CA2189428A1 - Fabric cleaning compositions containing subtilisin bpn' variants - Google Patents

Abstract

The present invention relates to fabric cleaning compositions comprising subtilisin BPN' variants, wherein the BPN' variant comprises one or more amino acid positions having a different amino acid than that occurring in wild-type subtilisin BPN' (i.e., substitution) at specifically identified positions, whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'.

Description

WO 95/29979 1 ~ ~ G~l FABRIC CLEANING COMPOSITIONS CONTAINING SUBTILISIN BPN' ~ARIANTS
TECHNICAL FIELD
The present invention relates to fabric cleaning cu" "~o~ "s 5 cul"~ ,i"g protease enzymes which are subtilisin variants.
BACKGROUND
Enzymes make up the largest class of naturally occurring proteins.
Each class of enzyme senerally catalyzes (acCe:l~ldt~s a reaction without being consumed) a different kind of chemical reaction. One class of 1û enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins. This ability to hydrolyze proteins has been taken advantage of by illC~I~JUldlilly naturally occurring and protein d"yi"e~l~d 15 proteases as an additive to laundry detergent pfelualdliùl~s~ Many stains on clothes are ~Jlule:illdceous and wide-specificity proteases can suu~Ldllli~l'y improve removal of such stains.
Unfortunately, the efficacy level of these proteins in their natural, bacterial ~"~/;,u""":"l, frequentiy does not translate into the relatively 20 unnatural wash environment. Specifically, protease ,l Idl d~,lel ialic:l such as thermal stability, pH stability, oxidative stability and substrate specificity are not l~e~,essd,ily optimized for utilization outside the natural environment of the enzyme.
The amino acid sequence of the protease d~ ""i"es the 25 ei Idl dU~ li~ of the protease. A change of the amino acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, d~,u~lldi~ly upon the location, nature and/or ll~dyl t~ s of the change in the amino acid sequence. Several a~u,u~a.,l,~
have been taken to alter the wild-type amino acid sequence of proteases in 30 an attempt to improve their properties, with the goal of increasing the affiCdCy of the protease in the wash environment. These d,u,u,~a.,l,~s include altering the amino acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
Despite the variety of d,up,ua,,l,~s described in the art, there is a 35 continuing need for c~",~osiliu"s c~,~,,u,i~i"g effective variants of proteases useful for cleaning fabric surfaces.
SUBSTITUTE SHEET (~ULE 26) W095129979 2 ~ $~ 4 2 ~ cl~9l ~
Obiects of the Present Invention It is an object of the present inventibn to provide fabric cleaning uu"~,uo~ iu"~ ~",,u,i~i,~,g subtilisin enzymb variants.
SUMMARY
The present invention relates to Cu"~,uOSi~ 15 .,u",~ ,i"9 subtilisin BPN' variants for cleaning fabric surfaces. The BPN' variants useful in these culn,uoaiIiu,~s comprise at least one, two or three amino acid positions having a different amino acid than that occurring in wild-type subtilisin BPN' (i.e., c~ Ihstit~ Ition) at :~,ue~.iru,~y identified positions, whereby the BPN'variant has decreased ddaul~uliul~ to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'.
DESCRIPTION
1. Subtilisin Variants Useful In Fabric Cleanina Cu",uo~iIiu"s This invention relates to fabric cleaning cull,~ait;on~ cu,,,,uli~ lg a subtilisin enzyme, in particular BPN~, that has been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme. The modified subtilisin enzymes (he,t~ dll~r, "BPN' variants") useful in the ,ulll~uu~iliulls of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. Certain of these BPN' variants are described in co-pending ~ I U S.S.N.
08/121,437, filed S~rt~her 15, 1993 by Brode et al.
The subtilisin enzymes useful in the ,~",~urj~iliu,~s of this invention belong to a class of enzymes known as proteases A protease is a catalyst for the cleavage of peptide bonds. One type of protease is a serine protease. A serine protease is distinguished by the fact that there is an essential serine residue at the active site.
The obs~ d~ that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme cul~c~llildliu,l is well documented. it 3û would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning ~ ' )5, the rate of hydrolysis would increase with increasing surface cu~ l ,l, dliOI~. This has been shown to be the case. (Brode, P.F. Ill and D. S. Rauch, L~NGMUIR, "Subtilisin BPN':
Activity on an l~,lll ' ' Substrate", Vol. 8, pp. 1325-1329 (1992~). In fact, a linear dtpe".lt:,)ce of rate upon surface cur,~,~"~,dliUII was found forinsoluble substrates when the surface Cul~ ldliO~l of the enzyme was varied. (Rubingh, D. N. and M. D. Bauer, ~Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface," in POLYMER SOWTIONS, BLENDS
SU~STITUTE SHEE~ (RULE 26) 2 ~ 8 ~ 4 2 ~ P~llv~ 9, AND INTERFACES, Ed. by 1. Noda and D. N. Rubingh, Elsevier, p. 464 (1992)).
Surprisingly, when seeking to apply this principle in the search for variant proteases which give better fabric cleaning p~trolllldl~ce, we did not find that enzymes which adsorb more give better pe,ru""d"~,e. In fact, we 5 surprisingly d~ ""i,~ed the opposite to be the case~ ult:ds~d ddaul~.liol) by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i.e., better cleaning ,uc:, ru, ll~dl IU~).
While not wishing to be bound by theory, it is believed that improved perFulllldllue:~ when COlll,Udlill9 one variant to another, is a result of the fact 1û that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed. At c~llllualdLle enzyme solution COllCl~ ld~iulla, this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher cu, ,~,e"l, dliOI~ of enzyme to the surface.
The mutations described herein are designed to change (i.e., decrease) the a~aul,uliù,~ of the enzyme to surface-bound soils. In BPN~, the amino acids from position 199 to position 220 form a large exterior loop on the enzyme molecule. It has been discovered that this loop plays a significant role in the a~aul,uLiul, of the enzyme molecule to a surface-bound 2û peptide, and specific mutations in this loop have a significant effect on this adau"u~iull. While not wishing to be bound by theory, it is believed that this loop is important to the d-lau"u~iu" of the BPN~ molecule for at least two reasons. First, the amino acids which comprise this exterior loop can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of this loop to the active-site and binding pocket of the BPN~
molecule gives it a role in the catalytically productive a~u,,uliu,, of the enzyme to surface-bound substrates (pe,uli.les/y, utt:i,, soils).
As used herein, "variant" means an enzyme having an amino acid sequence which differs from that of wild-type.
3û As used herein, "mutant BPN~ gene" means a gene coding for a BPN~
variant.
As used herein, "wild-type subtilisin BPN~ refers to a subtilisin enzyme l~:ultl,~ t,d by SEQ ID NO:1. The amino acid sequence for subtilisin BPN~ jS further described by Wells, J. A., E. Ferrari, D. J. Henner, D. A. Estell and E. Y. Chen, NUCLEIC ACIDS RESEARCH, Vol. Il, 7911-7925 (1983), i".,~, ,uu, dl~d herein by reference.
SUBSTITUTE SHEET (RULE 26) wos~nss7s ~ 28 ` ~ 01r~l ~

As used herein, the term "wiid-type amino acid sequence"
e~cu~ claaes SEQ ID NO:1 as well as SEQ ID NO:1 havins ".~.. Iif;- ,~ ns to the amino acid sequence other than at any of positions 1 99-22û.
As used herein, "more llydlupl.' amino acid" refers to any other amino acid having greater hy~lu~JII' ~.y than 2 subject amino acid with reference to the II~Jlupl~' 'y table below. The following h~lu~JII" '.y table (Table 1 ) lists amino acids in des~,~r,.li. ,~ order of increasing hy.lluyl ' 'y (see Hopp, T.P., and Woods, iCR., "Prediction of Protein Anti~enic D~ " "i"." Ita from Arrlino Acid Sequences", PK - _ ~ OF THE
1û NATIONAL ACADEMY OF SCIENCE USA, Vol. 78, pp. 3824-3828, 1981, i~ .w, ,uu, ~l~d herein by reference).

Arrlir o Acid H~dl uul, ~.y Value ~rp . he -_.
Tyr - .
Leu, lle - .
Val -' .
Met -Cys , Ala, His - .
Thr - .
Pro, Gly Gln, Asn .
Ser Arg+, Lys+, Glu-, Asp~
Table 1 also indicates which amino acids carry a charge (this ~,I,c"c,.,Ia,i~, being based on a pH of from about 8-9). The positively charged amino acids are Arg and Lys, the negatively charged amino acids are Glu and Asp, and the remaininy amino acids are neutral. In a preferred ~,,,i~u,li,,,a,,t of the present invention, the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp).
Therefore, for example, the statement ~substitute Gln with an equally or more l~yd~u~ll;lic amino acid which is neutral or has a negative charge"
means Gln would be Cllhstitllt~d with Asn (which is equally hydrophilic to Gln), or Ser, Glu or Asp (which are more hydrophilic than Gln); each of which are neutral or have a negative charge, and have a greater hy~ll u~ ' iy value as compared to Gln. Likewise, the statement "substitute SUBSTITUTE Sl IEET (RULE 26~

~18~g2~ ' wo ssnss7s , 1~ /0 ~6~
Pro with a more hydrophilic amino acid which is neutral or has a negative charge" means Pro would be s~ Ih5titl ItPd with Gln, Asn, Ser, Glu or Asp.
A. Variants comprisina at least one amino acid s~ ~hstitl ~ion In one e",Lodi",e"L of the present inYention, the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequence at one or more of positions 199, 200, 201, 202, 203, 204, 205, 5 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 218, 219 or 220 is sllhstit~ltPd whereby the BPN~ variant has d~,,,t:ased ad~.,",Iio,~ to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'. Preferably, the positions having a Cllhstitllt~d amino acid are 199, 200, 201, 202, 205, 207, 208, 209, 210, 211, 212 or 215; more 10 preferably, 200, 201, 202, 205 or 207.
Preferably, the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 201 is Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 202 is Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 203 is Met, Cys, His, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 204 is Glu.
Preferably, the substituting amino acid for position 205 is Leu, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 206 Pro, Asn or Ser.
Preferably, the substituting amino acid for position 207 is Asp or Glu.
Preferably, the substituting amino acid for position 208 is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 209 is lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 210 is Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 211 is Ala, Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 212 is Gln, Ser, Asp or Glu.
SUBSTITUTE SHEET (RULE 26) ... ... .... ..... _ . . ... ... .. . . .. .

WO 95/29979 2 1 8 9 4 2 8 ~ - 6 . ~ ).. 'C l~9l ~
Preferably, the substitutin3 ami~o,~acid for position 213 is Trp, Phe, Tyr, Leu, lle, Val, Met, Cys, Ala, His', Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 214 is Phe, Leu, lle, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu.
Preferably, the substituting amino acid for position 215 is Thr, Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 218 is Glu.
Preferably, the substituting amino acid for position 219 is Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 220 is Pro, Gly, Gln, Asn, Asp or Glu.
More preferably, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 219 and 220 is, with reference to Table 1, is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin BPN'.
More preferably still, the substituting amino acid for any of positions 199, 200, 201, 202, 203/ 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 219 and 220 is Asp, or Glu; and the substituting amino acid for positions 204 or 218 is Glu.
B. Variants c~" Ivl i:,i"~ at least two amino acid sl Ihstit~ ~tions In another .,~l~o~ "l of the present invention, the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequenoe at two or more of positions 199, 200, 201, 202, 203, 204, 205, 206,207,208,209,210,211,212,213,214,215,216,217,218,2190r220 j5 Cll' " Itc-r~ whereby the BPN' variant has decreased ad~.n~' ) to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'. Preferably, the positions having a substituting amino acid are 199, 200, 201, 202, 205, 207, 208, 209, 210, 211, 212, or215; more preferably, positions 200, 201, 202, 205 or 207.
Preferably, the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 201 is Gly, Gln, Asn, Ser, Asp or Glu.
SUBSTITUTE SHEET (RULE 26) W0 9S129979 2~ 8 ~ 4 ~ 8 . ~ o l~]

Preferably, the substituting amino acid for position 202 is Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 203 is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 204 is Asp or Glu.
Preferably, the substituting amino acid for position 205 is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 206 is Pro, Asn, Ser, Asp, or Glu.
Preferably, the substituting amino acid for position 207 is Asp or Glu.
Preferably, the substituting amino acid for position 208 is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 209 is lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 210 is Ala, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 211 is Ala, Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 212 is Gln, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, or Asp.
Preferably, the substituting amino acid for position 214 is Phe, Leu, lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 215 is Thr, Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 217 is Leu, lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 218 is Gln, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 219 is Pro, Gln, Asn, Ser, Asp or Glu.
35 Preferably, the substituting amino acid for position 220 is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
More preferably, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, SU~STITUTE SHEET (RULE 26) ,,, ,, , _ ,,, _ _ .. ..... . .. ... ... .. . ........ . . .

W095129979 ~1~9428 ~ r~".~ IO~C91 215, 216, 217, 218, 219 or 220 is, with reference to Table 1, is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisinBPN'.
More preferably still, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 218, 219 or 220 is Asp and Glu; for position 217 is Leu, Asp, or Glu; and for position 213 is Asp.
C. Variants Cul, ,ul i;,i"a at least three amino acid sl Ihstitl ~tions In another e",~u,li",e"l of the present invention, the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequence of three or more of positions 199, 20û, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 and 220, is sllhsti~ whereby the BPN' Yariant has decreased d~au"Jliu" to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'. Preferably, the positions havin~ a substituting amino acid are 199, 200, 201, 202, 205, 207, 208, 209, 210, 211, 212, or 215;
more preferably positions 200, 201, 202, 205 or 207.
Preferably, the substitutin3 amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 201 is Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 202 is Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 203 Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
PreFerably, the substituting amino acid for position 204 is selected from the group consisting of Asp or Glu.
Preferably, the substituting amino acid for position 205 is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 206 is Pro, Asn, Ser, Asp, or Glu.
Preferably, the substituting amino acid for position 207 is Asp or Glu.
Preferably, the substituting amino acid for position 208 is Pro, Gly, Gln, Asn, Ser, Asp or Glu.
SUBSTlTUTE SHEET (RULE 26) ~ WO 95/29979 ~ 1 8 ~ ~ ~ 8 Y~ C o1c~l Preferably, the substiluting amino acid for position 209 is lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 210 is Ala, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 211 is Ala, Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 212 is Gln, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 214 is Phe, Leu, lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 215 is Thr, Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 217 is Leu, lle, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 218 is Gln, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 219 is Pro, Gln, Asn, Ser, Asp or Glu.
Preferably, the substituting amino acid for position 220 is Pro, Gly, Gln, Asn, Ser Asp or Glu.
More preferably, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 or 220 is, with reference to Table 1, is neutral or negatiYely charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisinBPN'.
More preferabiy still, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 218, 219 or 220 is Asp or Glu; for position 217 is Leu, Asp, or Glu;
and for position 213 is Asp.
SUBSTITUTE SHEET (RULE 26 WO 95/29979 2 1 8 ~ ~ ~ 8 P~ 'Q ' '11 ~
,.1'û,, ~'' D. Pr~ud,dliu,, of enzvme variants Example 1 Mutant BPN' Genes A phagemid (pSS-5) containing the wild type subtilisin BPN' gene 5 (rr '~ u~, C. and J. A. Wells, (1989), ~Protein Cnyi~e~lillg of Disulfide Bonds in Subtilisin BPN', BIOCHEMISTRY, Vol. 28, pp. 48û7-4815) is lldl~arUllll~d intû C..l~eri,,l,id coG ung-strain CJ236 and a single stranded uracil-cûntaining DNA template is prûduced using the VCSM13 helper phage (Kunkel, T.A., J.D. Roberts and R.A. Zakour, "Rapid and efficient 10 site-specific mutagenesis without phenûtypic selection", METHODS IN
ENZYMOLOGY, Vol. 154, pp. 367-382, (1987); as modified by Yuckenber~, P.D., F. Witney, J. G~i~selco~ and J. McClary, "Site-directed in vitrû
mutagenesis using urac71-containing DNA and phagemid vectors", DlREcTED
MUTAGENESIS - A PRACTICAL APPROAcH, ed. M.J. ~ ?ile:la~l~, pp. 27-48, 15 (1g91 ); both of which are i, ,~,u, ,uu, dl~d herein by reference). A single primer site-directed mutagenesis ,llù~lir; ~ Jn of the method of Zoller and Smith (Zoller, M.J., and M. Smith, "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment ûf DNA", NucLElc ACIDS REsrARcH, Vol.
20 10, pp. 6487~500, (1982), ill-,ul~Jûl~ d herein by reference) is used tû
produce all mutants (basically as presented by Yuckenberg, et a/., 1991, abûve). Oligor~ leotides are made using an Applied Biosystem Inc. 380B
DNA synthesizer. Mutagenesis reaction products are ~,d,~fu,,,,ed intû
G~ id coli âtrain MM294 (American Type Culture Collection E Coli.
25 33625). All mutants are confirmed by DNA sequencin3 and the isolated DNA is t,d":-fù""ed into the Bacillus subtilis 6A,ul~,a~iul~ strain BG2036 (Yan~, M. Y., E. Ferrari and D. J. Henner, (1984), "Cloning of the Neutral Protease Gene of Bacillus subtillis and the Use of the Cloned Gene to Create an In Vitro-derived Deletion Mutation", JouRNAL OF BACTERIOLOGY, 30 Vol. 160, pp. 15-21). For some of the mutants a modified pSS-5 with a frameshift-stop codon mutation at amino acid 217 is used to produce the uracil template. Oligor~ lPotid~s are designed to restore the proper reading frame at position 217 and also encoded for random sl~hstit~tinns at positions 199, 20û, 201, 2û2, 2û3, 204, 205, 206, 207, 208, 209, 210, 211, 35 212, 213, 214, 215, 216, 217, 218, 219 and 22û (equimolar andlor variable miAtures of all four r~ otides for all three bases at these codons).
Mutations that correct for the frameshift-stop and produce a functional SUBSTITUTE SHEET (RULE 26) 218g428 WO 95/29979 F~IIIJ~ C~C IJ9I

enzyme are identified by their ability to digest casein. The random 51 IhStitl Itions are u~ . " ,i"ed by DNA sequencing.
Example 2 r~ rlld~iun The Bacillus subtilis cells (BE2û36) containing a subtilisin mutant of interest are grown to mid-log phase in a one liter culture of LB-glucose broth and inoculated into a Biostat ED fermenter (B. Braun Biotech, Inc., Allentown, Pennsylvania) in a total volume of 10 liters. The rt:llll~lltd~iVII
media contains Yeast Extract, starch, antifoam, buffers'and trace minerals 10 (see FERMENTATION: A PRACTICAL APPROACH, Ed. B. McNeil and L. M.
Harvey, 1990). The broth is kept at a constant pH of 7.0 during the r~ Ld~iu~1 nun. Clll~ldlll,ullt~ ,al is added for antibiotic selection of mutagenized plasmid. The cells are grown overni9ht at 37C to an A600 of about 60 and harvested.
Example 3 Purification The rt:lllle:ll~dt;UII broth is taken through the following steps to obtain pure enzyme. The broth is cleared of ~acillus subtilis cells by c~:,lt,i'u~ti~n, and clarified by removing fine particulates with a 100K cutoff 20 Ill~lllbldl~. This is followed by cû(lctlllLld~iull on a 10K cutoff Ill~lllbldlle, and flow dialysis to reduce the ionic strength and adjust the pH to 5.5 using 0.025M MES buffer (2-(N-,,,u,,ul,r'' ,o)ethanesulfonic acid). The enzyme is further purified by loading it onto either a cation exchange ,l ll UllldlO~ld~ul Iy column or an afffinity ad:,u~,u~iull ulllullld~u~u,ld,u~lr column and eluting it from 25 the column with a NaCI or a propylene glycol gradient (see Scopes, R. K, PROTEIN PURIFICATION PRINCIPLEs AND PRACTICE, Springer-Verlag, New York (1984), i,~co,,uu,d~t,d herein by reference).
The pNA assay (DelMar, E.G., C. Largman, J.W. Brodrick and M.C.
Geokas, ANAL. BIOCHEM., Vol. 99, pp. 316-320, (1979), illWl~uuld~d herein 30 by reference) is used to determine the active enzyme e.u"c~"' dtiUI~ for fractions collected during gradient elution. This assay measures the rate at which p-rl;~lUdl ''' l~ is released as the enyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-11" Udll'" ' (sMPF-pNA). The rate of production of yellow color from the 35 hydrolysis reaction is measured at 410 nm on a ~ut,-.~loul,uLu,,,_'~.. and is,uluuul~iorldl to the active enzyme col~ct~ ldLiull. In addition, db:~llJdlll,e measurements at 280 nm are used to detemmine the total protein SUE,STITUTE SHEET (RULE 26) .. _ .. ... . _ . _ .. . . .. . _ _ . _ _ WO9S/29979 ~ 2g ~ ` r~ .691 ~
cu, 1~ dLiul ,. The active enzymeltûtal-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution.
To avoid autolysis of the enzyme during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the 5 ClllUllldlU~U,ld,UIly column. Upon c~lll,ul~Li~ll of the purification procedure the purity of the stock enzyme solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel ele~,~,u,ul,u,~:,is) and the absolute enyme c~,~ce,,L~dliu,~ is d~L~""i"ed via an active site titration method using trypsin inhibitor type Il-T: turkey egg white purchased from Sigma Chemical 1û Company (St. Louis, Missouri). The measured conversion factors will show which changes made in the enzyme molecule at the various positions result in the enzyme variant having increased activity over the wild-type, a3ainst the soluble substrate pNA.
In pl~idldL;~JIl for use, the enzyme stock solution is eluted through a 15 Sephadex-G25 (Pl Idl ",a.,ia, Fi~ ;, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer in the enyme stock solution is ex.,l,d"~ed for 0.1 M Tris buffer (Tris(h~d,u~,,,t,ll,yl-d,,,i,,u,,,~il,c,,,e) containing O.û1M CaC12 and pH
adjusted to 8.6 with HCI. All e,c~i~,i",t:"i~ are carried out at pH 8.6 in Tris 20 buffer ~l ,c:", lo~ d at 25C.
E. Chdl dl..lel i~d~iUI~ Qf enzvme variants Example 4 Model Surface r, ~Udl dtiUI I
Aminopropyl controlled pore glass (CPG) purchased from CPG Inc.
25 (Fairfield, New Jersey) is used as a support for covalently attaching the sAAPF-pNA substrate purchased from Bachem, Inc. (Torrence, Califomia).
The reaction is carrTed out in dimethyl sulfoxide and (1-ethyl-3-[3-(dimethylamino)propyl] ,dlL- " "ide hy~iu~l~lu~i ie) (EDC) is used as a coupling agent. Upon cu,,,,uleLi il~ (monitored by pNA assay), the excess 30 solvent is removed, and the CPG:sMPF-pNA is rinsed with dimethyl sulfoxide (DMS0) and doubly-distilled water. This is followed by oven drying with a N2 purge at about 7ûC. The reaction scheme and plt:,UdldLi~n of the i""" ' " ' substrate are conducted as described by Brode, P.F. Ill, and D.S. Rauch, "Subtilisin BPN': Activity on an ll,,,l ' "
35 Substrate," LANGMUIR, Vol. 8, p. 1325-1329, (1992), i"-,~"uu,- 1ud herein by reference.
The CPG surface will have 62,ûOû ~ 7,ûûû pNA molecules/gm2. The surface area will remain unchanged from the value of 50.ûm2/g reported by SUeSTlTUTE SHEET (RULE 26~

~ WO 95/29979 218 9 4 2 8 ~ r~ c 1 9l CPG Inc. for the CPG as received. This suggests that the procedure used to add sMPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A).
Example 5 Surface Hvdrolvsis Assav - Using CPG:sMPF-pNA adaul uliu" of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single e,~.e,i,,,c:,,L A small volume of enzyme variant stock solution is added to a flask containing Tris buffer and CPG:sAAPF-pNA which has been 1û degassed. The flask is shaken on a wrist-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example every 2 minutes during the early stages of ad~o, uliù,) hydrolysis -e.g. the frst 20 minutes - and every 10 minutes towards the end of the exp~ n~"L). The CPG:sMPF-pNA is allowed to settle and the solution is sampled. Both the eAp~,i",t:"Lal procedure and the r~ of the adsu,u~iu,, and hydrolysis are conducted as described by Brode e~a/. 1992 above.
All enzymes are monitored for stability against autolysis and should show no dUUl~ idLJIe autolytic loss over the time course of this e~ ,i",~z"L.
Therefore, enyme ad~"uLiui~ can be d~te:""i"ed by measuring solution depletion. The difference between the initial enzyme variant COI)C~lltld~iull snd the cul~c~llLldLiul~ measured at each individual time point gives the amount of enzyme variant adsorbed. The amount of pNA hydrolyzed from the surface is measured by taking an dbsol LJdl ll e reading on an aliquot of the sample at 410 nm. The total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask. This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tris buffer at pH 8.6 when no enzyme is present. This base-hydrolysis ranges from 7-29% of the total hydrolysis dep~"di"~ on the efficiency of the 30 enzyme.
Example 6 Soluble Substrate Kinetic Analvsis The rates of hydrolysis of the soluble substrate sMPF-pNA are monitored by measuring the adso,l,d"-e increase as a function of time at 35 410 nm on a DU-70 ~ue, L~uul~uLu~ .. The enzyme u ull~llLIdLiull is held constant and is prepared to be in the range of 6-10 lla"u""~la( while the substrate Cul~C~IILIdLiul~ is varied from 9û-700 ~lM sMPF-pNA for each kinetic d~Lt~ illdLiul~ An dd~ulL~dlll.e data point is taken each second over SUBSTITUTE SHEET (RULE 26!
_ .. . . . . .. .... . ... .. _ _ .. _ ... _ .

WO 95129979 ~ ~, g ~ r~ J~ `4G~I
14 ~
a period of 900 seconds and the data are ~Idllarelled to a LOTusTM
spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts).
Analysis for kinetic pdldlll~Lela is conducted by the standard Lineweaver Burk analysis in which the data in the initial part of the run (generally the first minute) are fit to a linear l~:u~le~Sivll curve to ~ive vO. The vO and sO
data are ploUed in the standard inverse fashion to give KM and kcat F. ExamDIe BPN' variants BPN' variants of the present invention which have deul ea5ed ad:~ulf,liùl~ to and increased hydrolysis of surface bound substrates are exe~ in Table 2, below. In describing the specific mutations, the original amino acid occurring in wild-type is given first, the position number second, and the sl IhCtitl ItPd amino acid third.

Exam~le BPN' Variants --Single Mutation-Ala216Glu Al a2 1 6Asp Ala216Gly Val203Glu --Double Mutation--Ile205Leu + Ala216Glu Ile205Leu + Ala216Asp Pro210Ala + Gly215Thr T yr2 1~ Phe + Tyr2 1 7Asn Gln206Glu + Ala216Glu Ala216Glu + Try217Leu Gln2 0 6Glu + Tyr2 17Leu --Triple Mutation--Gln206Glu + Ala216Glu + Tyr217Leu Gln206Pro + Gly211Ala + Ala216Glu -Quadruple Mutation--Val203Glu + Gln206Glu + Ala216Glu f Tyr217Leu Val203Glu + Pro210Ala + Ala216Glu + Tyr217Leu --Quintuple Mutation--Val203Glu + Gln206Glu + Gly215Thr + Ala216Glu + Tyr217Leu Val203Glu + Pro210Ala + Gly215Thr + Ala216Glu + Tyr217Leu Il. Fabric Cleanina C~,,,uoc,iLiu,) Materials The fabric cleaning cu,,,p~ iù,)~ of the present invention also comprise, in addition to the BPN' variants described heleillL)e~ulel one or more cleaning cu,,,,uo~itiù,) materials cu,,,,udliL,le with the protease enzyme.
SU~STITUTE SHEET (RULE 26) 218~2~
WO9~i/299~9 r~ .'1691 The term "cleaning cor,l,uosilic" material", as used herein, means any liquid, soiid or gaseous material selected for the particular type of cleaning ~,ullluosiliol~ desired and the form of the product (e.g., liquid; granule; bar), which materials are also cu,,,udli~le with the BPN' variant used in the 5 cu",~,osi~iol~. The specific selection of cleaning ~",I~osi~io" materials are readily made by uul~sicl~, il l9 the fabric to be cleaned, and the desired fonm of the ~ uosiliull for the cleaning condition during use (e.g., through the wash detergent use). The term ''c~,,,,ud~i~le'', as used herein, means the cleaning culll,uo~iliù" materials do not reduce the proteolytic activity of the 1û BPN' variant to such an extent that the protease is not effective as desired during normal use situations. Specific cleaning u~lll,u~:.iliùll materials are e>~", ~ d in detail lle,~i"drler.
As used herein, "fabric cleaning ~,~",,uùsi~iull" refers to all forms for detergent cull,,uoai~iu,~s for cleaning fabrics, including but not limited to, 15 granular, liquid and bar forms. Preferred fabric cleaning cul,l,uc,~iliull~ are those in the liquid form.
As used herein, "effective amount of enzyme variant" refers to the quantity of enzyme variant necessary to achieve the enzymatic activity necessary in the specific cleaning cu,,,,uosiliu,~. Such effective amounts are 20 readily d~ lldilled by one ûf ordinary skill in the art and is based on many factors, such as the particular enzyme variant used, the cleaning ~"' 1, the specific ,u,,,,uoc,i~iu,, of the cleaning culll,uu.iliùll, and whether a liquid or dry (e.g., granular) culll,uoailiu" is required, and the like.
Preferably the cleaning c~,,,,uùsili~ns of the present invention comprise from 25 abûut 0.0001% to about 10% of ûne or more enzyme variants, more preferably from about 0.001% to about 1%, more preferab~y still from about 0.01% to about 0.1%. Several examples of various cleaning cu",,uosiliù,~s of the present invention are discussed in further detail below. All parts, p~lue~ dges and ratios used herein are by weight unless otherwise 3û specified.
The enzyme variants of the present invention can be used with various conventional i"~ di~"l:, to provide fully-formulated fabric laundering c~lll,uuailiol~s. Such cu",p~siliu"~, can be in the fonm of liquids, granules and the like. Such ~,ulll,uOailiùllS can be fommulated as modern 35 "~u"ce, l~ d" d~ U,tll ,~, which contain as much as 30%~0% by weight of surfactants.
The fabric cleaning culll,uoailiu~ls herein can optionally, and preferably, contain various anionic, nonionic, z~ ,iu"ic, etc., surfactants.
SUBSTITUTE SHEET (RULE 26~
_ _ ~ . ... ... . . .. .... . .. _ _ .. . . . .

wogsl29979 ~18g428 - 16 F~ 91 Such surfactants are typically present at levels of from about 5% to about 35% of the cu""~osi~iulls Nul' "ili"g examples of surfactants useful herein include the conYentional C11-C18 alkyl benzene sulfonates and primary and random alkyl sulfates, the C10-c18 secondary (2,3~ alkyl sulfates of the fommulas CH3(CH2)x(CHOSO3)-M+)CH3 and CH3(CH2)y(CHOSO3~M+) CH2CH3 wherein x and (y+1) are integers of at least about 7, preferably at least about 9, and M is a water-s~ " ,9 cation, especially sodium, the C10-C18 alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C10-c18 alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C10-C18 alkyl poly91ycosides, and their cu~ a,u~lldillg sulfated polyglycosides, C12-C18 alpha-sulfonated fatty acid esters, C12-C1g alkyl and alkyl phenol ". yl~tcs (especially ethoxylates and mixed ~llU~(y/l~u,uu~y)~ C12-C18 betaines and s~'' ' ' ,es ("sultaines"), C10-c18 amine oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy cdl~o~l-'-r (AEC) are preferred herein. (Use of such surfactants in cu,,,ui,,~ o,~ with the aforesaid amine oxide and/or betaine or sultaine surfactants is also preferred, dtpe"lli"y on the desires of the formulator.) Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C10-c18 N-methyl glucamides disclosed in US
Patent 5, 194,639, Connor et al., issued March 16, 1993, i~,u~,uul ' ' herein by reference.
A wide variety of other i"yl tl~ l ,t~ useful in fabric cleaning ll, can be included in the cu",~.u~i~iù"s herein, including other active i"u, ~di~ a, carriers, hydrotropes, ,u, uce~ ,y aids, dyes or pigments, âolvents for liquid formulations, etc. If an additional increment of sudsing is desired, suds boosters such as the C10-C16 ". ' ' can be i~uu~,uu~ ' into the ,u,,,,uu~ ,, typically at about 1% to about 10%
levels. The C10-C14 Illullut:tlldllOI and diethanol amides illustrate a typical class of such suds boosters. Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous. If desired, soluble magnesium salts such as M3CI2, MgSO4, and the like, can be addeb at levels of, typically, from about 0.1% to about 2%, to provide acldilio,)a"y sudsing.
The liquid fabric cleaning c~,,,uosi~iull~ herein can contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols e~ i by methanol, ethanol, propanol, and isou,u,ud"ùl are suitable. Monohydric alcohols are preferred for solubilizing surfactants, but SUBSTITUTE SHEET (RULE 26) W0 95/29979 2 I 8 9 ~ ~ ~ ' ' ` F~ 91 polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1,3-u,u,ud,,ediùl, ethylene glycol, glycerine, and 1,2-u,upd"euiùl) can also be used. The c~ ,u~ailiulla - may contain from about 5% to about 90%, typically from about 10% to about 5 50% of such carriers.
The fabric cleaning culll,uo:,iliu,,~ herein will preferably be formulatedsuch that during use in aqueous cleaning op~,dliù,,a, the wash water will have a pH between about 6.8 and about 11Ø Finished products thus are typically formulated at this range. Techniques for controlling pH at 10 ~-,u~ el~d~d usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.
When formulating the fabric cleaning cu,,,l.ùsi~iù,,:, of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight. Typical builders include the 1-10 micron 15 zeolites, pOIyUdl bUAyld~eS such as citrate and oxydisuccinates, layered silicates, ,~ ual-l Idl_5, and the like. Other conventional builders are listed in standard formularies.
Likewise, the formulator may wish to employ various additional enzymes, such as ~ cec~ lipases, amylases and proteases in such 20 culll~Jo~ ull~l typically at levels of from about 0.001% to about 1% by weight. Various fabric care enzymes are well-known in the laundry detergent art.
Various bleaching compounds, such as the pt:, dl bùr p~lbuldl~s and the like, can be used in such ~;OIlllJ~Jai~iUllS, typically at 25 levelsfromabout1%toabout15%byweight. If desired, suchcu,,,posiliu,,a can also contain bleach activators such as ltl,aa~ .ItSUidlllil~e, r,u,la,,ùyk,A~benzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1% to about 10% by weight.
Various soil release agents, especially of the anionic oligoester type, 30 various chelating agents, especially the d"lil,ù,uh~uhull ' and ethyk,.,e~id",i"~d;succinates, various clay soil removal agents, especially ~tIIUAYII~ tetraethylene pentamine, various dispersing agents, especidlly polyacrylates and pOI~dSudl ' , various bliylll~ la, especially anionic bliyll'~ , various suds suppressors, especially silicones and secondary 35 alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such .~,".~o~ ,"~ at levels ranging from about 1% to about 35% by weight. Standard formularies and published patents contain multiple, detailed des~ iù,)s of such conventional materials.
SUBSTITUTE SHEET (RULE 26~
_, _ _ .. .. .. ...... .. _ _ . .. .. . .. _ .. . _ .. . .

W0 95/29979 2 1 8 9 ~ 2 8 ~ gl ~

Enzyme stabilizers may also be used in the cleaning culllpOailiulls of the present invention. Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0.1%
5 to about 1%).
a. Granular fabric cleanina l,ullluoaiLiol~s The granular fabric cleaning cu",,u~silions of the present invention contain an effective amount of one or more enzyme Yariants of the present invention, preferably from about 0.001% to about 10%, more preferably from 10 about 0.005% to about 5%, more preferably from about 0.01% to about 1%
by weight of active enyme of the ~,u~,uoSi~iul~. In addition to one or more enzyme Yariants, the granular fabric cleaning c~ p~ailiul la typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent.
The granular fabric cleaning c~lll,uOailiol~ e",L,o-Ji",~l ,l of the present invention is illustrated by the following examples.
Examples 7-1 0 Granular Fabric Cleaninq C~l"~osi~iu~
Example No.
20Comoonent 7 8 9 10 Ala216Glu 0.10 0.20 0.03 0.05 Gln206Glu + Tyr217Leu - - 0.02 û.05 C13 linear alkyl benzene sulfonate 22.00 22.00 22.00 22.00 rlloa~ . (as sodium 23.00 23.00 23.00 23.00 25tripol~,ul IOa,u~ Idlt:a) Sodium carbonate 23.00 23.00 23.00 23.00 Sodium silicate 14.00 14.00 14.00 14.00 Zeolite 8.20 8.2û 8.20 8.20 Chelant (diethyld~"~,id",i"a- 0.40 0.40 0.40 0.40 30p~"~dac~ , acid) Sodium sulfate 5.50 5.50 5.50 5.50 Water balance to 100%
In Examples 7-8, the BPN' variants recited in Table 2, among others, are substituted for Ala216Glu, with substantially similar results.
In Examples 9-10, any ,Ulllbilld~;UII of the BPN' variants recited in Table 2, among others, are 5llhCti' l'~d for Ala216Glu and Gln206Glu +
Tyr217Leu, with substantially similar results.
SUBSTITUTE SHEET (RULE 26 ~189~2~
W095/29979 F~~ .'O~

Examples 11-14 Granular Fabric Cleanina Cc"":~05i~iu,~
Example No.
ComDonent 1 1 12 13 14 Gln206Glu + Ala216Glu + Tyr217Leu 0.10 0.20 0.03 0.05 Pro210Ala + Gly215Thr . - - 0.02 0.05 C12 alkyl benzene sulfonate 12.00 12.00 12.00 12.00 ZeoliteA(1-10,,,;~.,u,,,l'u,) 26.00 26.00 26.00 26.00 2butyl octanoic acid 4.00 4.00 4.00 4.00 10C12-C14 secondary (2,3) alkyl sulfate, 5.00 5.00 5.û0 5.00 Na salt Sodium citrate 5.00 5.00 5.00 5.00 Optical brightener 0.10 0.10 0.10 0.10 Sodium sulfate 17.00 17.00 17.00 17.00 Waterand minors balanceto 100%
In Examples 11-12, the BPN' variants recited in Tsble 2, among others, are s~hst~ ' ' for Gln206Glu + Ala216Glu + Tyr217Leu, with suua~d, 1~; "Jr similar results.
In Examples 13-14, any cullluilldliol~ of the BPN' variants recited in 20 Table 2, among others, are 5"~ " ~tPd for Gln206Glu + Ala216Glu +
Tyr217Leu and Pro210Aia + Gly215Thr, with sui,ald"~ similar results.
Examrles 15 and 16 Granular Fai~ric Cleanina COIlluùsi~;.Jlla Cul 11~01 It~l Ita Examole No.

Linearalkylbenzenesulphonate 11.4 10.70 Tallow alkyl sulphate 1.80 2.40 C14 15 alkyl sulphste 3.00 3.10 C14 1salcohol7timesethoxylated 4.00 4.00 Tallow alcohol 11 times ~l lu,.~ ,d 1.80 1.80 Dispersant 0.07 0.1 - Siiicone fluid 0.80 0.80 Trisodium citrate 14.00 15.00 - Citric acid 3.00 2.50 Zeolite 32.50 32.10 Maleic acid acrylic acid copolymer 5.00 5.00 Diethylene triamine penta methylene 1.00 0.20 pl lua,ul 10l li~. acid SUBSTITUTE SHEET (RULE 26) WO 9~i/29979 ~ ~ 8 9 4 ~ 8 PCIIUS95/0~.691 Ala216Glu +Tyr217Leu 0.30 0.30 Lipase 0.36 0.40 Amylase 0.30 0.30 Sodium silicate 2.00 2 50 Sodium sulphate 3.50 5.20 Polyvinyl pyrrolidone 0.30 0.50 Perborate 0.5 Phenol sulphonate 0.1 0.2 Peroxidase 0.1 0.1 Minors Up to 100 Upto 100 ExamPles 17 and 18 Granular Fabric Cleaninq Cu" lu~ ,iliol l~
ExamPle No.
Co, I IIJUI)~ 17 18 Sodium linear C12 alkyl benzene-sulfonate 6.5 8.0 Sodium sulfate 15.0 18.0 Zeolite A 26.0 22.0 Sodium "il, ilu~, i ' ' 5.0 5.0 Polyvinyl pyrrolidone 0.5 0-7 Te~,dac~ ,"lcne diamine 3.0 3.0 Boric acid 4.0 Perborate 0.5 Phenol sulphonate 0.1 0.2 ile205Leu + Ala216Glu 0.4 0.4 Fillers (e.g., silicates; Cdl UUI Idle:a, perfumes; Up to 100 Up to 100 water) Ex2mple 19 ComPact Granular Fabric Cleanina Cu",uo~iliu"
Cu,I,uu"~ ts Weiaht %
Alkyl Sulphate 8.0 Alkyl Ethoxy Suiphate 2.0 Mixture of C2s and C4s alcohol 3 and 7 times ethoxylated 6.0 ru~y~Jdl u~y fatty acid amide 2.5 Zeolite 1 7.0 Layered ' ' '_iL,.,~, 16.0 Carbonate 7.0 Maleic acid acrylic acid copolymer 5.0 Soil release polymer 0 4 SUBSTITUTE SHEET (RULE 26) -~ WO 95/29979 21 8 9 ~ ~ 8 ` r~ lC ~J9l Carboxymethyl cellulose 0.4 Poly (4-vinylpyridine) -N-oxide 0.1 Copolymer of ~i, ,yli,,,i~d~ule and Yinylpyrrolidone 0.1 PEG2000 0.2 Val203Glu + Gln206Glu + Ala216Glu + Tyr217Leu 0.5 Lipase 0.2 Cellulase 0.2 T~t,dut:~yl~ ylene diamine 6.0 P~l ~dl IJUI Idl~ : 22.0 Ethylene diamine disuccinic acid 0.3 Suds suppressor 3.5 Disodium-4,4'-bis (2-,,,u, ,ul 1~' ,o -4-anilino-s-triazin-6- 0.25 ylamino) stilbene-2,2'-disulphonate Disodium-4,4'-bis (2-sulfostyril) biphenyl 0.05 Water, Perfume and Minors Up to 100 ExamPle 20 Granular Fabric Cleanina C~,,,u~siLiu,, Com~onent Weiaht %
Linear alkyl benzene suiphonate 7.6 C16-C18 alkyl sulfate 1.3 C14 15 alcohol 7 times c;:lùxyldl~d 4.0 Coco-alkyl-dimethyl hydroxyethyl ammonium chloride 1.4 Dispersant 0 07 Silicone fluid 0.8 Trisodium citrate 5.0 Zeolite 4A 15.0 Maleic acid acrylic acid copolymer 4.0 Diethylene triamine penta methylene ph~,ulloni~ acid 0.4 Perborate 1 5.0 T~, ddc~'yl~,l. ,ylene diamine 5.0 Smectite clay 10.0 Poly (oxy ethylene) (MW 300,000) 0.3 Tyr214Phe + Tyr217Asn 0.4 Lipase 0.2 Amylase 0 3 Cellulase 0.2 Sodium silicate 3.0 Sodiurri carbonate 10.0 SU~STITUTE SHEET (RULE 26~

WO95/29979 2189428 ",, 5"
~22 Carboxymethyl cellulose 0.2 Bl iu~ 0.2 Water, perfume and minors Up to 100 ExamPle 21 Granular Fabric Cleanina Cclllvo~i~iu,, Component Weiaht %
Linear alkyl benzene sulfonate 6.92 Tallow alkyl sulfate 2.05 C14-1s alcohol 7 times ethoxylated 4.4 C12 1s alkyl ethoxy sulfate - 3 times ethoxylated 0.16 Zeolite 20.2 Citrate 5 5 Carbonate 1 5.4 Silicate 3.0 Maleic acid acrylic acid copolymer 4.0 Carboxymethyl cellulase 0.31 Soil release polymer 0.30 Val203Glu + Pro21 OAla + Gly21 5Thr + Ala216Glu + 0.2 Tyr21 7Leu Lipase 0.36 Cellulase 0. 13 Perborate tetrahydrate 11.64 Perborate monohydratQ 8.7 T~l, daCt:lyl~,:l ,ylene diamine 5.0 Diethylene tramine penta methyl ,ul IOa,u~)ul~iC acid 0.38 Magnesium sulfate 0.40 B~ a,~, 0.19 Perfume, silicone, suds suppressors 0.85 Minors Up to 100 b. Liauid fabric cleanina c~" ~vu:~iliul la Liquid fabric cleaning cu" ,,uu~iliul ,s of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1%, by weight of active enzyme of the ~,u~,uusiliul~.
Such liquid fabric cleaning cul"~,u:,iliu":, typically 8d-1iliull.,ll~' comprise an anionic surfactant, a fatty acid, a water-soluble detergency builder and water.
SU~STITUTE SEIEET (RULE 26) ~ W09~/29979 2~ 28 . . F~~ COI~9I

The liquid fabric cleaning composition ~ bo~ of the present invention is illustrated by the following examples.
Examples 22-26 Liauid Fabric Cleanina Cull,uosi~iu,,a Example No.
ComPonent 22 23 24 25 26 Gln206Glu + Ala216Glu +
Tyr217Leu 0.05 0.03 0.30 0.03 0.10 Pro210Ala + Gly215Thr - - - 0.01 0.20 10C12- C14 alkyl sulfate, Na 20.00 20.00 20.00 20.00 20.00 2-butyl octanoic acid 5.00 5.00 5.00 5.00 5.00 Sodium citrate 1.00 1.00 1.00 1.00 1.00 C10 alcohol ethoxylate (3) 13.00 13.00 13.00 13.00 13.00 ~dll ,d"old",i"e 2.50 2.50 2.50 2.50 2.50 15 Wd~ uv~rlenealvcol/cll,d"ol(100:1:1) balanceto100%
In Examples 22-24 the BPN' variants recited in Table 2, among others, are s~ Pd for Gln206Glu + Ala216Glu + Tyr217Leu, with suLald, ~ lly similar results.
In Examples 25-26, any CulllLlilldLiUl~ of the BPN' variants recited in 20 Table 2, among others, are s~ for Gln206Glu + Ala216Glu +
Tyr217Leu and Pro21 OAla + Gly21 5Thr, with suLa~d, lli~:'y similar results.
ExamPles 27-28 Liauid Fabric Cleaninq Cullluua;~iul,a Example No.
Comoonent 27 28 C12 14 alkenyl succinic acid 3.0 8.0 Citricacidmonohydrate 10.0 15.0 Sodium C12 15 alkyl sulphate 8.0 8.0 30Sodium sulfate of C12-1 s alcohol 2 times ~tl lu,~yl~tcd - 3.0 C12 15 alcohol 7 times ~;:,oxy~ J - 8.0 C12 15 alcohol 5 times ethoxylated 8.0 Diethylene triamine penta (methylene ~I~u ,,ul)ul ,i~, acid) 0.2 Oleic acid 1.8 35Ethanol 4.0 4.0 Pl U,Udl l~ iUI 2.0 2.0 Ala216Glu +Tyr217Leu 0.2 0.2 Polyvinyl,u,y,"' ' ~e 1.0 2.0 SUBSTITUTE SHEET (RULE 26~

W0 95/29979 2 ~ 8 9 4 2 8 !~ r~ 91 ~

Suds suppressor 0.15 0.15 NaOH up to pH 7.5 Perborate 0.5 Phenol sulphonate 0.1 0.2 F~ idase 0.4 0.1 Waters and minors up to 100 parts In each of Examples 27 and 28 herein, the BPN' variants recited in Table 2, among others, are s~lhstitl~t~d for Ala216Glu +Tyr217Leu, with substantially similar results.
ExamDles 29-31 Liquid Fabric Cleaninq C~ JOai~iu~ la Example No.
ComPonent 29 30 31 CitricAcid 7.10 3.00 3.00 Fatty Acid - 2.00 2.00 Ethanol 1.93 3.20 3.20 Boric Acid 2.22 3.50 3.50 Mu,,o~;:,d,,olc,,,,i,,e 0.71 1.09 1.09 1,2 F~u~d~diul 7.89 8.00 8.00 NaCumene Sulfonate 1.80 3.00 3.00 Narv""~ 0.08 0.08 0.08 NaOH 6.70 3.80 3.80 Silicon anti-foam agent 1.16 1.18 1.18 Ala216Glu 0.0145 Ala216Glu + Tyr217Leu - 0.0145 Gln206Glu + Ala216Glu + Tyr21 7Leu - - 0.0145 Lipase .200 .200 .2û0 Cellulase - 750 7.50 Soil release polymer 0.29 0.15 0.15 Anti-foaming agents 0.06 0.085 0.085 Brightener 36 0.095 Brightener 3 - 0.05 0.05 C12 alkyl benzenesulfonic acid 9.86 C12 15 alkyl polyethoxylate (2.5) sulfate 13.80 18.00 18.00 C12 glucose amide - 5.00 5.00 C12 13 alkyl polyethoxylate (9) 2.00 2.00 2.00 Water, perfume and minors balance to 100%
SUBSTITUTE SHEET (RULE 26!

WO 95/29979 2 1 8 g ~ 2 8 i ~

c. Bar fabric cleaninq cu" ~uosi~iol~s Bar fabric cleaning ~,u~ JOai~iullS of the present invention suitable for hand-washing soiled fabrics contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001% to 5 about 10%, more preferably from about 0.01% to about 1% by weight of the Cul I ~,uoSiLi~
The bar fabric cleaning c~,,,,uùsiliu,~ t~",i,u ii"~"~ of the present invention is illustrated by the following examples.
Examples 33-36 Bar Fabric Cleaninq C~ u~ailiulla Example No.
Com~onent 33 34 35 36 Val203Glu 0.3 - 0.1 0.02 Tyr214Phe + Tyr217Asn - 0-3 0.4 0.03 15C12-C16 alkyl sulfate, Na 20.0 20.0 20.0 20.00 C12-C14 N-methyl glucamide 5.0 5.0 5.0 5.00 C11-C13alkylben~enesulfonate,Na 10.0 10.0 10.0 10.00 Sodium carbonate 25.0 25.0 25.0 25.00 Sodium PYIU,VIIU~ . 7.0 7.0 7.û 7.ûO
20Sodium tripolyj,llo "ulIdt~ 7.0 7.0 7.0 7.00 ZeoliteA(0.1-.1011) 5.0 5.0 5.0 5.00 Cdl uuAy~ Icellulose 0.2 0.2 0.2 0.20 Polyacrylate (MW 1400) 0.2 0.2 0.2 0.20 Coconut ll~ull~lldl~oldllli~ie 5.0 5.0 5.0 5.0û
25Brightener, perfume 0.2 0.2 0.2 0.20 CaS04 1.0 1.0 1.0 1.ûO
MsSO4 1.0 1.0 1.0 1.00 Water 4.0 4.0 4.0 4.00 Filler~ balance to 100%
30 ~Can be selected from convenient materials such as CaCO3, talc, clay, siiicates, and the like.
In Example 33, the BPN' variants recited in Table 2, among others, are sl Ihctitl It~d for Val203Glu, with suiJaldr ,li :'y similar results.
In i_xample 34, the BPN' variants recited in Table 2, among others, 35 are s~h~tit~lt~d for Tyr214Phe + Tyr217Asn, with sui ald"" 'Iy similar results.
SUBSTlTUTi- SHEET (RULE 26 8g ~'~8 WO 95/29!~79 2 1 . .,u~ ~ c I'91 In Examples 3~-36, any culllbilldLiull of the BPN' variants recited in Table 2, among others, are .sllhctitlltpd for Val2û3Glu and Tyr214Phe +
Tyr217Asn, with substantially similar results.
While particular ~:IllLJodil,,~ a of the subject invention have been 5 disclosed, it will be obvious to those skilled in the art that various changesand ll,oJiti~,dliu"~ of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such ",u~iti.,alio"s that are within the scope of the invention.
1û

SUBSTITUTE SHEET ~RULE 26) j ~ ~
2189~28 WO 95129979 P~ 91 SEQUENCE LISTING
5 (1) GENERAL INFORMATION:
(i) APPLICANT: BRODE, PHILIP F
BARNETT, BOBBY L.
RUBINGH, DONN N.
0 GHOSH, CHANCh~AL K.
(il) TITLE OF INVENTION: SERINE PROTEASE cmN~ATN~Nr (Lii) NUMBER OF SEQUENCES: 1 (iV) uunnri~Ur~J~ r; ADDRESS:
A ADDRESSEE: THE PROCTER & GAMBLE COMPANY
B STREET: 11810 EAST MIAMI RIVER ROAD
C CITY ROSS
. D STATE: OH
E COUNTRY: USA
F ZIP: 45061 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC ihl~.
(C) OPERATING SYSTEM: PC DOS/MS-DOS
(D) SOFT~ARE: PatentIn Releaue #1.0, Version #1.25 (vi) CURRENT APPLICATION D
(A) APPLICATION NUMBER
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
( A ) NAME: ROOF, CARL J .
(B) REGISTRATION NUM3ER: 37,708 (c) REFERENCE/DOCXET NO. 5232 ~ix) ~rF~. lNr (A) TELEPNONE: 513-627-008 (B) TELEFAX: 513--627--D260 (2) lNr FOR SEQ ID NO:1:
(i) SEQUENCE ~nDn''l'FR~qTICS:
(A) LENGTH: 275 ~iino ~IcLdu ( B ) TYPE: amino ~cid (D) TOPOLOGY: lLne~r ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE L~ un.Lr.lUN: SEQ ID NO:1:
Al~ Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lyu Ala Pro A1A Leu 1 s lD lS
Eli- Ser Gln Gly Tyr Thr Gly S~r Asn Val Ly Val Al~ V~l Ile As Ser Gly }le A-p Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala SUBSTITUTE SHEET ~RULE 26) W0 95/29979 2 1 8 ~ ~ 2 8 ~ J~ ~28 ~ ''O IC~I ~
6er Met Val Pro Ser Glu Thr A~n Pro Phe Gln Asp Ann A~n Ser Hi~

Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gl Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Ya~ Ly~ Val Leu 0 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Al/l Ile Al~ Asn Asn Met Asp Yal Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala Al~ Leu Lys Ala Ala Val Asp Lys Al~ V~l Al~

Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Al~

V~l Gly Ala Val Asp ser Ser Asn Gln Arg Ala Ser Phe Ser Ser V~l Gly Pro Glu Leu Asp Val Met Ala E~ro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Asn LyY Tyr Gly Ala Tyr Asn Gly Thr Ser Met A1~ Ser Pro }li8 Val Al~ Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro A~n Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu A~n Thr Thr Thr Ly~

Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile A--n V~l Gln A1 Al~ Alil Gln SUBSTITUTE SHEET (RULE 26~

Claims (19)

1. Claims:
   A fabric cleaning composition comprising:
   (a) an effective amount of a BPN' variant comprising wild-type amino acid sequence wherein the wild-type amino acid sequence at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 218, 219 or 220 is substituted, characterized in that i. when a substitution occurs at position 199, the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu, ii. when a substitution occurs at position 200, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   iii. when a substitution occurs at position 201, the substituting amino acid for position 201 is Gly, Gln, Asn, Ser, Asp or Glu;
   iv. when a substitution occurs at position 202, the substituting amino acid for position 202 is Pro, Gln, Asn, Ser, Asp or Glu;
   v. when a substitution occurs at posiion 203, the substituting amino acid for position 203 is Met, Cys, His, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   vi. when a substitution occurs at position 204, the substituting amino acid for position 204 is Glu;
   vii. when a substitution occurs at posiion 205, the substituting amino acid for position 205 is Leu, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   viii. when a substitution occurs at position 206, the substituting amino acid for position 206 is Pro, Asn or Ser;
   ix. when a substitution occurs at posiion 207, the substituting amino acid for posiion 207 is Asp or Glu, x. when a substitution occurs at position 208, the substituting amino acid for position 208 is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   
   xi. when a substitution occurs at position 209, the substituting amino acid for position 209 is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   xii. when a substitution occurs at position 210, the substituting amino acid for position 210 is Gly, Gln, Asn, Ser, Asp or Glu;
   xiii. when a substitution occurs at position 211, the substituting amino acid for position 211 is Ala, Pro, Gln, Asn, Ser, Asp or Glu;
   xiv. when a substitution occurs at position 213, the substituting amino acid for position 212 is Gln, Ser, Asp or Glu;
   xv. when a substitution occurs at position 213, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   xvi. when a substitution occurs at position 214, the substituting amino acid for position 214 is Phe, Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu;
   xvii. when a substitution occurs at position 215, the substituting amino acid for position 215 is Thr, Pro, Gln, Asn, Ser, Asp or Glu;
   xvii when a substitution occurs at position 216, the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   xix. when a substitution occurs at position 218, the substituting amino acid for position 218 is Glu;
   xx. when a substitution occurs at position 219, the substituting amino acid for position 219 is Pro, Gln, Asn, Ser, Asp; or Glu; and xxi. when a substitution occurs at position 220, the substituting amino acid for position 220 is Pro, Gly, Gln, Asn, Asp or Glu;
   
   characterized in that the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'; and (b) one or more cleaning composition materials compatible with the BPN' variant
2. 2. The fabric cleaning composition of Claim 1, characterized in that a. when a substitution occurs at position 206, the substituting amino acid for position 206 is Asn or Ser;
   b. when a substitution occurs at position 211, the substituting amino acid for position 211 is Pro, Gln, Asn, Ser, Asp or Glu;
   c. when a substitution occurs at position 214, the substituting amino acid for position 214 is Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu; and d. when a substitution occurs at position 215, the substituting amino acid for position 215 is Pro, Gln, Asn, Ser, Asp or Glu.
3. 3. The fabric cleaning composition of Claim 2, characterized in that when position 216 is substituted, Gly is substituted for Ala at position 216.
4. 4. The fabric cleaning composition of Claim 2, characterized in that when a substitution occurs at one or more of positions 199, 200, 201, 202, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 219 or 220, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 219 or 220 is Asp or Glu; and when a substitution occurs at one or both of positions 204 or 208, the substituting amino acid for positions 204 or 218 is Glu; and wherein a substitution preferably occurs at one or more of positions of 199, 200, 201, 202, 205, 207, 208, 209, 210, 211, 212 or 215, more preferably atone or more of positions 200, 201, 202, 205 or 207.
5. 5. The fabric cleaning composition of Claim 1 having a single amino acid substitution characterized in that the substitution is:
   a. Glu for Ala at position 216;
   b. Asp for Ala at position 216; or c. Glu for Val at position 203.
6. 6. A fabric cleaning composition comprising:
   
   a) a BPN' variant comprising wild-type amino acid sequence in that the wild-type amino acid sequence at two or more of positions 199, 200. 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211. 212, 213, 214, 215, 216, 217, 218, 219 or 220 is substituted, characterized in that i. when a substitution occurs at position 199, the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   ii. when a substitution occurs at position 200, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu, iii. when a substitution occurs at position 201, the substituting amino acid for position 201 is Gly, Gln, Asn, Ser, Asp or Glu;
   iv. when a substitution occurs at position 202, the substituting amino acid for position 202 is Pro, Gln, Asn, Ser, Asp or Glu;
   v. when a substitution occurs at position 203, the substituting amino acid for position 203 is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   vi. when a substitution occurs at position 204, the substituting amino acid for position 204 is Asp or Glu;
   vii. when a substitution occurs at position 205, the substituting amino acid for position 205 is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   viii. when a substitution occurs at position 206, the substituting amino acid for position 206 is Pro, Asn, Ser, Asp, or Glu;
   ix. when a substitution occurs at position 207, the substituting amino acid for position 207 is Asp or Glu;
   x. when a substitution occurs at position 208, the substituting amino acid for position 208 is Pro, Gly, Gln, Asn or Ser;
   xi. when a substitution occurs at position 209, the substituting amino acid for position 209 is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   xii. when a substitution occurs at position 210, the substituting amino acid for position 210 is Ala, Gly, Gln, Asn, Ser, Asp or Glu;
   xiii. when a substitution occurs at position 211, the substituting amino acid for position 211 is Ala, Pro, Gln, Asn, Ser, Asp or Glu;
   xiv. when a substitution occurs at position 212, the substituting amino acid for position 212 is Gln, Ser, Asp or Glu;
   xv. when a substitution occurs at position 213, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   xvi. when a substitution occurs at position 214, the substituting amino acid for position 214 is Phe, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn or Ser;
   xvii. when a substitution occurs at position 215, the substituting amino acid for position 215 is Thr, Pro, Gln, Asn, Ser, Asp or Glu;
   xviii. when a substitution occurs at position 216, the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   xix. when a substitution occurs at position 217, the substituting amino acid for position 217 is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   xx. when a substitution occurs at position 218, the substituting amino acid for position 218 is Gln, Ser, Asp or Glu;
   xxi. when a substitution occurs at position 219, the substituting amino acid for position 219 is Pro, Gln, Asn, Ser, Asp or Glu; and xxii. when a substitution occurs at position 220, the substituting amino acid for position 220 is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
   characterized in that the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'; and (b) one or more cleaning composition materials compatible with the BPN' variant.
7. 7. The fabric cleaning composition of Claim 6, characterized in that the wild-type BPN' is substituted at two positions.
8. 8. The fabric cleaning composition of Claim 7 characterized in that the two substitutions are:
   a. Ala for Pro at position 210 and Thr for Gly at position 215;
   b. Phe for Tyr at position 214 and Asn for Tyr at position 217;
   c. Glu for Ala at position 216 and Leu for Tyr at position 217;
   d. Leu for Ile at position 205 and Glu for Ala at position 216;
   e. Leu for Ile at position 205 and Asp for Ala at position 216;
   f. Glu for Gln at position 206 and Glu for Ala at position 216;
   g. Asp for Ala at position 216 and Leu for Try at position 217;
   or h. Glu for Gln at position 206 and Leu for Try at position 217.
9. 9. The fabric cleaning composition of Claim 7, characterized in that a. when a substitution occurs at position 206, the substituting amino acid for position 206 is Glu, Asn or Ser;
   b. when a substitution occurs at position 210, the substituting amino acid for position 210 is Gly, Gln, Asn, Ser, Asp or Glu;
   c. when a substitution occurs at position 211, the substituting amino acid for position 211 is Pro, Gln, Asn, Ser, Asp or Glu;
   d. when a substitution occurs at position 214, the substituting amino acid for position 214 is Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu; and e. when a substitution occurs at position 215, the substituting amino acid for position 215 is Pro, Gln, Asn, Ser, Asp or Glu.
10. 10. The fabric cleaning composition of Claim 6, characterized in that when a substitution occurs at positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 217, 218, 219 or 220, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 217, 218, 219 or 220 is Asp or Glu; and when a substitution occurs at position 213, the substituting amino acid for position 213 is Asp; and wherein a substitution preferably occurs at two or more of positions 199, 200, 201, 202, 205, 207, 208, 209, 210, 211, 212, or 215, more preferably a substitution occurs at two or more of positions 200, 201, 202, 205 or 207.
11. 11. The fabric cleaning composition of Claim 6, characterized in that Glu or Asp is substituted for Ala at position 216 and Leu is substituted for Tyr at position 217.
12. 12. The fabric cleaning composition of Claim 6, characterized in that the wild-type BPN' is substituted at three positions.
13. 13. The fabric cleaning composition of Claim 12 wherein the three substitutions are:
    a. Pro substituted for Gln at position 206, Ala substituted for Gly at position 211, and Glu substituted for Ala at position 216;
    b. Val substituted for Ile at position 205, Ala substituted for Pro at position 210, and Glu substituted for Lys at position 213; or c. Glu substituted for Gln at position 206, Glu substituted for Ala at position 216, and Leu for Tyr at position 217.
14. 14. The fabric cleaning composition of Claim 12 characterized in that a. when a substitution occurs at position 206, the substituting amino acid for position 206 is Asn or Ser;
    b. when a substitution occurs at position 210, the substituting amino acid for position 210 is Gly, Gln, Asn, Ser, Asp or Glu;
    c. when a substitution occurs at position 211, the substituting amino acid for position 211 is Pro, Gln, Asn, Ser, Asp or Glu;
    d. when a substitution occurs at position 214, the substituting amino acid for position 214 is Leu, Ile, Val, Met, Cys, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu; and e. when a substitution occurs at position 215, the substituting amino acid for position 215 is Pro, Gln, Asn, Ser, Asp or Glu.
15. 15. The fabric cleaning composition of Claim 6, characterized in that the wild-type BPN' is substituted at four positions or five positions.
16. 16. The fabric cleaning composition of Claim 15, wherein the are:
    a. Glu substituted for Val at position 203, Glu substituted for Gln at position 206, Glu substituted for Ala at position 216, and Leu substituted for Tyr at position 217;
    b. Glu substituted for Val at position 203, Ala substituted for Pro at position 210, Glu substituted for Ala at position 216, and Leu substituted for Tyr at position 217;
    c. Glu substituted for Val at position 203, Glu substituted for Gln at position 206, Thr substituted for Gly at position 215, Glu substituted for Ala at position 216, and Leu substituted for Tyr at position 217; or d. Glu substituted for Val at position 203, Ala substituted for Pro at position 210, Thr substituted for Gly at position 215, Glu substituted for Ala at position 216, and Leu substituted for Tyr at position 217.
17. 17. The fabric cleaning composition of any of Claims 1 through 16, characterized in that said composition is in the form of a liquid.
18. 18. The fabric cleaning composition of any of Claims 1 through 16, wherein the composition comprises at least about 5% surfactant and at least about 5% builder, by weight of the
19. 19. A method for cleaning fabric, said method comprising contacting a fabric in need of cleaning with the composition of any of Claims 1 through 18.