrolase enzyme with ethyl acetate (3 % v/v), hydrogen peroxide (1200 ppm), and Triton X-100 (0.001%), in a sodium phosphate buffer (100 mM) for pH 7 and pH 8.

The bleaching effects were calculated as described above in Example 17. Figure 23 provides a graph showing the bleaching effects of the perhydrolase of the present invention tested at pH 7 and pH 8 on linen. EXAMPLE 19 Detergent Compositions In the following Example, various detergent compositions are exemplified. In GC821-2

these formulations, the enzymes levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions. The abbreviated component identifications therein have the following meanings:

LAS Sodium linear C^ \.\ alkyl benzene sulfonate.

TAS Sodium tallow alkyl sulfate. CxyAS Sodium Ciχ - Cjy alkyl sulfate.

CxyEz Clx - Cjy predominantly linear primary alcohol condensed with an average of z moles of ethylene oxide.

CxyAEzS C\_x - Cjy sodium alkyl sulfate condensed with an average of z moles of ethylene oxide. Added molecule name in the examples.

Nonionic Mixed ethoxylated/propoxylated fatty alcohol e.g. Plurafac LF404 being an alcohol with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5.

QAS R2.N+(CH3)2(C₂H4OH) with R₂ = C12-C14.

Silicate Amorphous Sodium Silicate (SiO2:Na2O ratio = 1.6-3.2:1).

Metasilicate Sodium metasilicate (SiO2:Na2O ratio = 1.0).

Zeolite A Hydrated Aluminosilicate of formula Nai2(Alθ2Siθ2)i2- 27H2O

SKS-6 Crystalline layered silicate of formula δ-Na2Si2θ5

Sulphate Anhydrous sodium sulphate. STPP Sodium Tripolyphosphate.

MA/AA Random copolymer of 4:1 acrylate/maleate, average molecular weight about 70,000-80,000.

AA Sodium polyacrylate polymer of average molecular weight 4,500. Polycarboxylate Copolymer comprising mixture of carboxylated monomers such as acrylate, maleate and methyacrylate with a MW ranging between 2,000-80,000 such as Sokolan commercially available from BASF, being a copolymer of acrylic acid, MW4,500.

BB1 3-(3,4-Dihydroisoquinolinium)propane sulfonate BB2 1 -(3 ,4-dihydroisoquinolinium)-decane-2-sulfate PB1 Sodium perborate monohydrate. PB4 Sodium perborate tetrahydrate of nominal formula NaBθ3.4H2θ.

Percarbonate Sodium percarbonate of nominal formula 2Na2Cθ3.3H2θ2 .

TAED Tetraacetyl ethylene diamine. NOBS Nonanoyloxybenzene sulfonate in the form of the sodium salt. DTPA Diethylene triamine pentaacetic acid. GC821-2

HEDP 1,1-hydroxyethane diphosphonic acid. DETPMP Diethyltriamine penta (methylene) phosphonate, marketed by Monsanto under the Trade name Dequest 2060.

EDDS Ethylenediamine-N,N'-disuccinic acid, (S,S) isomer in the form of its sodium salt

Diamine Dimethyl aminopropyl amine; 1,6-hezane ώamine; 1,3-propane diamine; 2-methyl-l,5-pentane diamine; 1,3-pentanediamine; 1- me yl-diaminopropane.

DETBCHD 5, 12- diethyl-l,5,8,12-tetraazabicyclo [6,6,2] hexadecane, dichloride, Mn(ϋ) salt

PAAC Pentaamine acetate cobalt(m) salt. Paraffin Paraffin oil sold under the tradename Winog 70 by Wintershall. Paraffin Sulfonate A Paraffin oil or wax in which some of the hydrogen atoms have been replaced by sulfonate groups.

Aldose oxidase Oxidase enzyme sold under the tradename Aldose Oxidase by Novozymes A/S

Galactose oxidase Galactose oxidase from Sigma Protease Proteolytic enzyme sold under the tradename Savinase, Alcalase, Everlase by Npvo Nordisk A/S, and the following from Genencor International, Inc: "Protease A" described in US RE 34,606 in Figures 1 A, IB, and 7, and at column 11, lines 11-37; "Protease B" described in US5,955,340 and US5,700,676 in Figures 1 A, IB and 5, as well as Table 1; and "Protease C" described in US6,312,936 and US 6,482,628 in Figures 1-3 [SEQ ID 3], and at column 25, line 12, "Protease D" being the variant 101 G/103 A 1041/159D/232V/236H/245R/248D/252K (BPN' numbering) described in WO 99/20723.

Amylase Amylolytic enzyme sold under the tradename Purafact Ox Am° described in WO 94/18314, WO96/05295 sold by Genencor; Natalase^®, Termamyl®, Fungamyl® and Duramyl®, all available from Novozymes A/S.

Lipase Lipolytic enzyme sold under the tradename Lipolase Lipolase Ultra by Novozymes A/S and Lipomax by Gist-Brocades. Cellulase Cellulytic enzyme sold under the tradename Carezyme, Celluzyme and or Endolase by Novozymes A S.

Pectin Lyase Pectaway® and Pectawash® available from Novozymes A/S.

PVP Polyvinylpyrrolidone with an average molecular weight of 60,000 PVNO Polyvinylpyridine-N-Oxide, with an average molecular weight of 50,000.

PVPVI Copolymer of vinylimidazole and vinylpyrrolidone, with an average molecular weight of 20,000. GC821-2

Brightener 1 Disodium 4,4^l-bis(2-sulphostyryl)biphenyl. Silicone antifoam Polydimethylsiloxane foam controller with siloxane-oxyalkylene copolymer as dispersing agent with a ratio of said foam controller to said dispersing agent of 10:1 to 100:1. Suds Suppressor 12% Silicone/silica, 18% stearyl alcohol,70% starch in granular form. SRP 1 Anionically end capped poly esters. PEG X Polyethylene glycol, of a molecular weight of x. PVP K60 ® Vinylpyrrolidone homopolymer (average MW 160,000) Jeffamine ® ED-2001 Capped polyethylene glycol from Huntsman Isachem ® AS A branched alcohol alkyl sulphate from Enichem MME PEG (2000) Monomethyl ether polyethylene glycol (MW 2000) from Fluka Chemie AG. DC3225C Silicone suds suppresser, mixture of Silicone oil and Silica from Dow Corning. TEPAE Tetreaethylenepentaamine ethoxylate. BTA Benzotriazole. Betaine (CH₃)₃N⁺CH_COO^' Sugar Industry grade D-glucose or food grade sugar CFAA C₁₂-C_j4 alkyl N-methyl glucamide TPKFA Ci2-C₁₄ topped whole cut fatty acids. Clay A hydrated aluminumu silicate in a general formula Al2θ3SiO₂-xH₂O. Types: Kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite. MCAEM Esters in the formula of R'O_x [(R²)_m (R³)„]_p PH Measured as a 1% solution in distilled water at 20°C.

EXAMPLE 20 Liquid Laundry Detergents The following liquid laundry detergent compositions of the present invention are prepared.

GC821-2

EXAMPLE 21 Hand-Dish Liquid Detergent Compositions The following hand dish liquid detergent compositions of the present invention are GC821-2

prepared.

GC821-2

The pH of Compositions (I)-(VI) is about 8 to about 11

EXAMPLE 22 Liquid Automatic Dishwashmg Detergent The following liquid automatic dishwashing detergent compositions of the present are prepared.

STPP 16 16 18 16 16 Potassium Sulfate - 10 8 - 10 l,2 propanediol 6.0 0.5 2.0 6.0 0.5 Boric Acid 4.0 3.0 3.0 4.0 3.0 CaCh dihydrate 0.04 0.04 0.04 0.04 0.04 Nonionic 0.5 0.5 0.5 0.5 0.5 Protease B 0.03 0.03 0.03 0.03 0.03 Amylase 0.02 - 0.02 0.02 - Aldose Oxidase - 0.15 0.02 - 0.01 Galactose Oxidase - - 0.01 - 0.01 PAAC 0.01 - - 0.01 - DETBCHD - 0.01 - - 0.01 Perhydrolase 0.1 0.03 0.05 0.03 0.06 MCAEM 5.0 3.0 12.0 8.0 1.0 (C₁₄-C₁₅E₁₂ Acetate) GC821-2

I II III IV Balance to 100% perfume / dye and/or water

EXAMPLE 23 Laundry Compositions The following laundry compositions of present invention, which may be in the form of granules or tablet, are prepared.

II III IV Base Product Ci4-Cι₅AS or TAS 8.0 5.0 3.0 3.0 3.0 LAS 8.0 - 8.0 - 7.0 C12-C15AE3S 0.5 2.0 1.0 - - Ci2-C₁₅E5 or E3 2.0 - 5.0 2.0 2.0 QAS - - - 1.0 1.0 Zeolite A 20.0 18.0 11.0 - 10.0 SKS-6 (dry add) - - 9.0 - - MA/AA 2.0 2.0 2.0 - - AA - - - - 4.0 3Na Citrate 2H₂O - 2.0 - - - Citric Acid (Anhydrous) 2.0 - 1.5 2.0 - DTPA 0.2 0.2 - - - EDDS - - 0.5 0.1 - HEDP - - 0.2 0.1 - PB1 3.0 4.8 - - 4.0 Percarbonate _ _ 3.8 5.2 _ 21-2

I II III rv V

NOBS 1.9 - - - -

NACA OBS - - 2.0 - -

TAED 0.5 2.0 2.0 5.0 1.00

BB1 0.06 - 0.34 - 0.14

BB2 - 0.14 - 0.20 -

Anhydrous Na Carbonate 15.0 18.0 8.0 15.0 15.0

Sulfate 5.0 12.0 2.0 17.0 3.0 Silicate - 1.0 - - 8.0

Protease B 0.033 0.033 - - -

Protease C - - 0.033 0.046 o.03:

Lipase - 0.008 - - -

Amylase 0.001 - - - 0.00

Cellulase - 0.0014 - - -

Pectin Lyase 0.001 0.001 0.001 0.001 0.00

Aldose Oxidase 0.03 - 0.05 - -

PAAC - 0.01 - - 0.05

Perhydrolase 0.03 0.05 1.0 0.06 0.1

MCAEM** 2.0 5.0 12.0 3.5 6.8

Balance to 100% Moisture and/or Minors* > Perfume / Dye, Brightener / SRPl / Na Carboxymethylcellulose/ Photobleach / MgS0 / PVPVI/ Suds suppressor /High Molecular PEG/Clay. , ** MCAEM is selected from the group consisting of C 9-C₁₁E₂.₅ Acetate, [Ci2H25N(CH₃χCH₂CH2θAc)2]⁺ Cl^", (CHs^NCH∑CHzOCHzCHzOAc, or mixtures thereof.. GC821-2

EXAMPLE 24 Liquid Laundry Detergents The following liquid laundry detergent formulations of the present invention are prepared. I I II III rv V LAS 11.5 11.5 9.0 - 4.0 - C12-C15AE2.85S - - 3.0 18.0 - 16.( C14-C15E 2.5 s 11.5 11.5 3.0 - 16.0 - C 12-C13E9 - - 3.0 2.0 2.0 1.0 C 12-C13E 7 3.2 3.2 - - - - CFAA - - - 5.0 - 3.0 TPKFA 2.0 2.0 - 2.0 0.5 2.0 Citric Acid 3.2 3.2 0.5 1.2 2.0 1.2 (Anhydrous) Ca formate 0.1 0.1 0.06 0.1 - - Na formate 0.5 0.5 0.06 0.1 0.05 CO! Na Culmene 4.0 4.0 1.0 3.0 1.2 - Sulfonate Borate 0.6 0.6 - 3.0 2.0 3.0 Na hydroxide 6.0 6.0 2.0 3.5 4.0 3.0 Ethanol 2.0 2.0 1.0 4.0 4.0 3.0 1,2 Propanediol 3.0 3.0 2.0 8.0 8.0 5.0 Mono- 3.0 3.0 1.5 1.0 2.5 1.0 ethanolamine TEPAE 2.0 2.0 - 1.0 1.0 1.0 PB1 - 4.5 - 2.8 - Protease A 0.03 0.03 0.01 0.03 0.02 0.0 GC821-2 π in IV Lipase - - - 0.002 - - Amylase - . - - - 0.002 - Cellulase - - - - - 0.00 Pectin Lyase 0.005 0.005 - - - Aldose Oxidase 0.05 - - 0.05 - 0.02 Galactose oxidase - 0.04 Perhydrolase 0.03 0.05 0.01 0.03 0.08 0.02 MCAEM 3.2 4.6 1.8 3.5 6.2 2.8 (C 12-C15 E₆ Acetate) PAAC 0.03 0.03 0.02 - - - DETBCHD - - - 0.02 0.01 - SRP l 0.2 0.2 - 0.1 - - DTPA - - - 0.3 - - PVNO - - - 0.3 - 0.2 Brightener 1 0.2 0.2 0.07 0.1 ^' - - Silicone antifoam 0.04 0.04 0.02 0.1 0.1 0.1 Balance to 100% perfume / dye, and/or water

EXAMPLE 25 Compact High-Density Dishwashing Detergents The following compact high density dishwashing detergent of the present invention are prepared: I II III IV V VI

STPP - 45.0 45.0 - - 40.0 GC821-2

I II Ill rv V VI

3Na Citrate 2H₂O 17.0 - - 50.0 40.2 -

Na Carbonate 17.5 14.0 20.0 - 8.0 33.6

Bicarbonate - - - 26.0 - -

Silicate 15.0 15.0 8.0 - 25.0 3.6

Metasilicate 2.5 4.5 4.5 - - -

PB1 - - 4.5 - - -

PB4 - - - 5.0 - -

Percarbonate - - - - - 4.8

BB1 - 0.1 0.1 - 0.5 -

BB2 0.2 0.05 - 0.1 - 0.6

Nonionic 2.0 1.5 1.5 3.0 1.9 5.9

HEDP 1.0 - - - - -

DETPMP 0.6 - - - - -

PAAC 0.03 0.05 0.02 - - -

Paraffin 0.5 0.4 0.4 0.6 - -

Protease B 0.072 0.053 0.053 0.026 0.059 0.01

Amylase 0.012 - 0.012 - 0.021 0.006

Lipase - 0.001 - 0.005 - -

Pectin Lyase 0.001 0.001 0.001 - - -

Aldose Oxidase 0.05 0.05 0.03 0.01 0.02 0.01

Perhydrolase 0.072 0.053 0.053 0.026 0.059 0.01

MCAEM 3.5 2.8 1.6 7.5 4.2 0.8

(C 12-C13 E 6.5

Acetate)

BTA 0.3 0.2 0.2 0.3 0.3 0.3

Polycarboxylate 6.0 - - - 4.0 0.9 GC821-2

I II III IV V VI

Perfume 0.2 0.1 0.1 0.2 0.2 0.2

Balance to 100% Moisture and/or Minors*

^♦Brightener / Dye / SRPl / Na Carboxymethylcellulose/ Photobleach / MgSO / PVPVI/ Suds suppressor /High Molecular PEG/Clay.

The pH of compositions (ϊ) through (VI) is from about 9.6 to about 11.3,

EXAMPLE 26 Tablet Detergent Compositions The following tablet detergent compositions of the present invention are prepared by compression of a granular dishwashing detergent composition at a pressure of 13KN/cm2 using a standard 12 head rotary press.

I II III IV V VI VII VIII STPP - 48.8 44.7 38.2 - 42.4 46.1 36.0 3Na Citrate 2H₂O 20.0 - - - 35.9 - - - Na Carbonate 20.0 5.0 14.0 15.4 8.0 23.0 20.0 28.0 Silicate 15.0 14.8 15.0 12.6 23.4 2.9 4.3 4.2 Lipase 0.001 - 0.01 - 0.02 - - - Protease B 0.042 0.072 0.042 0.031 - - - - Protease C - - - - 0.052 0.023 0.023 0.029 Perhydrolase 0.01 0.08 0.05 0.04 0.052 0.023 0.023 0.029 MCAEM 2.8 6.5 4.5 3.8 4.6 2.8 2.8 2.8 (C 12-Cl₃ E 6.5 Acetate) Amylase 0.012 0.012 0.012 - 0.015 - 0.017 0.002 GC821-2

I π πi IV VI VII VIII Pectin Lyase 0.005 0.002 Aldose Oxidase 0.03 0.02 0.02 0.03 PB1 3.8 7.8 8.5 Percarbonate 6.0 - - 6.0 5.0 BB1 0.2 - 0.5 - 0.3 0.2 BB2 - 0.2 - 0.5 0.1 0.2 Nonionic 1.5 2.0 2.0 2.2 1.0 4.2 4.0 6.5 PAAC 0.01 0.01 0.02 _ DETBCHD 0.02 0.02 TAED - - - - - 2.1 - 1.6 HEDP 1.0 - - 0.9 - 0.4 0.2 - DETPMP 0.7 - - - - - - - Paraffin 0.4 0.5 0.5 0.5 - - 0.5 - BTA 0.2 0.3 0.3 0.3 0.3 0.3 0.3 - Polycarboxylate 4.0 - - - 4.9 0.6 0.8 - PEG 400-30,000 - - - - - 2.0 - 2.0 Glycerol - - - - - 0.4 - 0.5 Perfume _ _ _ 0.05 0.2 0.2 0.2 0.2 Balance to 100% Moisture and/or Minors*

^♦Brightener / Dye / SRPl / Na Carboxymethylcellulose/ Photobleach / MgS0₄ / PVPVI/ Suds suppressor /High Molecular PEG/Clay.

The pH of Compositions (I) through 7(VEI) is from about 10 to about 11.5. The tablet weight of Compositions 7(1) through 7(VHT) is from about 20 grams to about 30 grams.

EXAMPLE 27 GC821-2

Liquid Hard Surface Cleaning Detergents The following liquid hard surface cleaning detergent compositions of the present invention are prepared. I II III rv V VI VII

C9-C11E5 2.4 1.9 2.5 2.5 2.5 2.4 2.5

C12-C14E5 3.6 2.9 2.5 2.5 2.5 3.6 2.5

C7-C9E6 - - - - 8.0 - -

Cl2-Cl₄E21 1.0 0.8 4.0 2.0 2.0 1.0 2.0

LAS - - - 0.8 0.8 - 0.8

Sodium culmene sulfonate 1.5 2.6 - 1.5 1.5 1.5 1.5

Isachem ® AS 0.6 0.6 - - - 0.6 -

Na2CO3 0.6 0.13 0.6 0.1 0.2 0.6 0.2

3Na Citrate 2H₂O 0.5 0.56 0.5 0.6 0.75 0.5 0.75

NaOH 0.3 0.33 0.3 0.3 0.5 0.3 0.5

Fatty Acid 0.6 0.13 0.6 0.1 0.4 0.6 0.4

2-butyl octanol 0.3 0.3 - 0.3 0.3 0.3 0.3

PEG DME-2000® 0.4 - 0.3 0.35 0.5 - -

PVP 0.3 0.4 0.6 0.3 0.5 - -

MME PEG (2000) ® - - - - - 0.5 0.5

Jeffamine ® ED-2001 - 0.4 - - 0.5 - -

PAAC - - - 0.03 0.03 0.03 -

DETBCHD 0.03 0.05 0.05 - - - -

Protease B 0.07 0.05 0.05 0.03 0.06 0.01 0.04

Amylase 0.12 0.01 0.01 - 0.02 - 0.01

Lipase - 0.001 - 0.005 - 0.005 -

Perhydrolase 0.07 0.05 0.08 0.03 0.06 0.01 0.04 GC821-2 i π in iv v vi vπ

MCAEM (C12-C15E8 3.5 5.6 4.8 5.3 3.6 8.0 4.7

Acetate)

Pectin Lyase 0.001 - 0.001 - - - 0.002

PB1 - 4.6 - 3.8 -

Aldose Oxidase ^' 0.05 - 0.03 - 0.02 0.02 0.05

Balance to 100% perfume / dye, and/or water

The pH of Compositions (I) through (VII) is from about 7.4 to about 9.5.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention. Those of skill in the art readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It is readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically GC821-2

disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed maybe resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Claims

GC821-2 CLAIMS

What is claimed is: 1. An isolated perhydrolase, wherein said perhydrolase exhibits a perhydrolysis to hydrolysis ratio that is greater than 1.

2. The perhydrolase of Claim 1 , wherein said perhydrolase is M. smegmatis perhydrolase.

3. An isolated perhydrolase, wherein said perhydrolase is at least approximately about 35% homologous to said M. smegmatis perhydrolase of Claim 2.

4. The perhydrolase of Claim 2, wherein said perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2.

5. An isolated perhydrolase having immunological cross-reactivity with said perhydrolase of Claim 2.

6. The perhydrolase of Claim 2, wherein said perhydrolase is at least a portion of said M. smegmatis perhydrolase, wherein said perhydrolase has a perhydrolysis to hydrolysis ration that is greater than 1.

7. The perhydrolase of Claim 2, wherein said perhydrolase is a structural homologue of said M. smegmatis perhydrolase, in which the active site is homologous to at least one amino acid selected from the group consisting of SI 1, D192, and H195 of the M. smegmatis perhydrolase. GC821-2

8. An isolated perhydrolase variant having an amino acid sequence comprising at least one modification of an amino acid made at a position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2.

9. The perhydrolase variant of Claim 8, wherein said at least one modification is made at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said modified amino acid is selected from the group consisting of Cys7, Asp 10, Serl 1, Leul2, Thrl3, Trpl4, Trpl6, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Vall25, Prol38, Leul40, Prol46, Prol48, Trpl49, Phel50, Ilel53, Phel54, Thrl59, Thrl86, Hel92, Ilel94, and Phel96.

10. The perhydrolase variant of Claim 8, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of Ml, K3, R4, 15, L6, C7, D10, Sll, L12, T13, W14, W16, G15, V17, P18, V19, D21, G22, A23, P24, T25, E26, R27, F28, A29, P30, D31, V32, R33, W34, T35, G36, L38,

Q40, Q41, D45, L42, G43, A44, F46, E47, V48, 149, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, 160, D61, D62, P63, T64, D65, P66, R67, L68, N69, G70, A71, S72, Y73, S76, C77, L78, A79, T80, L82, P83, L84, D85, L86, V87, N94, D95, T96, K97, Y99F100, RlOl, R102, P104, L105, D106, 1107, A108, L109, GI 10, Ml 11, SI 12, VI 13, L114, VI 15, T116, Q117, VI 18, L119, T120, S121, A122, G124, V125, G126,

T127, T128, Y129, P146, P148, W149, F150, 1153, F154, 1194, and F196. GC821-2

11. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in peracid hydrolysis compared to the wild-type perhydrolase.

12. The perhydrolase variant of Claim 11 , wherein said change in peracid hydrolysis is a decrease.

13. The perhydrolase variant of Claim 11 , wherein said change in peracid hydrolysis is an increase.

14. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.1 or less, in comparison with wild-type perhydrolase.

15. The perhydrolase variant of Claim 14, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in

M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of R4, L12, G15, P18, R27, W34L38, A44, E51, G52, L53, S54, T58, R67, L68, S72, A79, T80, D85, L86, V87, N94, K97, RlOl, VI 18, L119, G124, G126, and 1194.

16. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.2 or less, in comparison with wild-type perhydrolase.

17. The perhydrolase variant of Claim 16, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID GC821-2

NO:2, wherein said at least one substitution is selected from the group consisting of R4, 15, D10, L12, W14, G15, P18, V19, T25, R27, W34, L38, A44, 149, E50, E51, G52, L53, S54, A55, R56, T58, N59, D62, T64, D65, R67, L68, N69, S72, S76, C77, A79, T80, D85, L86, V87, N94, K97, RlOl, L82, P83, L86, V87, N94, T96, FlOO, RlOl, L109, Ml 11, Ll 14, VI 18, Ll 19, A122, G124, G126, T127, Y129, W149, and 1194.

18. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.3 or less, in comparison with wild-type perhydrolase.

19. The perhydrolase variant of Claim 18, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of R4, 15, D10, L12, W14, G15, L12, P18, V19, G22, A23, T25, E26, R27, W34, G36, L38,

Q41, L42, G43, A44, 149, E50, E51, G52, L53, S54, A55, R56, T57, N59, T58, D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, T96, K97, Y99, FlOO, RlOl, R102, P104, L109, Gl lO, Mill, L114, VI 18, L119, A122, G124, V125, G126, T127, Y129, W149, F154, and 1194.

20. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.4 or less, in comparison with wild-type perhydrolase.

21 The perhydrolase variant of Claim 20, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID GC821-2

NO:2, wherein said at least one substitution is selected from the group consisting of R4, 15, L6, DIO, SI 1, L12, W14, G15, W16, P18, V19, G22, A23, T25, E26, R27, F28, W34, T35, G36, L38, Q41, L42, G43, A44, D45, E47, 149, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, T58, 160, D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76, C77, A79, T80, L82, P83, D85, L86, V87, N94, P66, T96, K97, Y99, FlOO, RlOl, R102, P104, 1107, L109, GI 10, Ml 11, SI 12, Ll 14, VI 18, Ll 19, S121, A122, G124, V125, G126, T127, Y129, W149, F150,.F154, 1194, and F196.

22. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.5 or less, in comparison with wild-type perhydrolase.

23. The perhydrolase variant of Claim 22, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GllO, G124, G43, L109, Ll 19, L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, Tl 16, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, Ll 19, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58,

T80, VI18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, FlOO, RlOl, L109, Ml11, Ll14, Ll19, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, GllO, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, Ll14, Ll19, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56,

R67, S54, T57, VI18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, GllO, G124, G36, G43, G52, G70, 1107, 15, 160, L109, GC821-2

L119, L53, L6, L68, L82, Mi l l, P104, P66, R102, R67, Sll, SI 12, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

24. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.6 or less, in comparison with wild-type perhydrolase.

25. The perhydrolase variant of Claim 24, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GllO, G124, G43, L109, Ll 19, L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, Tl 16,

T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, L119, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, FlOO, RlOl, L109, Mill, L114, L119, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, GI 10, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, Ll 14, Ll 19, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI 18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, GllO, G124, G36, G43, G52, G70, 1107, 15, 160, L109, L119, L53, L6, L68, L82, Mi l l, P104, P66, R102, R67, Sll, SI 12, S121, S54, S72, T25,

T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, 1107, 1194, GC821-2

K97, L105, L109, Ll 14, Ll 19, L38, L68, L78, L82, L84, Ml 11, N69, N94, P104, P63, P66, R102, R27, Sll, SI 12, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, VI 13, V125, V19, W16, Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

26. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.7 or less, in comparison with wild-type perhydrolase.

27. The perhydrolase variant of Claim 26, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GI 10, G124, G43, L109,

Ll 19, L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, Tl 16, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, Ll 19, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87,

N94, T96, FlOO, RlOl, L109, Ml 11, Ll 14, Ll 19, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, GI 10, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI 18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, GllO, G124, G36, G43, G52, G70, 1107, 15, 160, L109,

L119, L53, L6, L68, L82, Mi l l, P104, P66, R102, R67, Sll, SI 12, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, GC821-2

D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, 1107, 1194, K97, L105, L109, Ll 14, Ll 19, L38, L68, L78, L82, L84, Ml 11, N69, N94, P104, P63, P66, R102, R27, SI 1, SI 12, S54, S72, Tl 16, T120, T127, T13, T25, T57, T80, T96, VI 13, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, 149, 15, 160, L105, L109, L12, L38, L42, L53, L84, L86, Mill, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54, S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

28. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 0.8 or less, in comparison with wild-type perhydrolase.

29. The perhydrolase variant of Claim 28, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GI 10, G124, G43, L109, Ll 19, L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, Tl 16, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, L119, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, D10, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87, N94, T96, FlOO, RlOl, L109, Ml 11, Ll 14, Ll 19, W149, Yld29, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, GllO, G124, G126, G22, G36, G43, G52, G70,

149, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI 18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, GC821-2

E26, E47, E51, F150, F196, F28, GllO, G124, G36, G43, G52, G70, 1107, 15, 160, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, Sll, SI 12, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, 1107, 1194, K97, L105, L109, L114, L119, L38, L68, L78, L82, L84, Mill, N69, N94, P104, P63, P66, R102, R27, Sll, SI 12, S54, S72, T116, T120, T127, T13, T25, T57, T80, T96, VI 13, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, 149, 15, 160, L105, L109, L12, L38, L42, L53, L84, L86, Mil 1, N59, P146, P24, P66, Q41, R102, R27, R56, SI 12, S121, S54, S72, Tl 16, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61, D65, D85, D95, E47, E51, F150, F196, F46, GllO, G126, G36, G43, G52, 1107, 1194, 149, 15, 160, 189, Ll 14, L42, L53, L68, L78, L84, Ml 11, N59, N94, P146, P24, P30, P63, P66, P83, Ql 17, RlOl, R4, SI 12, S121, S72, Tl 16, T120, T127, T13, T57, T96, VI 13, V125, V17, V19, V32, V87, W149, Y129, Y73, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215, and L216.

30. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis of about 1.5 or greater, in comparison with wild-type perhydrolase.

31. The perhydrolase variant of Claim 30, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46, GI 10, G124, G43, L109, GC821-2

Ll 19, L42, L68, L78, L82, L84, N59, P66, RlOl, R27, R4, R67, SI 12, S54, S76, Tl 16, T120, T25, V125, V48, W149, Y73, A44, A79, D85, E51, G124, G126, G15, G52, 1194, K97, Ll 19, L12, L38, L53, L68, L86, N94, P18, RlOl, R27, R4, R67, S54, S72, T58, T80, VI 18, V87, W34, R4, 15, DIO, L12, W14, V19, T25, W34, 149, E50, E51, L53, S54, A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83, L86, V87,

N94, T96, FlOO, RlOl, L109, Ml 11, Ll 14, Ll 19, W149, Y129, A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, GllO, G124, G126, G22, G36, G43, G52, G70, 149, K97, L109, L114, L119, L12, L38, L42, L53, L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, VI 18, V125, W14, W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150, F196, F28, GI 10, G124, G36, G43, G52, G70, 1107, 15, 160, L109, L119, L53, L6, L68, L82, Mill, P104, P66, R102, R67, Sl l, SI 12, S121, S54, S72, T25, T35, T57, T58, VI 18, V125, V19, W149, W16, A108, A122, A23, A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126, G22, G36, G52, 1107, 1194, K97, L105, L109, Ll 14, Ll 19, L38, L68, L78, L82, L84, Ml 11, N69, N94, P104, P63, P66, R102, R27, SI 1, SI 12, S54, S72, Tl 16, T120, T127, T13, T25, T57, T80, T96,

VI 13, A122, A29, A71, A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124, G126, G15, G36, G70, 149, 15, 160, L105, L109, L12, L38, L42, L53, L84, L86, Mill, N59, P146, P24, P66, Q41, R102, R27, R56, SI 12, S121, S54, S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149, W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61, D65, D85, D95, E47, E51, F150, F196, F46, GllO, G126, G36, G43, G52, 1107, 1194, 149, 15, 160, 189, Ll 14, L42, L53, L68, L78, L84, Ml 11, N59, N94, P146, P24, P30, P63, P66, P83, Ql 17, RlOl, R4, SI 12, S121, S72, T116, T120, T127, T13, T57, T96, VI 13, V125, V17, V19, V32, V87, W149, Y129, and Y73, Y99, A108, A44, C7, DIO, D106, D31, D61, D85, E26, E51, FlOO, F28, F46, GI 10, G22, G36, G43, G52, G70, 1107, 1153, 149, 15, 189, K3,

L105, L53, L6, L78, L86, Ml, N69, P104, P146, P18, P24, P30, P83, Q117, Q40, Q41, R102, R27, R33, R4, S121, S72, S76, T120, T128, T13, T35, T80, T96, VI 15, VI 18, GC821-2

V32V48, V87, W34, G190, V191, G193, T197, E198, A199, R202, D203, G205, V206, A209, E210, Q211, S214, and L215.

32. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a ratio of peracid hydrolysis between about 1.2 and about 1.5, in comparison with wild-type perhydrolase.

33. The perhydrolase variant of Claim 32, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, C7, D106, D31, D61, D85, E26, E50, E51, FlOO, F150, F28, F46, GllO, G126, G22, G70, 1107, K3, L105, L42, L6, L78, Mill, N59, N69, P104, P146, P148, P18, P30, P63, Q117, Q40, Q41, R102, R27, R33, R4, S54, S76, T116, T120, T128, T64, T80, T96,

VI 13, VI 15, VI 18, W34, and Y73.

34. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is at least about 1.2.

35. The perhydrolase variant of Claim 34, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of C7,

D10, L12, G15, P18, V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, 149, E51, L53, S54, A55, T57, D61, P63, T64, D65, P66, GC821-2

R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80, L82, P83, D85, L86, D95, K97, RlOl, T103, P104, L105, D106, 1107, L109, Mill, VI 13, Q117, VI 18, S121, G124, V125, G126, T127, P148, F150, 1153, F154, and F196.

36. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.8 or less.

37. The perhydrolase variant of Claim 36, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A108, A122, A23, A29, A44, A55, A71, A79, C7, C77, D10, D106, D21, D45, D61, D62, D65, D85, E26, E47, E50, E51, FlOO, F150, F154, F196, F28, F46, GI 10, G124, G126, G15, G22, G36, G52, G70, 1107, 1153, 1194, 149, 15, 160, 189, K3, K97, L105,

L109, Ll 14, Ll 19, L12, L38, L42, L53, L6, L68, L78, L82, L84, K86, Ml, Ml 11, N59N94, P146, P18, P24, P30, P66, P83, Q40, Q41, RlOl, R102, R27, R33, R4, R56, R67, Sl l, SI 12, S54, S72, S76, T103, T116, T120, T127, T128, T13, T25, T35, T57, T64, T80, T96, VI 13, VI 15, VI 18, V125, V17, V19, V32, V48, V87, W13, W149, W16, W34, Y129, Y73, and Y99.

38. The perhydrolase variant of Claim 8, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A108,

A122, A23, A29, A44, A55, A71, A79, C7, C77, D10, D106, D21, D31, D45, D61, D62, D65, D85, E26, E47, E50, E51, FlOO, F150, F154F196, F28, F46, GI 10, G124, G126, GC821-2

G15, G22, G36, G43, G52, G70, 1107, 1153, 1194, 149, 15, 160, 189, K3, K97, L105, L109, Ll 14, Ll 19, L12, L38, L42, L53, L6, L68, L78, L82, L84, L86, Ml, Ml 11, N59, N69, N94, P104, P146, P148, P18, P24, P30, P63, P66, P83, Ql 17, Q40, Q41, RlOl, R102, R27, R33, R4, R56, R67, Sll, SI 12, S121, S54, S72, S76, T103, T116, T120, T127, T128, T13, T25, T35, T57, T58, T64, T80, T96, VI 13, VI 15, VI 18, V125, V17, V19, V32, V48, V87, W14, W149, W16, W34,. Y129, Y73, and Y99.

39. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 1.2 and about 2.

40. The perhydrolase variant of Claim 39, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of C7, D10, L12, G15, P18, V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, 149, E51, L53, S54, A55, T57, D61, P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80, L82, P83, D85, L86, D95, K97, RlOl, T103, P104, L105, D106, 1107, L109, Mi ll, VI 13, Q117, VI 18, S121, G124, V125, G126, T127, P148, F150, 1153, F154, F196, G190, E198, A199, R202, D203, V206, A209, E210, Q211, and V212.

41. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 2 and about 2.5. GC821-2

42. The perhydrolase variant of Claim 41 , wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A44, C7, DIO, D85, D95, E26, E47, 1107, L12, L42, P104, P148, S54, Q40, Q117, D203, V206, E210.

43. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 2.5 and about 3.

44. The perhydrolase variant of Claim 43 , wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID

NO:2, wherein said at least one substitution is selected from the group consisting of A44, C7, 1107, K97, L12, L78, P104, Q40, and V125.

45. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is between about 3.0 and about 5.

46. The perhydrolase variant of Claim 45, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ED

NO:2, wherein said at least one substitution is selected from the group consisting of D10, D85, L53, L78, and S54. GC821-2

47. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.1 or less.

48. The perhydrolase variant of Claim 47, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, and W34.

49. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.2 or less.

50. The perhydrolase variant of Claim 49, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID

NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GllO, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, PI 8, R102, R33,

R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, and Y73. GC821-2

51. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.3 or less.

52. The perhydrolase variant of Claim 51 , wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GllO, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GI 10, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, GllO, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Ml 11, N94, P146, Q41, R102, R27, R4, R56, Sll, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, and Y129. .

53. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.4 or less.

54. The perhydrolase variant of Claim 53, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID

NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GllO, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, GC821-2

L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GllO, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, DIO, D62, F150, GI 10, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Ml 11, N94, P146, Q41, R102, R27, R4, R56, Sll, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, GI 10, G126, G22, 1194, 189, K97, Ll 14, L84, N59, P146, P83, R102, R27, R33, R4, R56, SI 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18, V125, V19, and V87.

55. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.5 or less.

56. The perhydrolase variant of Claim 55, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GI 10, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, Gl lO, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, GllO, G22, G70, 1153, 1194, 160, 189, K97, L114, L119, L12, L38, L6, L68, L82, Mi l l, N94, P146, Q41, R102, R27, R4, R56, Sll, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, GC821-2

E51, FlOO, F150, F154, GllO, G126, G22, 1194, 189, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120, T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, DIO, D23, E26, E50, E51, F150, GI 10, G126, G15, G36, 1107, 149, 15, K97, L109 ,L119, L12 L38, L6, L68, L84, L86, Ml 11, N59, P146, P24, Q40, RlOl, R102, R27, R33, R4, R56, SI 12, S72, S76, T127, T25, T35, T80, T96, VI 15,

V32, V87, W34, and Y129 .

57. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.6 or less.

58. The perhydrolase variant of Claim 57, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GllO, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sl l, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GllO, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34,

Y129, Y73, A122, A23, A44, C7, D10, D62, F150, Gl lO, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Ml 11, N94, P146, Q41, R102, R27, R4, R56, SI 1, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, GllO, G126, G22, 1194, 189, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, SI 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18,

V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, GllO, G126, G15, G36, 1107, 149, 15, K97, L109 ,L119, L12 L38, L6, L68, L84, L86, Mill, N59, P146, P24, GC821-2

Q40, RlOl, R102, R27, R33, R4, R56, SI 12, S72, S76, T127, T25, T35, T80, T96, VI 15, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, gl26, G36, G52, 1107, 15, 189, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, SI 12, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, V115N118, V32, V48, V87, W149, and Y73.

59. The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.7 or less.

60. The perhydrolase variant of Claim 59, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GllO, G52, G70, 1107, 1194, 15, K97, L12, L53, L6,

L86, N94, P83, R102, R4, R56, SI 1, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GI 10, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, GllO, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Ml 11, N94, P146, Q41, R102, R27, R4, R56, Sll, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, Gl lO, G126, G22, 1194, 189, K97, L114, L84, N59, P146, P83, R102, R27, R33, R4, R56, SI 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, GllO, G126, G15, G36,

1107, 149, 15, K97, L109 ,L119, L12 L38, L6, L68, L84, L86, Mi ll, N59, P146, P24, Q40, RlOl, R102, R27, R33, R4, R56, SI 12, S72, S76, T127, T25, T35, T80, T96, VI 15, GC821-2

V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, gl26, G36, G52, 1107, 15, 189, L109, Ll 14, Ll 19, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, SI 12, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, VI 15N118, V32, V48, V87, W149,Y73, A122, A23, A29, A71, A79, C7, D61, D62, D85, E26, E51, FlOO, F28, F46, GI 10, G126, G52, G7Q, 1107, 149, 15, 160, 189, L109, Ll 14, L12, L38, L68, L82, L86, Ml 11, N59, N94, P83, R102, R33, R4, SI 12, S72, S76, T103, Tl 16, T128, T25, T35, T57, T58, T64, V19, V32, V48, V87, Y129, Y73, and Y99.

....

61. . _The perhydrolase variant of Claim 8, wherein said variant perhydrolase exhibits a change in perhydrolysis, such that the ratio of variant perhydrolase perhydrolysis to wild-type perhydrolase perhydrolysis is about 0.8 or less.

62. The perhydrolase variant of Claim 36, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ED

NO:2, wherein said at least one substitution is selected from the group consisting of A23, A55, D10, D62, F150, F196, F28, GllO, G52, G70, 1107, 1194, 15, K97, L12, L53, L6, L86, N94, P83, R102, R4, R56, Sll, S54, T120, T13, T25, T80, VI 15, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62, F150, F154, GllO, G22, G52, G70, 1194, K3, K97, L105, L12, L38, L53, L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, SI 12, S54, T127, T13, T35, T64, T80, T96, VI 18, V48, W149, W16, W34, Y129, Y73, A122, A23, A44, C7, D10, D62, F150, GI 10, G22, G70, 1153, 1194, 160, 189, K97, Ll 14, Ll 19, L12, L38, L6, L68, L82, Ml 11, N94, P146, Q41, R102, R27, R4, R56, Sl l, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129, A55, C77, E51, FlOO, F150, F154, GllO, G126, G22, 1194, 189, K97, L114, L84, N59, P146, P83,

R102, R27, R33, R4, R56, SI 12, S54, S72, S76, T120, T127, T13, T25, T57, T96, VI 18, V125, V19, V87, A23, A55, D10, D23, E26, E50, E51, F150, GllO, G126, G15, G36, GC821-2

1107, 149, 15, K97, L109 ,L119, L12 L38, L6, L68, L84, L86, Ml 11, N59, P146, P24, Q40, RlOl, R102, R27, R33, R4, R56, SI 12, S72, S76, T127, T25, T35, T80, T96, VI 15, V32, V87, W34, Y129, A108, A44, A55, D21, D62, F150, gl26, G36, G52, 1107, 15, 189, L109, Ll 14, Ll 19, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66, P83, R27, SI 12, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96, VI 15,V118, V32, V48, V87,

W149,Y73, A122, A23, A29, A71, A79, C7, D61, D62, D85, E26, E51, FlOO, F28, F46, GI 10, G126, G52, G70, 1107, 149, 15, 160, 189, L109, Ll 14, L12, L38, L68, L82, L86, Mill, N59, N94, P83, R102, R33, R4, SI 12, S72, S76, T103, T116, T128, T25, T35, T57, T58, T64, V19, V32, V48, V87, Y129, Y73, Y99, A108, A122, A29, A55, C77, DIO, D106, D45, D61, D62, D65, D85, E47, E50, FlOO, F150, F28, F46, GllO, G124, ^' G126, G15, G36, 1153, 1194, 15, 160, 189, K3, K97, L105, L109, Ll 14, Ll 19, L38, L42, L68, L84, L86, Ml, N59, P24, P30, P83, RlOl, R27, R4, R56, SI 12, S54, S76, T103, Tl 16, T120, T127, T128, T13, T35, T64, VI 13, V17, V19, V32, V48, V87, Y129, Y73, and Y99.

63. A perhydrolase variant, wherein said variant exhibits greater perhydrolysis activity and decreased peracid hydrolysis activity as compared to wild-type perhydrolase.

64. The perhydrolase variant of Claim 8, wherein said perhydrolase exhibits perhydrolysis activity ratio of at least about 1.2, and peracid hydrolysis activity ratio of about 0.8 or less, as compared to wild-type perhydrolase.

65. The perhydrolase variant of Claim 64, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID

NO:2, wherein said at least one substitution is selected from the group consisting of A29, A44, A55, A71, A79, C7, D10, D106, D31, D85, E26, E47, F150, F154, F196, F28, GC821-2

G124, G126, G36, G43, 1153, L109, L42, L53, L109, L42, L53, L109, L42, L53, L68, L82, L86, Mill, N69, P104, P148, P18, P63, P66, P83, Q117, Q40, RlOl, R67, S54, S121, S72, S76, T25, T64, VI 15, and V19.

66. The perhydrolase variant of Claim 8, wherein said perhydrolase exhibits perhydrolysis activity ratio of at least about 1.2, a peracid hydrolysis activity ratio of about 0.8 or less, and a protein concentration ratio of at least 0.5, as compared to wild- type perhydrolase.

67. The perhydrolase variant of Claim 66, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A29, A44, A71, A79, C7, D85, E26, E47, E51, F150, F154, F196, F28, G124, G126, G36, 1153, L109, L12, L53, L68, L82, M111, N69, P104, P148, P18, P63, P66, P83, Q117,

Q40, RlOl, R67, S121, S54, S72, S76, T25, T64, V125, and V19.

68. A perhydrolase variant wherein said variant perhydrolase exhibits an increase in expression of said perhydrolase variant, as compared to the expression of wild-type perhydrolase.

69. The perhydrolase variant of Claim 68, wherein said modification comprises at least one substitution at an amino acid position equivalent to a position in M. smegmatis perhydrolase comprising the amino acid sequence set forth in SEQ ID NO:2, wherein said at least one substitution is selected from the group consisting of A2, 15, C7, F8, Sll, L12, T13, W14, W16, V17, P18, V19, E20, G22, A23, P24, T25, A29, P30, V32, T35, G36, V37, A39, F46, E47, S54, A55, R56, T58, 160, D61, D62, P63, T64, GC821-2

P66, R67, L68, N69, G70, S72, Y73, L74, P75, S76, C77, L78, A79, T80, L82, P83, L84, L86, 189, T93, T96, K97, A98, Y99, FlOO, RlOl, R102, T103, P104, L105, D106, 1107, A108, L109, GllO, SI 12, VI 13, L114, VI 15, T116, Q117, VI 18, L119, T120, S121, A122, G124, V125, G126, T127, T128, Y129, P130, P132, K133, L135, V136, S138, P141, L142, A143, M145, H147, W149, F150, Q151, 1153, G157, Q159, T161, T162,

L164, A165, R166, V167, Y168, A170, L171, A172, M175, K176, P178, A182, G183, S184, VI 85, 1186, T188, 1194, F196, V191, N201, L208, A209, Q211, Q213, S214, L215, and L216.

70. An isolated protein comprising a homolog of M. smegmatis perhydrolase, wherein said homolog is a protein within the SGNH-hydrolase family of proteins.

71. An isolated protein having at least about 35% identity with the amino acid sequence of M. smegmatis perhydrolase, in which the protein comprises at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78,

H81, P83, M90, K97, GllO, L114, L135, F180, G205, Sl l, D192, and H195.

72. An isolated protein having at least about 38% identity with the amino acid sequence of M. smegmatis perhydrolase, wherein said protein exhibits perhydrolysis activity.

73. A homolog of M. smegmatis perhydrolase, wherein said homolog is a perhydrolase comprising at least one motif selected from the group consisting of GDSL- GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT.

74. The homolog of Claim 72, wherein said homolog exhibits perhydrolysis. GC821-2

75. The homolog of Claim 72, wherein said homolog exhibits a perhydrolysis to hydrolysis ratio that is great than about 1.

76. The homolog of Claim 72, wherein said homolog is immunologically cross-reactive with antibodies raised against said M. smegmatis perhydrolase.

77. The homolog of Claim 72, wherein antibodies raised against said homolog cross-react with said M. smegmatis perhydrolase.

78. An isolated protein having at least about 35% identity with the amino acid sequence of at least one M. smegmatis perhydrolase homolog, wherein said protein exhibits perhydrolysis activity.

79. An isolated protein having perhydrolase activity, wherein said protein is in the form of a multimer in solution.

80. The isolated protein of Claim 79, wherein said protein is a perhydrolase that comprises a dimer.

81. The isolated protein of Claim 79, wherein said protein is a perhydrolase that comprises an octamer.

82. An isolated protein having perhydrolase activity, wherein said protein is in the form of a multimer in solution and said protein is selected from the group consisting of M. smegmatis perhydrolase, M. smegmatis perhydrolase homologs, and M. smegmatis perhydrolase variants. GC821-2

83. The isolated protein of Claim 79, wherein said protein is selected from the group consisting of modified serine hydrolases and modified cysteine hydrolases, wherein said modified serine hydrolases or said modified cysteine hydrolases comprise increased perhydrolase activity as compared to unmodified serine hydrolases or unmodified cysteine hydrolases

84. An isolated protein having perhydrolase activity, wherein said protein comprises at least one motif selected from the group consisting of GDSL-GRTT, GDSL- ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT.

85. The isolated protein of Claim 84, wherein said protein is obtained from a member of the Rhizobiales.

86. The isolated protein of Claim 84, wherein said protein is obtained from a member of the genus Mycobacterium.

87. An isolated gene identified using at least one primer selected from the group consisting of SEQ ID NOS :21-69.

88. A method for identifying a perhydrolase, comprising the steps of: a) identifying source of said perhydrolase; b) analyzing said source to identify sequences comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT; c) expressing said sequences identified in step b) to produce said perhydrolase; and d) testing said perhydrolase for perhydrolysis activity. GC821-2

89. The method of Claim 88, wherein said analyzing step is an amplification step wherein the primer sequences set forth in SEQ ED NOS :21-69 are used to amplifying said sequences comprising at least one motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT.

90. The method of Claim 88, wherein said source is selected from the group consisting of environmental sources and metagenomic sources.

91. The protein identified using the method of Claim 88.

92. An isolated nucleic acid sequence encoding the protein of Claim 91.

93. The method of Claim 88, wherein said protein exhibits a perhydrolysis to hydrolysis ratio that is greater than about 1.

94. The method of Claim 88, wherein said protein exhibits a perhydrolysis activity that is at least about 0.2, compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase.

95. The method of Claim 88, wherein said protein comprises at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, GllO, L114, L135, F180, G205, Sll, D192, and H195.

96. The method of Claim 88, wherein said analyzing step comprises searching at least one amino acid database. GC821-2

97. The method of Claim 88, wherein said analyzing step comprises searching at least one nucleic acid database to identify nucleic acid sequences encoding the amino acid sequences of said perhydrolase.

98. The method of Claim 96, wherein said source is selected from the group consisting of environmental sources and metagenomic sources.

99. An isolated nucleic acid sequence encoding the protein of Claim 96.

100. An isolated nucleic acid sequence identified using the method of Claim

96.

101. The method of Claim 96, wherein said protein exhibits a perhydrolysis to hydrolysis ratio that is greater than about 1,

102. The method of Claim 96, wherein said protein exhibits a perhydrolysis activity that is at least about 0.2, compared to the perhydrolysis activity exhibited by M. smegmatis perhydrolase.

103. The method of Claim 96, wherein said protein comprises at least three residues selected from the group consisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, GI 10, Ll 14, L135, F180, G205, Sll, D192, and H195, as set forth in SEQ ID NO:2.

104. A variant perhydrolase of Claim 1 , wherein said variant perhydrolase has an altered substrate specificity as compared to wild-type M. smegmatis perhydrolase. GC821-2

105. The variant perhydrolase of Claim 104, wherein said variant has altered para nitrophenyl caproate (PNC) activity, as compared to wild-type M. smegmatis perhydrolase.

106. A variant perhydrolase of Claim 1 , wherein said variant perhydrolase has an altered pi as compared to wild-type M. smegmatis perhydrolase.

107. The variant perhydrolase of Claim 106, wherein said variant perhydrolase comprises at least one positively charged mutation.

108. The variant perhydrolase of Claim 106, wherein said variant perhydrolase comprises at least one negatively charged mutation.

109. A variant perhydrolase of Claim 1 , wherein said variant has increased stability, as compared to wild-type M. smegmatis perhydrolase.

110. The variant perhydrolase of Claim 109, wherein said stability is selected from the group consisting of thermostability, enzymatic stability, and chemical stability.

111. The perhydrolase of Claim 1 , wherein said variant exhibits at least one altered surface property.

112. The perhydrolase of Claim 111, wherein said variant comprises mutations at least one substitution at sites selected from the group consisting of the residues set forth in Table 15-1.

113. The perhydrolase of Claim 1 , wherein said perhydrolase is a variant GC821-2

perhydrolase having at least one improved property as compared to wild-type perhydrolase.

114. An expression vector comprising a polynucleotide sequence encoding the perhydrolase variant of Claiml 13.

115. A host cell comprising said expression vector of Claim 114.

116. The host cell of Claim 115, wherein said host is selected from the group consisting of Bacillus sp., Streptomyces sp., Escherichia, and Pantoea sp.

117. A perhydrolase produced by said host cell of Claim 116.

118. A composition comprising at least a portion of the isolated perhydrolase of Claim 1.

119. The composition of Claim 118, wherein said perhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2.

120. The composition of Claim 118, wherein said perhydrolase is encoded by the polynucleotide sequence set forth in SEQ ID NO:l.

121. The polynucleotide sequence of Claim 1218, wherein said sequence comprises at least a portion of SEQ ID NO: 1.

122. An expression vector comprising the polynucleotide sequence of Claim

121. GC821-2

123. A host cell comprising said expression vector of Claim 122.

124. The host cell of Claim 123, wherein said host is selected from the group consisting of Bacillus sp., Streptomyces sp., Escherichia, and Pantoea sp.

125. A perhydrolase produced by said host cell of Claim 124.

126. A variant perhydrolase, wherein said perhydrolase comprises at least one substitution corresponding to the amino acid positions in SEQ ID NO:2, and wherein said variant perhydrolase has better performance in at least one property, compared to wild- type M. smegmatis perhydrolase.

127. An isolated polynucleotide comprising a nucleotide sequence (i) having at least about 70% identity to SEQ ID NO: 1 , or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence set forth in SEQ ED NO: 1, under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence set forth in SEQ ID NO: 1.

128. A vector comprising the polynucleotide of Claim 127.

129. A host cell transformed with the vector of Claim 128.

130. A polynucleotide comprising a sequence complementary to at least a portion of the sequence set forth in SEQ ID NO: 1.

131. A method of producing an enzyme having perhydrolase activity, GC821-2

comprising: (a) transforming a host cell with an expression vector comprising a polynucleotide having at least 70% sequence identity to SEQ ID NO:l; (b) cultivating said transformed host cell under conditions suitable for said host cell to produce said perhydrolase; and (c) recovering said perhydrolase.

132. The method of Claim 131, wherein said host cell is selected from the group consisting of Streptomyces, Pantoea, Escherichia, and Bacillus species.

133. A probe comprising a 4 to 150 polynucleotide sequence substantially identical to a corresponding fragment of SEQ ED NO:l, wherein said probe is used to detect a nucleic acid sequence coding for an enzyme having perhydrolase activity.

134. A cleaning composition comprising: a) at least 0.0001 weight percent of said perhydrolase of Claim 1 ; b) a molecule comprising an ester moiety; and c) optionally, an adjunct ingredient.

135. A cleaning composition comprising: a) at least 0.0001 weight percent of said perhydrolase of Claim 1 ; b) a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, said peroxygen source being selected from the group consisting of: i. a per-salt; ii. an organic peroxyacid; iii. urea hydrogen peroxide; GC821-2 iv. a carbohydrate and carbohydrate oxidase mixture, and v. mixtures thereof; c) from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety, and d) optionally, an adjunct ingredient.

136. A cleaning composition comprising: a) from about 0.0001 to about 1 weight percent of said perhydrolase of Claim 8; b) a material selected from the group consisting of a peroxygen source, hydrogen peroxide and mixtures thereof, said peroxygen source being selected from the group consisting of: vi. a per-salt vii. an organic peroxyacid; viii. urea hydrogen peroxide; ix. a carbohydrate and carbohydrate oxidase mixture, and x. mixtures thereof; c) from about 0.01 to about 50 weight percent of a molecule comprising an ester moiety d) optionally, an adjunct ingredient.

137. A cleaning composition according to Claim 136, said composition comprising an adjunct ingredient.

138. A cleaning composition according to Claim 137, wherein said adjunct ingredient is selected from the group consisting of surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme GC821-2

stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, pigments and mixtures thereof.

139. A cleaning composition according to Claim 137, wherein: a) said perhydrolase exhibits a perhydrolysis to hydrolysis molar ratio that is greater than about 0.1 ; b) said per-salt is selected from the group consisting of alkalimetal perborate, alkalimetal percarbonate, alkalimetal perphosphates, alkalimetal persulphates and mixtures thereof; c) said carbohydrate is selected from the group consisting of mono- carbohydrates, di- carbohydrates, tri- carbohydrates, oligo- carbohydrates and mixtures thereof; d) said carbohydrate oxidase is selected from the group consisting of aldose oxidase (IUPAC classification EC 1.1.3.9), galactose oxidase (IUPAC classification ECl.1.3.9), cellobiose oxidase (IUPAC classification ECl.1.3.25), pyranose oxidase (IUPAC classification EC 1.1.3.10), sorbose oxidase (IUPAC classification ECl.1.3.11) hexose oxidase (IUPAC classification ECl.1.3.5). glucose oxidase (IUPAC classification EC 1.1.3.4) and mixtures thereof; and e) said molecule comprising an ester moiety has the formula:

R¹O_x [(R²)_m (R³)„]p (i) wherein R is a moiety selected from the group consisting of H, substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and GC821-2 heteroaryl; (ii) each R is an alkoxylate moiety, (iii) R is an ester-forming moiety having the formula: R⁴CO- wherein R⁴ is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl,; (iv) x is 1 when R¹ is H; when R is not H, x is an integer that is equal to or less than the number of carbons in R¹; (v) p is an integer that is equal to or less than x; (vi) m is an integer from 0 to 50; and (vii) n is at least 1

140. The cleaning composition of Claim 139, wherein: a) R¹ is an C2-C32 substituted or unsubstituted alkyl or heteroalkyl moiety; b) each R is independently an ethoxylate or propoxylate moiety; and c) m is an integer from 1 to 12.

141. The cleaning composition of Claim 140, wherein R is an ester-forming moiety having the formula: R⁴CO- wherein R⁴ is: a) a substituted or unsubstituted alkyl, alkenyl or alkynyl moiety comprising from 1 to 22 carbon atoms; or b) a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl or heteroaryl moiety comprising from 4 to 22 carbon atoms.

142. The cleaning composition of Claim 137, wherein the molecule comprising the ester moiety has the formula:

GC821-2

wherein: a) R is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, said R moiety that comprises an amine moiety being selected from the group consisting of substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; b) each R² is an alkoxylate moiety; c) R is an ester-forming moiety having the formula: R CO- wherein R may be H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; d) x is 1 when R¹ is H; when R¹ is not H, x is an integer that is equal to or less than the number of carbons in R ; e) p is an integer that is equal to or less than x • f) m is an integer from 0 to 12; and g) n is at least 1.

143. The cleaning composition of Claim 136, wherein said molecule comprising an ester moiety has a weight average molecular weight of less than 600,000 Daltons.

144. The cleaning composition of Claim 143, wherein said adjunct ingredient is selected from the group consisting of surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing GC821-2

aids, pigments and mixtures thereof.

145. A method of cleamng, said method comprising the steps of: a) contacting a surface and/or an article comprising a fabric with the cleaning composition of Claim 136 and/or a composition comprising the cleaning composition of Claim 136; and b) optionally washing and/or rinsing said surface or material.

146. A method of cleaning, said method comprising the steps of: a) contacting a surface and or an article comprising a fabric with the cleaning composition of Claim 137 and/or a composition comprising the cleaning composition of Claim 137; and b) optionally washing and/or rinsing said surface or material.

147. A bleaching composition comprising the perhydrolase of Claim 1.

148. The bleaching composition of Claim 147, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

149. A bleaching composition comprising the perhydrolase variant of Claim 8.

150. The bleaching composition of Claim 149, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, GC821-2

endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wa l degrading enzymes, hemicellulases, and cellulases.

151. A bleaching composition comprising the perhydrolase variant of Claim 113.

152. The bleaching composition of Claim 151, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall degrading enzymes, hemicellulases, and cellulases.

153. A bleaching composition comprising the perhydrolase variant of Claim 132.

154. The bleaching composition of Claim 153, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall degrading enzymes, hemicellulases, and cellulases.

155. A bleaching composition comprising the perhydrolase of Claim 3.

156. The bleaching composition of Claim 155, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall GC821-2

degrading enzymes, hemicellulases, and cellulases.

157. A disinfecting composition comprising the perhydrolase of Claim 1.

158. The disinfecting composition of Claim 157, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall degrading enzymes, hemicellulases, and cellulases.

159. A disinfecting composition comprising the perhydrolase variant of Claim 8.

160. The disinfecting composition of Claim 159, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall degrading enzymes, hemicellulases, and cellulases.

161. A disinfecting composition comprising the perhydrolase variant of Claim

113.

162. The disinfecting composition of Claim 161, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall degrading enzymes, hemicellulases, and cellulases. GC821-2

163. A disinfecting composition comprising the perhydrolase variant of Claim 132.

164. The disinfecting composition of Claim 163, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall degrading enzymes, hemicellulases, and cellulases.

165. A disinfecting composition comprising the perhydrolase variant of Claim

166. The disinfecting composition of Claim 165, further comprising at least one additional enzymes or enzyme derivatives selected from the group consisting of proteases, amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases, endoglycosidases, lysozyme, bacterial cell wall degrading enzymes, fungal cell wall degrading enzymes, hemicellulases, and cellulases.