US20110251073A1 - Compositions And Methods Comprising Variant Microbial Proteases - Google Patents
Compositions And Methods Comprising Variant Microbial Proteases Download PDFInfo
- Publication number
- US20110251073A1 US20110251073A1 US12/996,538 US99653809A US2011251073A1 US 20110251073 A1 US20110251073 A1 US 20110251073A1 US 99653809 A US99653809 A US 99653809A US 2011251073 A1 US2011251073 A1 US 2011251073A1
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- Prior art keywords
- subtilisin
- protease
- variant
- detergent
- property
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38681—Chemically modified or immobilised enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
- C11D2111/10—Objects to be cleaned
- C11D2111/12—Soft surfaces, e.g. textile
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
Definitions
- the present invention provides methods for protein engineering and variant proteases. Specifically, the invention provides methods utilizing site evaluation libraries. The present invention also provides subtilisin variants suitable for various uses.
- Serine proteases are a subgroup of carbonyl hydrolases comprising a diverse class of enzymes having a wide range of specificities and biological functions.
- Much research has been conducted on the subtilisins, due largely to their usefulness in cleaning and feed applications. Additional work has been focused on the adverse environmental conditions (e.g., exposure to oxidative agents, chelating agents, extremes of temperature and/or pH) which can adversely impact the functionality of these enzymes in various applications. Nonetheless, there remains a need in the art for enzyme systems that are able to resist these adverse conditions and retain or have improved activity over those currently known in the art.
- proteins are modified in order to obtain desired protein properties.
- the nucleotide sequence of a cloned gene encoding a protein is mutated and the modified gene is expressed to produce mutants, which are screened for activities of interest. Often, the mutant properties are compared with the properties of wild-type protein.
- NP-hard problems are problems for which efficient polynomial-time algorithms are not currently known, and if any NP-hard problem could be solved, all NP-hard problems could be solved (See e.g., Pierce and Winfree, Protein Engineer., 15:779-782 [2002]).
- Current strategies for building and screening libraries generally involve generating protein sequence diversity randomly across the whole sequence or in controlled random fashion at defined positions within the protein.
- Saturation mutagenesis (Estell et al., in World Biotech Report 1984, vol. 2: USA, Online Publications, London [1984], pages 181-187; and Wells et al., Gene 34:315-323 [1985]) is one technique that can be used to search protein space for mutations that optimize several properties in a protein.
- Several groups have developed strategies for identifying sites to be changed by saturation mutagenesis (Reetz et al., Agnew. Chem. Int. Edn., 44:4192-4196 [2005]; Kato et al., J. Mol. Biol., 351:683-692 [2005]; and Sandberg et al., Proc. Natl. Acad. Sci., 90:8367-8371 [1993]), but no general system for site identification has been proposed.
- the present invention provides methods for protein engineering. Specifically, the invention provides methods utilizing site evaluation libraries. In particular, the present invention provides means to use information obtained about a number of desired properties, in order to rationally and efficiently design libraries that will optimize those properties. In some embodiments, the present invention provides means to design libraries that are improved for at least two desired properties.
- the present invention provides means to identify positions within an amino acid sequence of a protein that are relevant in improving desired properties of the protein.
- the present invention provides means to determine which mutations are desirable in order to produce proteins with these desired properties, as well as improved properties.
- the present invention provides means to identify amino acid positions and mutations that have improvements of a particular percentage better than the wild-type protein (e.g., better than 110% of the wild-type for one property).
- the present invention provides means to identify mutations that provide at least one much improved property and at least one additional property that is not significantly worse than the wild-type protein (e.g., better than 110% of wild-type for one property, yet not worse than 90% of wild-type for another property).
- libraries are constructed based on this information.
- the libraries are constructed using all of the identified mutations, while in some other embodiments, the libraries are constructed using a subset of the identified mutations. Indeed, it is not intended that the libraries be constrained to any particular number and/or type of mutations.
- the present invention provides methods for protein engineering comprising the steps of: providing a library of protein variants; testing the library of protein variants for at least one property of interest in a test of interest; identifying a range of values for said the at least one property of interest; identifying a minimum within the range of values that is associated with a favorable outcome in the test of interest; and providing a plurality of protein variants having at least one mutation above said minimum in the range of the at least one property of interest, thereby providing a library of protein variants comprising at least one mutation, and wherein the library is enriched in members having a favorable outcome in the test of interest.
- the favorable outcome corresponds to a value of greater than about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of a maximal value observed in the test set forth in the first step above.
- more than one test of interest is used in the methods of the present invention.
- the protein is an enzyme.
- the enzyme is selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.
- the present invention also provides methods for protein engineering comprising the steps of: providing a library of protein variants; testing the library of protein variants for at least two properties of interest in a test of interest; identifying a range of values for the at least two properties of interest; identifying a minimum within the range of values that is associated with a favorable outcome in the test of interest; and providing a plurality of protein variants above the minimum of the range of the at least two properties of interest, thereby providing a library of protein variants enriched in members having the favorable outcome in the test of interest.
- the favorable outcome corresponds to a value of greater than about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of a maximal value observed in the test set forth in the first step above.
- the protein is an enzyme.
- the enzyme is selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.
- the present invention also provides methods for protein engineering comprising the steps of: providing a wild-type protein and a library of protein variants of the wild-type protein; testing the library of protein variants and the wild-type protein for at least one property of interest in a test of interest; identifying a range of values for the at least one property of interest; identifying a minimum within the range of values that is associated with a favorable outcome in the test of interest; identifying the protein variants having a favorable outcome as compared to the results obtained for the wild-type, wherein the favorable outcome is an improved property of interest; and providing a plurality of protein variants above the minimum of the range of the at least one property of interest, thereby providing a library of improved protein variants enriched in members having the favorable outcome in the test of interest.
- the methods further comprise the step of determining the performance index, wherein the performance index is determined by dividing the value obtained for each of the improved protein variants and the value obtained for the wild-type protein. In some particularly preferred embodiments, the methods further comprise the step of identifying the improved protein variants, wherein the improved protein variants achieve performance index values greater than 1.1 in the test of interest.
- the protein is an enzyme. In some particularly preferred embodiments, the enzyme is selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases. In some alternative embodiments, the protein is selected from antibodies and growth factors.
- the wild-type protein is a mature form an enzyme selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.
- the property of interest is selected from charge, wash performance, hard surface cleaning performance, solubility, thermal stability, storage stability, detergent stability, substrate binding, enzyme inhibition, expression level, reaction rate, and substrate degradation.
- the wild-type protein and the protein variant are components of at least one detergent composition.
- wash performance is tested in a detergent composition formulated into a powdered or liquid detergent having a pH of between 5 and 12.0.
- the present invention also provides methods for producing an improved variant of a parent protein within a protein fold, comprising: assaying multiple variants of a test protein within the protein fold spanning a range of a property of interest in an assay of interest; identifying a minimum within the range of the property of interest that is associated with a favorable outcome in the assay of interest; assaying a parent protein of the protein fold in the assay of interest; and producing an improved variant of the parent protein by introducing an amino acid substitution is the parent protein such that the improved variant is above the minimum of the range of the property of interest.
- the parent protein and the test protein are different.
- the methods further comprise the step of determining the performance index, wherein the performance index is determined by dividing the value obtained for the improved protein variant and the value obtained for the parent protein.
- the test proteins and the parent proteins are enzymes.
- the enzymes are selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.
- the test and parent proteins are selected from antibodies and growth factors.
- the parent protein is a mature form an enzyme selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.
- the property of interest is selected from charge, wash performance, hard surface cleaning performance, thermal stability, solubility, storage stability, detergent stability, substrate binding, enzyme inhibition, expression level, reaction rate, and substrate degradation.
- the test and parent proteins are components of at least one detergent composition.
- the improved protein variant is a component of a detergent composition.
- wash performance is tested in a detergent composition formulated into a powdered or liquid detergent having a pH of between about 5 and about 12.0.
- the present invention also provides isolated subtilisin variants of a Bacillus subtilisin, wherein the subtilisin variant is a mature form having proteolytic activity and comprising a substitution at one or more positions selected from positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 65, 66, 67, 71, 72, 73, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112,
- amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:2.
- the one or more positions are selected from: 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 65, 66, 67, 71, 72, 73, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 122,
- amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:2.
- the one or more positions are selected from: 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 21, 22, 23, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 44, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 62, 65, 66, 67, 71, 73, 77, 78, 79, 81, 82, 83, 84, 85, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 102, 105, 106, 110, 111, 112, 113, 114, 115, 116, 122, 123, 124, 125, 126, 128, 129, 131, 133, 134, 135, 136, 137, 139, 141,
- subtilisin variant set forth as SEQ ID NO:2.
- the subtilisin variant further comprises a substitution at one or more positions selected from 18, 52, 72, 117, 119, 127, 144, 185, 209 and 213, and wherein the positions are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN′ set forth as SEQ ID NO:2.
- the substitution comprises one or more of the positions: A001C, A001D, A001E, A001F, A001G, A001H, A001I, A001K, A001L, A001M, A001N, A001Q, A001R, A001S, A001V, A001W, Q002A, Q002D, Q002E, Q002G, Q002S, S003A, S003D, S003F, S003G, S003I, S003K, S003L, S003M, S003N, S003P, S003Q, S003R, S003T, S003V, S003W, S003Y, P005C, P005E, P005G, P005N, P005Q, P005T, Y006A, Y006C, Y006E, Y006F, Y006G, Y006K, Y006K, Y00
- the substitution comprises a combination selected from: Y021H-A045V-Y217E, Y021W-S101E-G128R-Y217Q, Y021H-Y217E, Y021H-A045V-S101N-Y217Q, Y021H-A045I-Y217E, Y021H-A045I-S101E-Y217Q, Y021W-A045I-S101E-Y217E, D036N-S101E-Y217L, Y021H-A045V-Y217Q, Y021H-A045I-S101E-Y217L, Y021H-A045I-Y217E, Y021H-Y217Q, Y021W-S101E-Y217L, Y021W-A045V-S101E-Y217L, Y021H-Y217Q
- the substitution comprises a combination selected from: S024S-V028V-M050M-A092A-Q103E-A114G-V246V, S024S-V028V-M050V-A092A-Q103Q-A114A-V246V, S024S-V028V-M050V-A092A-Q103Q-A114G-V246V, S024H-V028V-M050V-A092A-Q103Q-A114A-V246T, S024H-V028V-M050V-A092A-Q103E-A114A-V246V, S024H-V028V-M050V-A092A-Q103Q-A114G-V246V, S024H-V028V-M050V-A092A-Q103Q-A114G-V246V, S024H-V028V-M050V
- the substitution comprises a combination selected from: S024S-V028T-M050V-A092G-Q103E-A114G-V246T, S101N-M119H, M119N-K213N-Y217L, Y217L, M119N-K213N, K213N, K213N-Y217Q, S101E-K213N-Y217E, S101E-M119N-Y217L, Y217Q, S101N-M119N-K213N-Y217E, S101N-M119N-K213N-Y217L, Y021H-A045I-S101E-Y217L, S101E-Y217L, A045I-Y217Q, S101S-M119M-K213K-Y217L, Y021H-A045I-S101E-Y217L, Y045I-Y217Q, S101S
- the present invention further provides the variants set forth in the Tables included in the Examples, as well as compositions comprising these variants.
- the present invention provides isolated nucleic acids encoding the subtilisin variant, expression vectors comprising the nucleic acids, and host cells comprising the expression vector.
- the present invention provides cleaning compositions comprising the subtilisin variant.
- the cleaning composition is a laundry detergent.
- the laundry detergent is a cold water detergent, a low pH detergent, or a compact detergent.
- the present invention provides methods for producing a subtilisin variant of a Bacillus subtilisin, comprising: transforming a host cell with an expression vector comprising a nucleic acid encoding the subtilisin variant; and cultivating the transformed host cell under conditions suitable for the production of the subtilisin variant.
- the methods further comprise the step of harvesting the produced subtilisin variant.
- the host cell is a Bacillus species, and in a subset of these embodiments, the Bacillus species is B. subtilis .
- the present invention provides a method of cleaning, comprising the step of contacting a surface and/or an article comprising a fabric with a cleaning composition comprising an isolated subtilisin variant.
- the present invention provides methods for protease engineering comprising the steps of: a) providing a plurality of site evaluation libraries (SELs) each comprising a plurality of protease variants having distinct substitutions at an identical amino acid position of the protease; b) testing the protease variants of the SELs and a standard protease in a test of a property of interest; c) determining a performance index (PI) for each of the protease variants for the test; d) identifying two or more of the amino acid positions as non-restrictive positions, wherein at least one of the plurality of protease variants in each of two of the SELs has a PI greater than 0.5; and f) providing a multiple mutation library comprising a plurality of multiply-substituted protease variants each comprising substitutions in the two or more non-restrictive positions.
- SELs site evaluation libraries
- PI performance index
- the test comprises two or more different assays selected from the group consisting of stain removal assays (microswatch), LAS stability assays, detergent stability assays, and specific activity assays.
- the protease is selected from the group consisting of a bacterial serine protease, a bacterial subtilisin, and a bacterial neutral metalloprotease.
- the present invention provides methods for producing a multiply substituted subtilisin variant of a Bacillus subtilisin, comprising: testing a plurality of singly-substituted subtilisin variants in a first test of a first property and a second test of a second property, wherein the property of a parent subtilisin is given a value of 1.0 in each test, a favorable first or second property has a value greater than 1.0, and an unduly unfavorable first or second property has a value less than about 0.80 or in some preferred embodiments, less than about 0.60; identifying a substitution in at least one of the singly-substituted subtilisin variants that is associated with a favorable first property and which is not associated with an unduly unfavorable second property; identifying a substitution in at least one of the singly-substituted subtilisin variants that is associated with a favorable second property and which is not associated with an unduly unfavorable first property; and introducing the
- the methods further comprise testing the multiply-substituted subtilisin variant in the first test and the second test, wherein an improved subtilisin variant achieves a value of greater than 1.0 in both of the first and second tests, or a value of greater than 1.0 in the first test and a value of 0.80 to 1.0 in the second test.
- the methods further comprise producing the improved subtilisin variant(s).
- the first and second properties are negatively correlated.
- a favorable first or second property has a value greater than about 1.2.
- an unduly unfavorable first or second property has a value less than about 0.40.
- the first property is stability, and the second property is wash performance.
- the stability comprises stability in detergent and wash performance comprises blood milk ink (BMI) wash performance in detergent.
- the parent bacterial subtilisin is a wild type mature form of a B. amyloliquefaciens subtilisin BPN′ having an amino acid sequence set forth as SEQ ID NO:2.
- the parent bacterial subtilisin is a wild type of a B. lentus GG36 subtilisin having an amino acid sequence set forth as SEQ ID NO:562, or a BPN′′ comprising a Y217L substitution (SEQ ID NO:565), alone or in combination with other modifications.
- wash performance is tested in a powder or liquid detergent composition having a pH of between 5 and 12.0. It is not intended that the steps be limited to the exact order listed above, as any suitable order finds use in the present invention. However in some preferred embodiments, the substitutions are in positions in a parent subtilisin having a solvent accessible surface (SAS) of greater than about 50% or greater than about 65%.
- SAS solvent accessible surface
- FIG. 1A provides a map of pHPLT-VAAc1
- FIG. 1B provides a map of pHPLT-BPN′.
- FIG. 2A depicts BMI cleaning performance of a BPN′-Y217L CCL in North American laundry detergent as a function of charge change.
- FIG. 2B depicts BMI cleaning performance of a GG36 CCL in North American laundry detergent as a function of charge change.
- FIG. 3A depicts BMI cleaning performance of a BPN′-Y217L CCL in Western European liquid laundry detergent as a function of charge change.
- FIG. 3B depicts BMI cleaning performance of a GG36 CCL in Western European liquid laundry detergent as a function of charge change.
- FIG. 4A depicts BMI cleaning performance of a BPN′-Y217L CCL in Japanese powdered laundry detergent as a function of charge change.
- FIG. 4B depicts BMI cleaning performance of a GG36 CCL in Japanese powdered laundry detergent as a function of charge change.
- FIG. 5A depicts baked egg yolk cleaning performance of a BPN′-Y217L CCL in automatic dish washing detergent as a function of charge change.
- FIG. 5B depicts baked egg yolk cleaning performance of a GG36 CCL in automatic dish washing detergent as a function of charge change.
- FIG. 6 depicts LAS/EDTA stability as a function of net charge change relative to parent BPN′-Y217L, for a library containing 80 variants.
- the present invention provides methods for protein engineering. Specifically, the invention provides methods utilizing site evaluation libraries.
- the problem to be solved is to identify at least one protein sequence that meets or exceeds the minimum value required for a number of properties. This requires knowledge of mutations that are good for a particular property, as well as knowledge of those mutations that are bad for any of the desired properties.
- the present invention provides means to meet the goal by identifying those positions in the protein that can be altered to improve the primary property and keep the values for other properties within desired limits.
- the present invention provides means to evaluate all positions in a protein for all the properties of interest by building “site evaluation libraries” at each site.
- these libraries contain 9-19 mutations at each position, and are used to evaluate each position for use in engineering the protein and constructing libraries.
- Each property is measured relative to the parent enzyme and an apparent free energy difference for each mutant vs. wild type is calculated.
- delta delta G (“i.e., ⁇ G”) apparent values are then used to determine additivity.
- the Gibbs Free Energy for a process represents the maximum amount of work that can be performed by a system.
- the change in Free energy relative to the parent enzyme ( ⁇ G) is given as follows;
- ⁇ G ⁇ RT ln ( k variant /k parent )
- ⁇ G app ⁇ RT ln ( P variant /P parent )
- P variant is the performance value for the variant and P parent is the performance value for the parent enzyme under the same conditions.
- the ⁇ G app values may be expected to behave in a similar fashion as to ⁇ G for data distributions and additivity. However, since ⁇ G is the maximum amount of work that can be carried out by the variant compared to the parent enzyme, the quantity ⁇ G app will generally underestimate the ⁇ G and lead to results that appear synergistic in that the properties of two additive positions may be greater than the value predicted by adding their ⁇ G app values together.
- the methods of the present invention used to design efficient libraries that were used to engineer multiple properties in parallel. Although certain enzymes are described herein, the methods apply to any protein of interest for engineering.
- Site evaluation libraries SELs
- SELs Site evaluation libraries
- the methods were built as described herein by introducing from 12 to 19 substitutions at each of the positions. The resulting mutations were analyzed using activity and stability assays. The wild type amino acid is listed as a reference point for every position.
- measurements were used to determine the mean and standard deviation of ⁇ G app for the parent enzyme.
- the parent mean ( ⁇ parent ) was normalized to 0, and the standard deviation ( ⁇ parent ) for ⁇ G app was determined
- the site evaluation data were tested for evidence of correlation between properties.
- the ⁇ G app values for each property were plotted versus each other property and correlation coefficients were calculated. The two activity measurements on protein substrates were correlated.
- deleterious mutations for any property are correlated with deleterious mutations for every other property, regardless of correlations of the properties. Only a small number of positions (5-10%) have mutations that are bad for all properties. These positions define the “fold” and are conserved in evolution. The implication of this is that although identification of beneficial mutations for any property requires a truly predictive screen for that property, identification of mutations likely to be deleterious for any property can be accomplished using ANY screen.
- a simplified protein engineering strategy is to build SELs and screen using a simple activity and/or stability screen. The deleterious mutations are identified and those positions that have few deleterious mutations are used to build libraries and combinatorial mutations to improve multiple properties.
- picking sites that are on the surface of the protein, have few interactions and are variable in sequence alignments provides a high proportion of productive sites.
- Sites that are on the interior of the molecule, have many contacts and are strongly conserved in evolution will have a high probability of having deleterious mutations and should be avoided. It is contemplated that any suitable method for analyzing sequence and/or structural information will find use in the present invention, including but not limited to computer and/or electronic methods and/or programs.
- the methods provide pairwise comparisons of the numbers of variants with more than 5% wt activity and less than 5% activity for each of two properties, along with correlation coefficients for the two properties.
- the site evaluation library data are used for combinatorial library design.
- Traditional directed evolution builds random libraries and screens large numbers of library for single properties, combines these and repeats the process.
- Bloom et al. Curr. Opin. Struct. Biol., 15:447-452 [2005]
- Bloom et al. Proc. Natl. Acad. Sci. USA 103:5869-5874 [2006]
- Guo et al. Proc. Natl. Acad. Sci. USA 101:9205-9210 [2004]
- the accumulation of positive mutations for one property commonly leads to decreases in other properties.
- the cleaning composition of the present invention are advantageously employed for example, in laundry applications, hard surface cleaning, automatic dishwashing applications, as well as cosmetic applications such as dentures, teeth, hair and skin.
- the enzymes of the present invention are ideally suited for laundry applications.
- the enzymes of the present invention may be employed in both granular and liquid compositions.
- the variant proteases of the present invention also find use cleaning additive products.
- low temperature solution cleaning applications find use.
- the additive product may be, in its simplest form, one or more proteases.
- the additive is packaged in dosage form for addition to a cleaning process. Any suitable single dosage form also finds use with the present invention, including but not limited to pills, tablets, gelcaps, or other single dosage units such as pre-measured powders or liquids.
- filler(s) or carrier material(s) are included to increase the volume of such composition. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like.
- Suitable filler or carrier materials for liquid compositions include, but are not limited to 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. In some embodiments, the compositions contain from about 5% to about 90% of such materials. Acidic fillers find use to reduce pH. Alternatively, the cleaning additives include adjunct ingredients as more fully described below.
- the present cleaning compositions and cleaning additives require an effective amount of at least one of the protease variants provided herein, alone or in combination with other proteases and/or additional enzymes.
- the required level of enzyme is achieved by the addition of one or more protease variants of the present invention.
- the present cleaning compositions will comprise at least about 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 of the variant proteases of the present invention.
- the cleaning compositions herein are typically 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 neat pH from about 3.0 to about 9.0 or even from about 3 to about 5.
- 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, etc., and are well known to those skilled in the art.
- Suitable low pH cleaning compositions typically have a neat pH of from about 3 to about 5, and are typically free of surfactants that hydrolyze in such a pH environment.
- surfactants include sodium alkyl sulfate surfactants that comprise at least one ethylene oxide moiety or even from about 1 to about 16 moles of ethylene oxide.
- Such cleaning compositions typically comprise a sufficient amount of a pH modifier, such as sodium hydroxide, monoethanolamine or hydrochloric acid, to provide such cleaning composition with a neat pH of from about 3 to about 5.
- Such compositions typically comprise at least one acid stable enzyme.
- the compositions are liquids, while in other embodiments, they are solids.
- the pH of such liquid compositions is typically measured as a neat pH.
- the pH of such solid compositions is measured as a 10% solids solution of said composition wherein the solvent is distilled water. In these embodiments, all pH measurements are taken at 20° C.
- the variant protease(s) when the variant protease(s) is/are employed in a granular composition or liquid, it is desirable for the variant protease to be in the form of an encapsulated particle to protect the variant protease from other components of the granular composition during storage.
- encapsulation is also a means of controlling the availability of the variant protease during the cleaning process.
- encapsulation enhances the performance of the variant protease(s) and/or additional enzymes.
- the variant proteases of the present invention are encapsulated with any suitable encapsulating material known in the art.
- the encapsulating material typically encapsulates at least part of the catalyst for the variant protease(s) of the present invention.
- the encapsulating material is water-soluble and/or water-dispersible.
- the encapsulating material has a glass transition temperature (Tg) of 0° C. or higher. Glass transition temperature is described in more detail in WO 97/11151.
- the encapsulating material is selected from consisting of carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes, and combinations thereof.
- the encapsulating material When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. Typically, the encapsulating material is a starch. Suitable starches are described in EP 0 922 499; U.S. Pat. No. 4,977,252; U.S. Pat. No. 5,354,559, and U.S. Pat. No. 5,935,826.
- the encapsulating material is a microsphere made from plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof; commercially available microspheres that find use include those supplied by EXPANCEL® (Stockviksverken, Sweden), and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (PQ Corp., Valley Forge, Pa.).
- plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof; commercially available microspheres that find use include those supplied by EXPANCEL® (Stockviksverken, Sweden), and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (
- variant proteases of the present invention find particular use in the cleaning industry, including, but not limited to laundry and dish detergents. These applications place enzymes under various environmental stresses.
- the variant proteases of the present invention provide advantages over many currently used enzymes, due to their stability under various conditions.
- wash conditions including varying detergent formulations, wash water volumes, wash water temperatures, and lengths of wash time, to which proteases involved in washing are exposed.
- detergent formulations used in different geographical areas have different concentrations of their relevant components present in the wash water.
- a European detergent typically has about 4500-5000 ppm of detergent components in the wash water
- a Japanese detergent typically has approximately 667 ppm of detergent components in the wash water.
- detergents typically have about 975 ppm of detergent components present in the wash water.
- a low detergent concentration system includes detergents where less than about 800 ppm of detergent components are present in the wash water.
- Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.
- a medium detergent concentration includes detergents where between about 800 ppm and about 2000 ppm of detergent components are present in the wash water.
- North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water. Brazil typically has approximately 1500 ppm of detergent components present in the wash water.
- a high detergent concentration system includes detergents where greater than about 2000 ppm of detergent components are present in the wash water.
- European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.
- Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. As mentioned above, Brazil typically has approximately 1500 ppm of detergent components present in the wash water. However, other high suds phosphate builder detergent geographies, not limited to other Latin American countries, may have high detergent concentration systems up to about 6000 ppm of detergent components present in the wash water.
- concentrations of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition (“low detergent concentration geographies”), for example about 667 ppm in Japan, to between about 800 ppm to about 2000 ppm (“medium detergent concentration geographies”), for example about 975 ppm in U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm (“high detergent concentration geographies”), for example about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.
- low detergent concentration geographies for example about 667 ppm in Japan
- intermediate detergent concentration geographies for example about 975 ppm in U.S. and about 1500 ppm in Brazil
- high detergent concentration geographies for example about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.
- concentrations of the typical wash solutions are determined empirically. For example, in the U.S., a typical washing machine holds a volume of about 64.4 L of wash solution. Accordingly, in order to obtain a concentration of about 975 ppm of detergent within the wash solution about 62.79 g of detergent composition must be added to the 64.4 L of wash solution. This amount is the typical amount measured into the wash water by the consumer using the measuring cup provided with the detergent.
- the temperature of the wash water in Japan is typically less than that used in Europe.
- the temperature of the wash water in North America and Japan is typically between 10 and 30° C. (e.g., about 20° C.), whereas the temperature of wash water in Europe is typically between 30 and 60° C. (e.g., about 40° C.).
- Water hardness is usually described in terms of the grains per gallon mixed Ca 2+ /Mg 2+ .
- Hardness is a measure of the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) in the water. Most water in the United States is hard, but the degree of hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts per million (parts per million converted to grains per U.S. gallon is ppm # divided by 17.1 equals grains per gallon) of hardness minerals.
- European water hardness is typically greater than 10.5 (for example 10.5-20.0) grains per gallon mixed Ca 2+ /Mg 2+ (e.g., about 15 grains per gallon mixed Ca 2+ /Mg 2+ ).
- North American water hardness is typically greater than Japanese water hardness, but less than European water hardness.
- North American water hardness can be between 3 to 10 grains, 3-8 grains or about 6 grains.
- Japanese water hardness is typically lower than North American water hardness, usually less than 4, for example 3 grains per gallon mixed Ca 2+ /Mg 2+ .
- the present invention provides variant proteases that show surprising wash performance in at least one set of wash conditions (e.g., water temperature, water hardness, and/or detergent concentration).
- the variant proteases of the present invention are comparable in wash performance to other subtilisin proteases.
- the variant proteases of the present invention exhibit enhanced wash performance as compared to subtilisin proteases currently commercially available.
- the variant proteases provided herein exhibit enhanced oxidative stability, enhanced thermal stability, and/or enhanced chelator stability.
- the variant proteases of the present invention find use in cleaning compositions that do not include detergents, again either alone or in combination with builders and stabilizers.
- the cleaning compositions comprise at least one variant protease of the present invention at a level from about 0.00001% to about 10% by weight of the composition and the balance (e.g., about 99.999% to about 90.0%) comprising cleaning adjunct materials by weight of composition.
- the cleaning compositions of the present invention comprises at least one variant protease at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% by weight of the composition and the balance of the cleaning composition (e.g., about 99.9999% to about 90.0%, about 99.999% to about 98%, about 99.995% to about 99.5% by weight) comprising cleaning adjunct materials.
- preferred cleaning compositions comprise one or more additional enzymes or enzyme derivatives which provide cleaning performance and/or fabric care benefits, in addition to one or more of the variant proteases provided herein.
- additional enzymes or enzyme derivatives include, but are not limited to other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g. laccases), and/or mannanases.
- Suitable proteases include those of animal, vegetable or microbial origin. In some particularly preferred embodiments, microbial proteases are used. In some embodiments, chemically or genetically modified mutants are included.
- the protease is a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases include subtilisins, especially those derived from Bacillus (e.g., subtilisin, lentus, amyloliquefaciens, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168).
- Additional examples include those mutant proteases described in U.S. Pat. Nos. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, all of which are incorporated herein by reference. Additional protease examples include, but are not limited to trypsin (e.g., of porcine or bovine origin), and the Fusarium protease described in WO 89/06270.
- protease enzymes include MAXATASE®, MAXACALTM, MAXAPEMTM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT® and PURAFECT® OXP (Genencor); ALCALASE®, SAVINASE®, PRIMASE®, DURAZYMTM, RELASE® and ESPERASE® (Novozymes); and BLAPTM (Henkel Kilandit GmbH auf Aktien, Duesseldorf, Germany.
- Various proteases are described in WO95/23221, WO 92/21760, and U.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625, U.S. Pat. No. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, and various other patents.
- any suitable lipase finds use in the present invention.
- Suitable lipases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are encompassed by the present invention.
- useful lipases include Humicola lanuginosa lipase (See e.g., EP 258 068, and EP 305 216), Rhizomucor miehei lipase (See e.g., EP 238 023), Candida lipase, such as C. antarctica lipase (e.g., the C. antarctica lipase A or B; See e.g., EP 214 761), a Pseudomonas lipase such as P.
- C. antarctica lipase e.g., the C. antarctica lipase A or B; See e.g., EP 214 761
- Pseudomonas lipase such as P.
- alcaligenes and P. pseudoalcaligenes lipase See e.g., EP 218 272), P. cepacia lipase (See e.g., EP 331 376), P. stutzeri lipase (See e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase [Dartois et al., Biochem. Biophys. Acta 1131:253-260 [1993]); B. stearothermophilus lipase [See e.g., JP 64/744992]; and B. pumilus lipase [See e.g., WO 91/16422]).
- B. subtilis lipase e.g., B. subtilis lipase [Dartois et al., Biochem. Biophys. Acta 1131:253-260 [
- cloned lipases find use in some embodiments of the present invention, including but not limited to Penicillium camembertii lipase (See, Yamaguchi et al., Gene 103:61-67 [1991]), Geotricum candidum lipase (See, Schimada et al., J. Biochem., 106:383-388 [1989]), and various Rhizopus lipases such as R. delemar lipase (See, Hass et al., Gene 109:117-113 [1991]), a R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 [1992]) and R. oryzae lipase.
- Penicillium camembertii lipase See, Yamaguchi et al., Gene 103:61-67 [1991]
- Geotricum candidum lipase See, Schimada
- cutinases Other types of lipolytic enzymes such as cutinases also find use in some embodiments of the present invention, including but not limited to the cutinase derived from Pseudomonas mendocina (See, WO 88/09367), and the cutinase derived from Fusarium solani pisi (See, WO 90/09446).
- lipases include commercially available lipases such as M1 LIPASETM, LUMA FASTTM, and LIPOMAXTM (Genencor); LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE PTM “Amano” (Amano Pharmaceutical Co. Ltd., Japan).
- the cleaning compositions of the present invention further comprise lipases at a level from about 0.00001% to about 10% of additional lipase by weight of the composition and the balance of cleaning adjunct materials by weight of composition.
- the cleaning compositions of the present invention also comprise, lipases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% lipase by weight of the composition.
- amylase alpha and/or beta
- suitable amylases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments.
- Amylases that find use in the present invention include, but are not limited to ⁇ -amylases obtained from B. licheniformis (See e.g., GB 1,296,839).
- Commercially available amylases that find use in the present invention include, but are not limited to DURAMYL®, TERMAMYL®, FUNGAMYL® and BANTM (Novozymes) and RAPIDASE® and MAXAMYL® P (Genencor).
- the cleaning compositions of the present invention further comprise amylases at a level from about 0.00001% to about 10% of additional amylase by weight of the composition and the balance of cleaning adjunct materials by weight of composition.
- the cleaning compositions of the present invention also comprise, amylases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% amylase by weight of the composition.
- any suitable cellulase finds used in the cleaning compositions of the present invention.
- Suitable cellulases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments.
- Suitable cellulases include, but are not limited to Humicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307).
- Especially suitable cellulases are the cellulases having color care benefits (See e.g., EP 0 495 257).
- Commercially available cellulases that find use in the present include, but are not limited to CELLUZYME® (Novozymes), and KAC-500(B)TM (Kao Corporation).
- cellulases are incorporated as portions or fragments of mature wild-type or variant cellulases, wherein a portion of the N-terminus is deleted (See e.g., U.S. Pat. No. 5,874,276).
- the cleaning compositions of the present invention further comprise cellulases at a level from about 0.00001% to about 10% of additional cellulase by weight of the composition and the balance of cleaning adjunct materials by weight of composition.
- the cleaning compositions of the present invention also comprise cellulases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% cellulase by weight of the composition.
- mannanase suitable for use in detergent compositions also finds use in the present invention.
- Suitable mannanases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments.
- Various mannanases are known which find use in the present invention (See e.g., U.S. Pat. No. 6,566,114, U.S. Pat. No. 6,602,842, and U.S. Pat. No. 6,440,991, all of which are incorporated herein by reference).
- the cleaning compositions of the present invention further comprise mannanases at a level from about 0.00001% to about 10% of additional mannanase by weight of the composition and the balance of cleaning adjunct materials by weight of composition.
- the cleaning compositions of the present invention also comprise, mannanases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% mannanase by weight of the composition.
- peroxidases are used in combination with hydrogen peroxide or a source thereof (e.g., a percarbonate, perborate or persulfate) in the compositions of the present invention.
- oxidases are used in combination with oxygen. Both types of enzymes are used for “solution bleaching” (i.e., to prevent transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed together in a wash liquor), preferably together with an enhancing agent (See e.g., WO 94/12621 and WO 95/01426).
- Suitable peroxidases/oxidases include, but are not limited to those of plant, bacterial or fungal origin.
- the cleaning compositions of the present invention further comprise peroxidase and/or oxidase enzymes at a level from about 0.00001% to about 10% of additional peroxidase and/or oxidase by weight of the composition and the balance of cleaning adjunct materials by weight of composition.
- the cleaning compositions of the present invention also comprise, peroxidase and/or oxidase enzymes at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% peroxidase and/or oxidase enzymes by weight of the composition.
- additional enzymes find use, including but not limited to perhydrolases (See e.g., WO 05/056782).
- mixtures of the above mentioned enzymes are encompassed herein, in particular one or more additional protease, amylase, lipase, mannanase, and/or at least one cellulase. Indeed, it is contemplated that various mixtures of these enzymes will find use in the present invention.
- the varying levels of the variant protease(s) and one or more additional enzymes may both independently range to about 10%, the balance of the cleaning composition being cleaning adjunct materials. The specific selection of cleaning adjunct 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 (e.g., through the wash detergent use).
- cleaning adjunct materials include, but are not limited to, surfactants, builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilizing systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, preservatives, anti-oxidants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments, and pH control agents (See e.g., U.S.
- an effective amount of one or more variant protease(s) provided herein are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal.
- cleaning compositions include cleaning compositions for such applications as cleaning hard surfaces, fabrics, and dishes.
- the present invention provides fabric cleaning compositions, while in other embodiments, the present invention provides non-fabric cleaning compositions.
- the present invention also provides cleaning compositions suitable for personal care, including oral care (including dentrifices, toothpastes, mouthwashes, etc., as well as denture cleaning compositions), skin, and hair cleaning compositions. It is intended that the present invention encompass detergent compositions in any form (i.e., liquid, granular, bar, semi-solid, gels, emulsions, tablets, capsules, etc.).
- compositions of the present invention preferably contain at least one surfactant and at least one builder compound, as well as one or more cleaning adjunct materials preferably selected from organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors.
- cleaning adjunct materials preferably selected from organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors.
- laundry compositions also contain softening agents (i.e., as additional cleaning adjunct materials).
- the compositions of the present invention also find use detergent additive products in solid or liquid form.
- the density of the laundry detergent compositions herein ranges from about 400 to about 1200 g/liter, while in other embodiments, it ranges from about 500 to about 950 g/liter of composition measured at 20° C.
- compositions of the invention preferably contain at least one surfactant and preferably at least one additional cleaning adjunct material selected from organic polymeric compounds, suds enhancing agents, group II metal ions, solvents, hydrotropes and additional enzymes.
- compositions comprising at least one variant protease of the present invention are a compact granular fabric cleaning composition, while in other embodiments, the composition is a granular fabric cleaning composition useful in the laundering of colored fabrics, in further embodiments, the composition is a granular fabric cleaning composition which provides softening through the wash capacity, in additional embodiments, the composition is a heavy duty liquid fabric cleaning composition.
- the compositions comprising at least one variant protease of the present invention are fabric cleaning compositions such as those described in U.S. Pat. Nos. 6,610,642 and 6,376,450.
- the variant proteases of the present invention find use in granular laundry detergent compositions of particular utility under European or Japanese washing conditions (See e.g., U.S. Pat. No. 6,610,642).
- the present invention provides hard surface cleaning compositions comprising at least one variant protease provided herein.
- the compositions comprising at least one variant protease of the present invention is a hard surface cleaning composition such as those described in U.S. Pat. Nos. 6,610,642, 6,376,450, and 6,376,450.
- the present invention provides dishwashing compositions comprising at least one variant protease provided herein.
- the compositions comprising at least one variant protease of the present invention is a hard surface cleaning composition such as those in U.S. Pat. Nos. 6,610,642 and 6,376,450.
- the present invention provides dishwashing compositions comprising at least one variant protease provided herein.
- the compositions comprising at least one variant protease of the present invention comprise oral care compositions such as those in U.S. Pat. Nos. 6,376,450, and 6,376,450.
- the formulations and descriptions of the compounds and cleaning adjunct materials contained in the aforementioned U.S. Pat. Nos. 6,376,450, 6,605,458, 6,605,458, and 6,610,642, find use with the variant proteases provided herein.
- the cleaning compositions of the present invention are formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448, 5,489,392, and 5,486,303, all of which are incorporated herein by reference.
- the pH of such composition is adjusted via the addition of a material such as monoethanolamine or an acidic material such as HCl.
- adjuncts illustrated hereinafter are suitable for use in the instant cleaning compositions.
- these adjuncts are incorporated 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, colorants, dyes or the like. It is understood that such adjuncts are in addition to the variant proteases of the present invention. 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 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, hydrogen peroxide, sources of hydrogen peroxide, 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 and/or pigments.
- suitable examples of such other adjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348, that are incorporated by reference.
- the aforementioned adjunct ingredients may constitute the balance of the cleaning compositions of the present invention.
- the cleaning compositions according to the present invention comprise a surfactant or surfactant system wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof.
- the cleaning compositions of the present invention comprise one or more detergent builders or builder systems.
- 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, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof.
- polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid
- polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,
- the cleaning compositions herein contain a chelating agent.
- Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof.
- the cleaning compositions provided herein 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.
- the cleaning compositions of the present invention 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 polyvinylimidazoles or mixtures thereof.
- the cleaning compositions of the present invention 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.
- the cleaning compositions comprise one or more detergent enzymes which provide cleaning performance and/or fabric care benefits.
- suitable enzymes include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ⁇ -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof.
- a typical combination is cocktail of conventional applicable enzymes including at least one protease, at least one lipase, at least one cutinase, and/or at least one cellulase in conjunction with at
- the enzymes used in the cleaning compositions are stabilized any suitable technique.
- the enzymes employed herein are 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.
- the cleaning compositions of the present invention 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.
- 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
- a sequestrate having defined stability constants for the cat
- compositions herein are catalyzed by means of a manganese compound.
- 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. Pat. No. 5,576,282.
- cobalt bleach catalysts useful herein are known, and are described, for example, in U.S. Pat. Nos. 5,597,936, and 5,595,967.
- cobalt bleach catalysts useful herein are known, and are described, for example, in U.S. Pat. Nos. 5,597,936, and 5,595,967.
- Such cobalt catalysts are readily prepared by known procedures, such as taught for example in U.S. Pat. Nos. 5,597,936, and 5,595,967.
- the compositions provided herein also suitably include a transition metal complex of a macropolycyclic rigid ligand (i.e., “MRL”).
- MRL macropolycyclic rigid ligand
- the compositions and cleaning processes herein are adjustable, 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 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 MRLs herein are a special type of ultra-rigid ligand that is cross-bridged such as 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane.
- Suitable transition metal MRLs are readily prepared by known procedures, such as taught for example in WO 00/332601, and U.S. Pat. No. 6,225,464.
- the cleaning compositions of the present invention are formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,516,448, 5,489,392, and 5,486,303, all of which are incorporated herein by reference.
- the cleaning compositions disclosed herein of find use in cleaning a situs (e.g., a surface, dishware, or fabric).
- a situs e.g., a surface, dishware, or fabric.
- the situs is optionally washed and/or rinsed.
- “washing” includes but is not limited to, scrubbing, and mechanical agitation.
- the cleaning compositions are typically employed at concentrations of from about 500 ppm to about 15,000 ppm in solution.
- 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 about 1:1 to about 30:1.
- the present invention also provides compositions and methods utilizing various surfactants and surfactant blends to assess and improve the performance of proteases in detergent compositions.
- various surfactants and surfactant blends to assess and improve the performance of proteases in detergent compositions.
- LAS linear alkylbenzene sulfonate
- AES alkylethoxy sulfate
- AE alcohol ethoxylate
- selecting a surfactant composition for optimum performance of a protease was not simply a matter of selecting the surfactant in which the protease was most stable. Rather, selecting an optimum surfactant composition was based on combining different surfactants in a ratio that provided the best overall cleaning results, wherein the mixture included surfactants that if used in the absence of other surfactants, would destabilize the protease.
- a protease is able to function better in the presence of a first surfactant when a second surfactant is present but less well if a third surfactant is present.
- a protease tolerates much higher levels of a first surfactant, depending on the ratio of a second and third surfactant.
- the present compositions and methods provide detergent composition comprising at least one protease and a mixture of surfactants in a ratio preselected to promote the activity of the protease, wherein the surfactants are not selected simply by measuring the stability of the protease in each surfactant.
- the protease is inactivated or exhibits reduced activity in the presence of a different ratio of the same surfactants, or in the presence of any subset of the surfactants but in the absence of at least one of the surfactants in the mixture.
- the protease is inactivated or exhibits reduced activity in the presence of any one of the surfactants, in the absence of other surfactant present in the mixture.
- the presence of a first surfactant allows the use of an increased amount of a second surfactant, wherein the same amount of the second surfactant in the absence of the first surfactant would inactivate or reduce the activity of the protease.
- a first type of surfactant e.g., a non-ionic or anionic detergent
- a second type of detergent permits the use of a second type of detergent, where the second type of detergent in the absence of the first type of surfactant would inactivate or reduce the activity of the protease.
- FNA Bacillus amyloliquefaciens subtilisin BPN′-Y217L
- FNA Bacillus amyloliquefaciens subtilisin BPN′-Y217L
- the protease was active in a composition comprising a relatively small amount of AE and a larger amount of either AES or LAS, or both.
- the protease was less active in a composition comprising only AES, only LAS, and particularly, only AE.
- the preferred composition was not one that included only the single surfactant in which the protease was most active, but rather one that included at least two, and preferably all three surfactants, any one of which would inactivate or reduce the activity of the protease if used individually at a sufficient level.
- the particular ratio of AES>LAS>AE appeared to produce the best result for this protease at the low temperature.
- the protease was active in a composition comprising only AES, or AES in combination with LAS and a relatively small amount of AE.
- the tolerance for AES increased at the higher temperature, making the inclusion of the other surfactants less important.
- 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 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.
- proteolytic activity refers to a protein or peptide exhibiting the ability to hydrolyze peptides or substrates having peptide linkages.
- Many well known procedures exist for measuring proteolytic activity Kalisz, “Microbial Proteinases,” In: Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology, [ 1988]).
- proteolytic activity may be ascertained by comparative assays which analyze the respective protease's ability to hydrolyze a commercial substrate.
- Exemplary substrates useful in the analysis of protease or proteolytic activity include, but are not limited to di-methyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111). Colorimetric assays utilizing these substrates are well known in the art (See e.g., WO 99/34011; and U.S. Pat. No. 6,376,450, both of which are incorporated herein by reference). The pNA assay (See e.g., Del Mar et al., Anal.
- Biochem., 99:316-320 [1979]) also finds use in determining the active enzyme concentration for fractions collected during gradient elution.
- This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPF-pNA).
- sAAPF-pNA succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide
- the rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration.
- absorbance measurements at 280 nm can be used to determine the total protein concentration.
- the active enzyme/total-protein ratio gives the enzyme purity.
- the genus Bacillus includes all species within the genus “ Bacillus ,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus , and B. thuringiensis . It is recognized that the genus Bacillus continues to undergo taxonomical reorganization.
- the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus , which is now named “ Geobacillus stearothermophilus .”
- Geobacillus stearothermophilus The production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus , although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus , and Virgibacillus.
- polynucleotide and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases.
- polynucleotides genes, gene fragments, chromosomal fragments, ESTs, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- polynucleotides comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches.
- the sequence of nucleotides is interrupted by non-nucleotide components.
- DNA construct and “transforming DNA” are used interchangeably to refer to DNA used to introduce sequences into a host cell or organism.
- the DNA may be generated in vitro by PCR or any other suitable technique(s) known to those in the art.
- the DNA construct comprises a sequence of interest (e.g., as an incoming sequence).
- the sequence is operably linked to additional elements such as control elements (e.g., promoters, etc.).
- the DNA construct may further comprise a selectable marker. It may further comprise an incoming sequence flanked by homology boxes.
- the transforming DNA comprises other non-homologous sequences, added to the ends (e.g., stuffer sequences or flanks).
- the ends of the incoming sequence are closed such that the transforming DNA forms a closed circle.
- the transforming sequences may be wild-type, mutant or modified.
- the DNA construct comprises sequences homologous to the host cell chromosome. In other embodiments, the DNA construct comprises non-homologous sequences. Once the DNA construct is assembled in vitro it may be used to: 1) insert heterologous sequences into a desired target sequence of a host cell, and/or 2) mutagenize a region of the host cell chromosome (i.e., replace an endogenous sequence with a heterologous sequence), 3) delete target genes, and/or 4) introduce a replicating plasmid into the host.
- expression cassette and “expression vector” refer to nucleic acid constructs generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell.
- the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
- the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
- expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell.
- the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into one or more cell types.
- Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, cassettes and the like.
- the polynucleotide construct comprises a DNA sequence encoding the protease (e.g., precursor or mature protease) that is operably linked to a suitable prosequence (e.g., secretory, etc.) capable of effecting the expression of the DNA in a suitable host.
- plasmid refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in some eukaryotes or prokaryotes, or integrates into the host chromosome.
- the term “introduced” refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, conjugation, and transduction (See e.g., Ferrari et al., “Genetics,” in Hardwood et al, (eds.), Bacillus , Plenum Publishing Corp., pages 57-72, [1989]).
- the terms “transformed” and “stably transformed” refers to a cell that has a non-native (heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained for at least two generations.
- a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
- DNA encoding a secretory leader i.e., a signal peptide
- a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
- a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
- operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
- gene refers to a polynucleotide (e.g., a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).
- homologous genes refers to a pair of genes from different, but usually related species, which 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).
- proteins are defined as having a common “fold” if they have the same major secondary structures in the same arrangement and with the same topological connections. Different proteins with the same fold often have peripheral elements of secondary structure and turn regions that differ in size and conformation. In some cases, these differing peripheral regions may comprise half the structure. Proteins placed together in the same fold category do not necessarily have a common evolutionary origin (e.g., structural similarities arising from the physics and chemistry of proteins favoring certain packing arrangements and chain topologies).
- homology refers to sequence similarity or identity, with identity being preferred. This homology is determined using standard techniques 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. Acad. Sci. USA 85:2444 [1988]; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis.); and Devereux et al., Nucl. Acid Res., 12:387-395 [1984]).
- an “analogous sequence” is one wherein the function of the gene is essentially the same as the gene based on the BPN′ protease. Analogous sequences are determined by known methods of sequence alignment. A commonly used alignment method is BLAST, although as indicated above and below, there are other methods that also find use in aligning sequences.
- PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the 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.
- BLAST BLAST algorithm
- Altschul et al. BLAST algorithm
- WU-BLAST-2 WU-BLAST-2 uses several search parameters, most of which are set to the default values.
- the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. However, the values may be adjusted to increase sensitivity.
- a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
- 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 starting sequence (i.e., the sequence of interest).
- a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
- recombinant includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified.
- recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.
- “Recombination,” “recombining,” and generating a “recombined” nucleic acid are generally the assembly of two or more nucleic acid fragments wherein the assembly gives rise to a chimeric gene.
- mutant DNA sequences are generated with site saturation mutagenesis in at least one codon. In another preferred embodiment, site saturation mutagenesis is performed for two or more codons. In a further embodiment, mutant DNA sequences have more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, or more than about 98% homology with the wild-type sequence. In alternative embodiments, mutant DNA is generated in vivo using any known mutagenic procedure such as, for example, radiation, nitrosoguanidine and the like. The desired DNA sequence is then isolated and used in the methods provided herein.
- 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.
- selection of cells by growth in the presence of a drug results in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug 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 (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template 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.
- the term “primer” refers to an oligonucleotide, whether occurring 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 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.
- 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 primers will depend on many factors, including temperature, source of primer and the use of the method.
- probe refers to an oligonucleotide (i.e., a sequence of 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 sequences.
- 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.
- target when used in reference to 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.
- PCR polymerase chain reaction
- the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
- 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 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.
- PCR polymerase chain reaction
- amplification reagents refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme.
- amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
- RT-PCR refers to the replication and amplification of RNA sequences.
- reverse transcription is coupled to PCR, most often using a one enzyme procedure in which a thermostable polymerase is employed, as described in U.S. Pat. No. 5,322,770, herein incorporated by reference.
- the RNA template is converted to cDNA due to the reverse transcriptase activity of the polymerase, and then amplified using the polymerizing activity of the polymerase (i.e., as in other PCR methods).
- restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
- restriction site refers to a nucleotide sequence recognized and cleaved by a given restriction endonuclease and is frequently the site for insertion of DNA fragments.
- restriction sites are engineered into the selective marker and into 5′ and 3′ ends of the DNA construct.
- “Homologous recombination” means the exchange of DNA fragments between two DNA molecules or paired chromosomes at the site of identical or nearly identical nucleotide sequences.
- chromosomal integration is homologous recombination.
- amino acid refers to peptide or protein sequences or portions thereof.
- protein peptide
- polypeptide are used interchangeably.
- protein of interest and “polypeptide of interest” refer to a protein/polypeptide that is desired and/or being assessed.
- the protein of interest is expressed intracellularly, while in other embodiments, it is a secreted polypeptide.
- these enzymes include the serine proteases of the present invention.
- the protein of interest is a secreted polypeptide which is fused to a signal peptide (i.e., an amino-terminal extension on a protein to be secreted). Nearly all secreted proteins use an amino-terminal protein extension which plays a crucial role in the targeting to and translocation of precursor proteins across the membrane. This extension is proteolytically removed by a signal peptidase during or immediately following membrane transfer.
- a polynucleotide is said to “encode” an RNA or a polypeptide if, in its native state or when manipulated by methods known to those of skill in the art, it can be transcribed and/or translated to produce the RNA, the polypeptide or a fragment thereof.
- the anti-sense strand of such a nucleic acid is also said to encode the sequences.
- a DNA can be transcribed by an RNA polymerase to produce RNA, but an RNA can be reverse transcribed by reverse transcriptase to produce a DNA.
- a DNA can encode a RNA and vice versa.
- “Host strain” or “host cell” refers to a suitable host for an expression vector comprising DNA according to the present invention.
- An enzyme is “overexpressed” in a host cell if the enzyme is expressed in the cell at a higher level that the level at which it is expressed in a corresponding wild-type cell.
- polypeptide proteins and polypeptide are used interchangeability herein.
- the 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used through out this disclosure. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
- a “prosequence” is an amino acid sequence between the signal sequence and mature protease that is necessary for the secretion of the protease. Cleavage of the pro sequence results in a mature active protease.
- signal sequence refers to any sequence of nucleotides and/or amino acids which may participate in the secretion of the mature or precursor forms of the protein.
- This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protein gene, which participate in the effectuation of the secretion of protein. They are often, but not universally, bound to the N-terminal portion of a protein or to the N-terminal portion of a precursor protein.
- the signal sequence may be endogenous or exogenous.
- the signal sequence may be that normally associated with the protein (e.g., protease), or may be from a gene encoding another secreted protein.
- One exemplary exogenous signal sequence comprises the first seven amino acid residues of the signal sequence from Bacillus subtilis subtilisin fused to the remainder of the signal sequence of the subtilisin from Bacillus lentus (ATCC 21536).
- hybrid signal sequence refers to signal sequences in which part of sequence is obtained from the expression host fused to the signal sequence of the gene to be expressed. In some embodiments, synthetic sequences are utilized.
- mature form of a protein or peptide refers to the final functional form of the protein or peptide.
- a mature form of the protease of the present invention includes at least the amino acid sequence identical to residue positions 1-275 of SEQ ID NO:2.
- precursor form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein.
- the precursor may also have a “signal” sequence operably linked, to the amino terminus of the prosequence.
- the precursor may also have additional polynucleotides that are involved in post-translational activity (e.g., polynucleotides cleaved therefrom to leave the mature form of a protein or peptide).
- Naturally occurring enzyme refers to an enzyme having the unmodified amino acid sequence identical to that found in nature.
- Naturally occurring enzymes include native enzymes, those enzymes naturally expressed or found in the particular microorganism.
- the terms “derived from” and “obtained from” refer to not only a protease produced or producible by a strain of the organism in question, but also a protease encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protease which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protease in question.
- a “derivative” within the scope of this definition generally retains the characteristic proteolytic activity observed in the wild-type, native or parent form to the extent that the derivative is useful for similar purposes as the wild-type, native or parent form.
- Functional derivatives of serine protease encompass naturally occurring, synthetically or recombinantly produced peptides or peptide fragments which have the general characteristics of the serine protease of the present invention.
- the term “functional derivative” refers to a derivative of a nucleic acid which has the functional characteristics of a nucleic acid which encodes serine protease.
- Functional derivatives of a nucleic acid which encode serine protease of the present invention encompass naturally occurring, synthetically or recombinantly produced nucleic acids or fragments and encode serine protease characteristic of the present invention.
- Wild type nucleic acid encoding serine proteases according to the invention include naturally occurring alleles and homologues based on the degeneracy of the genetic code known in the art.
- nucleic acids or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence, as measured using one of the following sequence comparison or analysis algorithms.
- optical alignment refers to the alignment giving the highest percent identity score.
- Percent sequence identity refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two optimally aligned polypeptide sequences are identical.
- substantially identical in the context of two nucleic acids or polypeptides thus refers to a polynucleotide or polypeptide that comprising at least about 70% sequence identity, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, preferably at least about 90%, preferably at least about 95%, preferably at least about 97%, preferably at least about 98%, and preferably at least about 99% sequence identity as compared to a reference sequence using the programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.
- One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide.
- polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive.
- a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative 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).
- equivalent refers to serine proteases enzymes that are encoded by a polynucleotide capable of hybridizing to the polynucleotide having the sequence as shown in SEQ ID NO:1, under conditions of medium to maximum stringency.
- an equivalent mature serine protease comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and/or at least about 99% sequence identity to the mature BPN′ serine protease having the amino acid sequence of SEQ ID NO:2.
- isolated refers to a material that is removed from its original environment (e.g., the natural environment if it is naturally occurring).
- the material is said to be “purified” when it is present in a particular composition in a higher or lower concentration than exists in a naturally occurring or wild type organism or in combination with components not normally present upon expression from a naturally occurring or wild type organism.
- a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
- such polynucleotides are part of a vector, and/or such polynucleotides or polypeptides are part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- a nucleic acid or protein is said to be purified, for example, if it gives rise to essentially one band in an electrophoretic gel or blot.
- isolated when used in reference to a DNA sequence, refers to a DNA sequence that has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (See e.g., Dynan and Tijan, Nature 316:774-78 [1985]). The term “an isolated DNA sequence” is alternatively referred to as “a cloned DNA sequence”.
- isolated when used in reference to a protein, refers to a protein that is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins.
- An isolated protein is more than about 10% pure, preferably more than about 20% pure, and even more preferably more than about 30% pure, as determined by SDS-PAGE. Further aspects of the invention encompass the protein in a highly purified form (i.e., more than about 40% pure, more than about 60% pure, more than about 80% pure, more than about 90% pure, more than about 95% pure, more than about 97% pure, and even more than about 99% pure), as determined by SDS-PAGE.
- combinatorial mutagenesis refers to methods in which libraries of variants of a starting sequence are generated.
- the variants contain one or several mutations chosen from a predefined set of mutations.
- the methods provide means to introduce random mutations which were not members of the predefined set of mutations.
- the methods include those set forth in U.S. patent application Ser. No. 09/699,250, filed Oct. 26, 2000, hereby incorporated by reference.
- combinatorial mutagenesis methods encompass commercially available kits (e.g., QuikChange® Multisite, Stratagene, San Diego, Calif.).
- library of mutants refers to a population of cells which are identical in most of their genome but include different homologues of one or more genes. Such libraries can be used, for example, to identify genes or operons with improved traits.
- starting gene refers to a gene of interest that encodes a protein of interest that is to be improved and/or changed using the present invention.
- MSA multiple sequence alignment
- the terms “consensus sequence” and “canonical sequence” refer to an archetypical amino acid sequence against which all variants of a particular protein or sequence of interest are compared. The terms also refer to a sequence that sets forth the nucleotides that are most often present in a DNA sequence of interest. For each position of a gene, the consensus sequence gives the amino acid that is most abundant in that position in the MSA.
- Consensus mutation refers to a difference in the sequence of a starting gene and a consensus sequence. Consensus mutations are identified by comparing the sequences of the starting gene and the consensus sequence resulting from an MSA. In some embodiments, consensus mutations are introduced into the starting gene such that it becomes more similar to the consensus sequence. Consensus mutations also include amino acid changes that change an amino acid in a starting gene to an amino acid that is more frequently found in an MSA at that position relative to the frequency of that amino acid in the starting gene. Thus, the term consensus mutation comprises all single amino acid changes that replace an amino acid of the starting gene with an amino acid that is more abundant than the amino acid in the MSA.
- initial hit refers to a variant that was identified by screening a combinatorial consensus mutagenesis library.
- initial hits have improved performance characteristics, as compared to the starting gene.
- the term “improved hit” refers to a variant that was identified by screening an enhanced combinatorial consensus mutagenesis library.
- the terms “improving mutation” and “performance-enhancing mutation” refer to a mutation that leads to improved performance when it is introduced into the starting gene.
- these mutations are identified by sequencing hits that were identified during the screening step of the method.
- mutations that are more frequently found in hits are likely to be improving mutations, as compared to an unscreened combinatorial consensus mutagenesis library.
- the term “enhanced combinatorial consensus mutagenesis library” refers to a CCM library that is designed and constructed based on screening and/or sequencing results from an earlier round of CCM mutagenesis and screening.
- the enhanced CCM library is based on the sequence of an initial hit resulting from an earlier round of CCM.
- the enhanced CCM is designed such that mutations that were frequently observed in initial hits from earlier rounds of mutagenesis and screening are favored. In some preferred embodiments, this is accomplished by omitting primers that encode performance-reducing mutations or by increasing the concentration of primers that encode performance-enhancing mutations relative to other primers that were used in earlier CCM libraries.
- performance-reducing mutations refer to mutations in the combinatorial consensus mutagenesis library that are less frequently found in hits resulting from screening as compared to an unscreened combinatorial consensus mutagenesis library.
- the screening process removes and/or reduces the abundance of variants that contain “performance-reducing mutations.”
- the term “functional assay” refers to an assay that provides an indication of a protein's activity.
- the term refers to assay systems in which a protein is analyzed for its ability to function in its usual capacity.
- a functional assay involves determining the effectiveness of the enzyme in catalyzing a reaction.
- target property refers to the property of the starting gene that is to be altered. It is not intended that the present invention be limited to any particular target property.
- the target property is the stability of a gene product (e.g., resistance to denaturation, proteolysis or other degradative factors), while in other embodiments, the level of production in a production host is altered. Indeed, it is contemplated that any property of a starting gene will find use in the present invention.
- a property affecting binding to a polypeptide refers to any characteristic or attribute of a nucleic acid that can be selected or detected. These properties include, but are not limited to, a property affecting binding to a polypeptide, a property conferred on a cell comprising a particular nucleic acid, a property affecting gene transcription (e.g., promoter strength, promoter recognition, promoter regulation, enhancer function), a property affecting RNA processing (e.g., RNA splicing, RNA stability, RNA conformation, and post-transcriptional modification), a property affecting translation (e.g., level, regulation, binding of mRNA to ribosomal proteins, post-translational modification).
- a binding site for a transcription factor, polymerase, regulatory factor, etc., of a nucleic acid may be altered to produce desired characteristics or to identify undesirable characteristics.
- polypeptide or grammatical equivalents thereof in the context of a polypeptide (including proteins), as used herein, refer to any characteristic or attribute of a polypeptide that can be selected or detected. These properties include, but are not limited to oxidative stability, substrate specificity, catalytic activity, thermal stability, alkaline stability, pH activity profile, resistance to proteolytic degradation, K M , k cat , k cat /k M ratio, protein folding, inducing an immune response, ability to bind to a ligand, ability to bind to a receptor, ability to be secreted, ability to be displayed on the surface of a cell, ability to oligomerize, ability to signal, ability to stimulate cell proliferation, ability to inhibit cell proliferation, ability to induce apoptosis, ability to be modified by phosphorylation or glycosylation, and/or ability to treat disease, etc.
- the term “screening” has its usual meaning in the art and is, in general a multi-step process.
- a mutant nucleic acid or variant polypeptide therefrom is provided.
- a property of the mutant nucleic acid or variant polypeptide is determined.
- the determined property is compared to a property of the corresponding precursor nucleic acid, to the property of the corresponding naturally occurring polypeptide or to the property of the starting material (e.g., the initial sequence) for the generation of the mutant nucleic acid.
- the screening procedure for obtaining a nucleic acid or protein with an altered property depends upon the property of the starting material the modification of which the generation of the mutant nucleic acid is intended to facilitate.
- the skilled artisan will therefore appreciate that the invention is not limited to any specific property to be screened for and that the following description of properties lists illustrative examples only. Methods for screening for any particular property are generally described in the art. For example, one can measure binding, pH, specificity, etc., before and after mutation, wherein a change indicates an alteration.
- the screens are performed in a high-throughput manner, including multiple samples being screened simultaneously, including, but not limited to assays utilizing chips, phage display, and multiple substrates and/or indicators.
- screens encompass selection steps in which variants of interest are enriched from a population of variants.
- these embodiments include the selection of variants that confer a growth advantage to the host organism, as well as phage display or any other method of display, where variants can be captured from a population of variants based on their binding or catalytic properties.
- a library of variants is exposed to stress (heat, protease, denaturation) and subsequently variants that are still intact are identified in a screen or enriched by selection. It is intended that the term encompass any suitable means for selection. Indeed, it is not intended that the present invention be limited to any particular method of screening.
- targeted randomization refers to a process that produces a plurality of sequences where one or several positions have been randomized.
- randomization is complete (i.e., all four nucleotides, A, T, G, and C can occur at a randomized position.
- randomization of a nucleotide is limited to a subset of the four nucleotides.
- Targeted randomization can be applied to one or several codons of a sequence, coding for one or several proteins of interest. When expressed, the resulting libraries produce protein populations in which one or more amino acid positions can contain a mixture of all 20 amino acids or a subset of amino acids, as determined by the randomization scheme of the randomized codon.
- the individual members of a population resulting from targeted randomization differ in the number of amino acids, due to targeted or random insertion or deletion of codons.
- synthetic amino acids are included in the protein populations produced.
- the majority of members of a population resulting from targeted randomization show greater sequence homology to the consensus sequence than the starting gene.
- the sequence encodes one or more proteins of interest.
- the proteins have differing biological functions.
- the incoming sequence comprises at least one selectable marker. This sequence can code for one or more proteins of interest. It can have other biological function. In many cases the incoming sequence will include a selectable marker, such as a gene that confers resistance to an antibiotic.
- modified sequence and “modified genes” are used interchangeably herein to refer to a sequence that includes a deletion, insertion or interruption of naturally occurring nucleic acid sequence.
- the expression product of the modified sequence is a truncated protein (e.g., if the modification is a deletion or interruption of the sequence).
- the truncated protein retains biological activity.
- the expression product of the modified sequence is an elongated protein (e.g., modifications comprising an insertion into the nucleic acid sequence).
- an insertion leads to a truncated protein (e.g., when the insertion results in the formation of a stop codon).
- an insertion may result in either a truncated protein or an elongated protein as an expression product.
- mutant sequence and “mutant gene” are used interchangeably and refer to a sequence that has an alteration in at least one codon occurring in a host cell's wild-type sequence.
- the expression product of the mutant sequence is a protein with an altered amino acid sequence relative to the wild-type.
- the expression product may have an altered functional capacity (e.g., enhanced enzymatic activity).
- mutagenic primer or “mutagenic oligonucleotide” (used interchangeably herein) are intended to refer to oligonucleotide compositions which correspond to a portion of the template sequence and which are capable of hybridizing thereto. With respect to mutagenic primers, the primer will not precisely match the template nucleic acid, the mismatch or mismatches in the primer being used to introduce the desired mutation into the nucleic acid library. As used herein, “non-mutagenic primer” or “non-mutagenic oligonucleotide” refers to oligonucleotide compositions which will match precisely to the template nucleic acid. In one embodiment of the invention, only mutagenic primers are used.
- the primers are designed so that for at least one region at which a mutagenic primer has been included, there is also non-mutagenic primer included in the oligonucleotide mixture.
- a mixture of mutagenic primers and non-mutagenic primers corresponding to at least one of the mutagenic primers it is possible to produce a resulting nucleic acid library in which a variety of combinatorial mutational patterns are presented. For example, if it is desired that some of the members of the mutant nucleic acid library retain their precursor sequence at certain positions while other members are mutant at such sites, the non-mutagenic primers provide the ability to obtain a specific level of non-mutant members within the nucleic acid library for a given residue.
- the methods of the invention employ mutagenic and non-mutagenic oligonucleotides which are generally between 10-50 bases in length, more preferably about 15-45 bases in length. However, it may be necessary to use primers that are either shorter than 10 bases or longer than 50 bases to obtain the mutagenesis result desired. With respect to corresponding mutagenic and non-mutagenic primers, it is not necessary that the corresponding oligonucleotides be of identical length, but only that there is overlap in the region corresponding to the mutation to be added. Primers may be added in a pre-defined ratio according to the present invention.
- the resulting library have a significant level of a certain specific mutation and a lesser amount of a different mutation at the same or different site
- by adjusting the amount of primer added it is possible to produce the desired biased library.
- by adding lesser or greater amounts of non-mutagenic primers it is possible to adjust the frequency with which the corresponding mutation(s) are produced in the mutant nucleic acid library.
- contiguous mutations refers to mutations which are presented within the same oligonucleotide primer.
- contiguous mutations may be adjacent or nearby each other, however, they will be introduced into the resulting mutant template nucleic acids by the same primer.
- discontiguous mutations refers to mutations which are presented in separate oligonucleotide primers. For example, discontiguous mutations will be introduced into the resulting mutant template nucleic acids by separately prepared oligonucleotide primers.
- wild-type sequence or “wild-type gene” are used interchangeably herein, to refer to a sequence that is native or naturally occurring in a host cell.
- the wild-type sequence refers to a sequence of interest that is the starting point of a protein engineering project.
- the wild-type sequence may encode either a homologous or heterologous protein.
- a homologous protein is one the host cell would produce without intervention.
- a heterologous protein is one that the host cell would not produce but for the intervention.
- amino acid position numbers refer to those assigned to the mature Bacillus amyloliquefaciens subtilisin sequence of SEQ ID NO:2.
- the invention is not limited to the mutation of this particular subtilisin but extends to precursor proteases containing amino acid residues at positions which are “equivalent” to the particular identified residues in the Bacillus amyloliquefaciens subtilisin.
- modification refers to any change or alteration in an amino acid sequence. It is intended that the term encompass substitutions, deletions, insertions, and/or replacement of amino acid side chains in an amino acid sequence of interest (e.g., a subtilisin sequence). It is also intended that the term encompass chemical modification of an amino acid sequence of interest (e.g., a subtilisin sequence).
- antibodies refers to immunoglobulins. Antibodies include but are not limited to immunoglobulins obtained directly from any species from which it is desirable to produce antibodies. In addition, the present invention encompasses modified antibodies. The term also refers to antibody fragments that retain the ability to bind to the epitope that the intact antibody binds and include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, anti-idiotype (anti-ID) antibodies. Antibody fragments include, but are not limited to the complementarity-determining regions (CDRs), single-chain fragment variable regions (scFv), heavy chain variable region (VH), light chain variable region (VL). Polyclonal and monoclonal antibodies are also encompassed by the present invention. Preferably, the antibodies are monoclonal antibodies.
- CDRs complementarity-determining regions
- scFv single-chain fragment variable regions
- VH heavy chain variable region
- VL light chain variable region
- Polyclonal and monoclonal antibodies are also encompassed by the present invention.
- oxidation stable refers to proteases of the present invention that retain a specified amount of enzymatic activity over a given period of time under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention, for example while exposed to or contacted with bleaching agents or oxidizing agents.
- the proteases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% proteolytic activity after contact with a bleaching or oxidizing agent over a given time period, for example, at least about 1 minute, about 3 minutes, about 5 minutes, about 8 minutes, about 12 minutes, about 16 minutes, about 20 minutes, etc.
- the stability is measured as described in the Examples.
- chelator stable refers to proteases of the present invention that retain a specified amount of enzymatic activity over a given period of time under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention, for example while exposed to or contacted with chelating agents.
- the proteases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% proteolytic activity after contact with a chelating agent over a given time period, for example, at least about 10 minutes, about 20 minutes, about 40 minutes, about 60 minutes, about 100 minutes, etc.
- the chelator stability is measured as described in the Examples.
- thermostability refers to proteases of the present invention that retain a specified amount of enzymatic activity after exposure to identified temperatures over a given period of time under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention, for example while exposed altered temperatures. Altered temperatures includes increased or decreased temperatures.
- the proteases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% proteolytic activity after exposure to altered temperatures over a given time period, for example, at least about 60 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, etc.
- the thermostability is determined as described in the Examples.
- enhanced stability in the context of an oxidation, chelator, thermal and/or pH stable protease refers to a higher retained proteolytic activity over time as compared to other serine proteases (e.g., subtilisin proteases) and/or wild-type enzymes.
- serine proteases e.g., subtilisin proteases
- diminished stability in the context of an oxidation, chelator, thermal and/or pH stable protease refers to a lower retained proteolytic activity over time as compared to other serine proteases (e.g., subtilisin proteases) and/or wild-type enzymes.
- serine proteases e.g., subtilisin proteases
- cleaning activity refers to the cleaning performance achieved by the protease under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention.
- cleaning performance is determined by the application of various cleaning assays concerning enzyme sensitive stains, for example grass, blood, milk, or egg protein as determined by various chromatographic, spectrophotometric or other quantitative methodologies after subjection of the stains to standard wash conditions.
- Exemplary assays include, but are not limited to those described in WO 99/34011, and U.S. Pat. No. 6,605,458 (both of which are herein incorporated by reference), as well as those methods included in the Examples.
- cleaning effective amount of a protease refers to the quantity of protease described hereinbefore that achieves a desired level of enzymatic activity in a specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, etc.
- cleaning adjunct materials means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel; or foam composition), which materials are also preferably compatible with the protease enzyme used in the composition.
- granular compositions are in “compact” form, while in other embodiments, the liquid compositions are in a “concentrated” form.
- enhanced performance in the context of cleaning activity refers to an increased or greater cleaning activity of certain enzyme sensitive stains such as egg, milk, grass or blood, as determined by usual evaluation after a standard wash cycle and/or multiple wash cycles.
- diminished performance in the context of cleaning activity refers to an decreased or lesser cleaning activity of certain enzyme sensitive stains such as egg, milk, grass or blood, as determined by usual evaluation after a standard wash cycle.
- subtilisin protease e.g., commercially available proteases
- OPTIMASETM protease Genencor
- PURAFECTTM protease products Genencor
- SAVINASETM protease Novozymes
- BPN′-variants See e.g., U.S. Pat. No.
- Re 34,606 RELASETM, DURAZYMETM, EVERLASETM, KANNASETM protease (Novozymes), MAXACALTM, MAXAPEMTTM, PROPERASETM proteases (Genencor; See also, U.S. Pat. No. Re 34,606, and U.S. Pat. Nos. 5,700,676; 5,955,340; 6,312,936; and 6,482,628), and B. lentus variant protease products (e.g., those described in WO 92/21760, WO 95/23221 and/or WO 97/07770).
- Exemplary subtilisin protease variants include, but are not limited to those having substitutions or deletions at residue positions equivalent to positions 76, 101, 103, 104, 120, 159, 167, 170, 194, 195, 217, 232, 235, 236, 245, 248, and/or 252 of BPN′.
- Cleaning performance can be determined by comparing the proteases of the present invention with those subtilisin proteases in various cleaning assays concerning enzyme sensitive stains such as grass, blood or milk as determined by usual spectrophotometric or analytical methodologies after standard wash cycle conditions.
- fabric cleaning compositions include hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the soaking and/or pretreatment of stained fabrics (e.g., clothes, linens, and other textile materials).
- non-fabric cleaning compositions include non-textile (i.e., fabric) surface cleaning compositions, including but not limited to dishwashing detergent compositions, oral cleaning compositions, denture cleaning compositions, and personal cleansing compositions.
- inorganic filler salts are conventional ingredients of detergent compositions in powder form.
- the filler salts are present in substantial amounts, typically about 17 to about 35% by weight of the total composition.
- the filler salt is present in amounts not exceeding about 15% of the total composition.
- the filler salt is present in amounts that do not exceed about 10%, or more preferably, about 5%, by weight of the composition.
- the inorganic filler salts are selected from the alkali and alkaline-earth-metal salts of sulfates and chlorides.
- a preferred filler salt is sodium sulfate.
- surfactant refers to any compound generally recognized in the art as having surface active qualities. Surfactants generally include anionic, cationic, nonionic, and zwitterionic compounds, which are further described, herein.
- the terms “contacting” and “exposing” refer to placing a surfactant and lipolytic enzyme in sufficient proximity an oily stain or oily soil to enable the enzyme and surfactant to at least partially decrease the amount of the stain or soil by producing fatty acids that are solubilized in the surfactant. Contacting may occur in a washing machine, a sink, on a body surface, etc.
- BPN′ e.g., reference protease
- BPN′ variants this assay was started using filtered culture supernatant from microliter plates grown 3-4 days at 33° C. with shaking at 230 rpm and humidified aeration. A fresh 96-well flat bottom microtiter plate (MTP) was used for the assay. First, 100 ⁇ L/well of 0.25 N HCl was placed in each well. Then, 50 ⁇ L of filtered culture broth was added. The light scattering/absorbance at 405 nm (use 5 sec mixing mode in the plate reader) was then determined, in order to provide the “blank” reading.
- MTP microtiter plate
- TCA trichloroacetic acid
- this assay was performed using filtered culture supernatant from microtiter plates grown approximately 3 days at 37° C. with shaking at 300 rpm and humidified aeration.
- 100 ⁇ L of a 0.25 M HCl solution was added to each well of a 96-well flat bottom microtiter plate.
- 25 ⁇ L aliquots of the filtered culture supernatants (containing the proteases) were added to wells.
- the light scattering/absorbance at 405 nm using the 5 sec mixing mode in the plate reader was then determined, in order to provide the “blank” reading.
- the equipment used was a Biomek FX Robot (Beckman Coulter) and a SpectraMAX (type 340; Molecular Devices) MTP Reader; the MTP's were from Costar (type 9017).
- the equipment used was a Biomek FX Robot (Beckman Coulter) and a SpectraMAX type 340 (Molecular Devices) MTP Reader; and the MTPs were type 9017 (Costar).
- a standard curve can be created by calibrating the TCA readings with AAPF assays of clones with known conversion factors.
- the TCA results are linear with respect to protein concentration from 50 to 500 protein per ml (ppm) and can thus be plotted directly against enzyme performance for the purpose of choosing good-performing variants.
- the reagent solutions used were: 100 mM Tris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer); 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl 2 and 0.005% TWEEN®-80 (Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388).
- suc-AAPF-pNA working solution 1 ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer and mixed well for at least 10 seconds.
- the assay was performed by adding 10 ⁇ l of diluted protease solution to each well, immediately followed by the addition of 190 ⁇ l 1 mg/ml suc-AAPF-pNA working solution. The solutions were mixed for 5 sec., and the absorbance change in kinetic mode (20 readings in 5 minutes) was read at 410 nm in an MTP reader, at 25° C.
- LAS and LAS/EDTA stability was measured after incubation of the test protease in the presence of LAS and LAS/EDTA respectively, as a function of residual activity determined using the AAPF assay.
- TRIS buffer (free acid): Sigma T-1378); 6.35 g is dissolved in about 960 ml water; pH is adjusted to 8.2 with 4N HCl. Final concentration of TRIS is 52.5 mM.
- a 0.063% LAS solution was prepared in 52.5 mM Tris buffer pH 8.2.
- the suc-AAPF-pNA working solution was prepared by adding 1 ml of 100 mg/ml suc-AAPF-pNA stock solution (in DMSO) to 100 ml (100 mM) TRIS buffer, pH 8.6. To dilute the supernatants, flat-bottomed plates were filled with dilution buffer and an aliquot of the supernatant was added and mixed well. The dilution ratio depended on the concentration of the protease controls in the growth plates (AAPF activity). The desired protein concentration was 80 ppm.
- the AAPF activity was then determined as described above.
- the stability of the samples was determined by calculating the ration of the residual and initial AAPF activity as follows:
- the reagents used were dodecyllbenzene sulfonate, sodium salt (DOBS, Sigma No. D-2525), TWEEN®-80 (Sigma No. P-8074), di-sodium EDTA (Siegfried Handel No. 164599-02), HEPES (Sigma No.
- H-7523 unstress buffer: 50 mM HEPES (11.9 ⁇ l+0.005% TWEEN®-80, pH 8.0, Stress buffer: 50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0, reference protease and protease variant culture supernatants, containing 200-400 ⁇ g/ml protein.
- V- or U-bottom MTP as dilution plates (Greiner 651101 and 650161 respectively), F-bottom MTP (Corning 9017) for unstress and LAS/EDTA buffer as well as for suc-AAPF-pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMS Incubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), sealing tape: Nunc (236366)
- the iEMS incubator/shaker (Thermo/Labsystems) was set at 29° C. Culture supernatants were diluted into plates containing unstress buffer to a concentration of ⁇ 25 ppm (master dilution plate). 20 ⁇ l of sample from the master dilution plate was added to plates containing 180 ⁇ l unstress buffer to give a final incubation concentration of 2.5 ppm. The contents were mixed and kept at room temperature and a AAPF assay was performed on this plate.
- the stain removal performance of the protease variants was determined in commercially available detergents. Heat inactivation of commercial detergent formulas serves to destroy the enzymatic activity of any protein components while retaining the properties of non-enzymatic components. Thus this method was suitable for preparing commercially purchased detergents for use in testing the enzyme variants of the present invention.
- Microswatches of 1 ⁇ 4′′ circular diameter were obtained from CFT. Single microswatches or two microswatches were placed vertically in each well of a 96-well MTP to expose the whole surface area (i.e., not flat on the bottom of the well).
- Microswatches containing blood milk and ink (BMI) of 0.25 inch circular diameter were obtained from CFT. Before cutting of the swatches, the fabric (EMPA 116) was washed with water. One microswatch was vertically placed in each well of a 96-well microliter plate in order to expose the whole surface area (i.e., not flat on the bottom of the well). The desired detergent solution was prepared as described herein. After equilibrating the Thermomixer at 25° C., 190 ⁇ l of detergent solution was added to each well of the MTP, containing microswatches. To this mixture, 10 ⁇ l of the diluted enzyme solution was added so that the final enzyme concentration was 1 ng/ml (determined from BCA assay).
- the MTP was sealed with tape and placed in the incubator for 30 minutes, with agitation at 1400 rpm. Following incubation under the appropriate conditions, 100 ⁇ l of the solution from each well was transferred into a fresh MTP. The new MTP containing 100 ⁇ l of solution/well was read at 405 nm using a MTP SpectraMax reader. Blank controls, as well as a control containing two microswatches and detergent but no enzyme were also included.
- the 96-well baked egg yolk substrate plates used in these assays were prepared from chicken egg yolks. Chicken egg yolks were separated from the whites, released from the membrane sac, and diluted 20% (vol/weight) with Milli-Q water. The diluted yolk was stirred for 15 min at room temperature using a magnetic stirrer. Five ⁇ L were carefully pipetted into the center of each well of a 96-well V-bottom plate (Costar #3894) using an 8-channel pipette. The plates were baked at 90° C. for 1 hour and cooled at room temperature. The baked egg yolk substrate plates were stored at room temperature and used within one week of preparation. Automatic dish detergents were prepared as described elsewhere in this document and pre-heated to 50° C.
- a 190 ⁇ L aliquot of detergent was added to each well of the 96-well plate using an 8-channel pipette.
- Ten ⁇ L of diluted enzyme was added to each well using a 96-channel pipetting device.
- the plate was carefully sealed with an adhesive foil sealer and incubated at 50° C. with shaking for 30 min.
- 120 ⁇ L of the reaction mixture was transferred to a new 96-well flat-bottom plate, and the absorbance/light scattering was determined at 405 nm.
- the absorbance/light scattering at 405 nm is proportional to egg yolk removal.
- the MTP was sealed with adhesive foil and placed in the incubator for 30 minutes, with agitation. Following incubation, 100 ⁇ l of the solution from each well was transferred into a fresh MTP. This MTP was read at 405 nm using a SpectraMax MTP reader. Blank controls, as well as controls containing microswatches and detergent but no enzyme were also included.
- This type of microswatch was pretreated using the fixation method described below.
- 8 liters of distilled water were mixed well with 80 ml of 30% hydrogen peroxide using a ladle.
- Forty pieces of EMPA 116 swatches were laid down in a fan-type distribution prior to being added into the solution to ensure uniform fixation.
- the swatches were swirled in the solution for a total of 30 minutes, continuously for the first five minutes and occasionally for the remaining 25 minutes.
- the solution was discarded and the swatches were rinsed 6 times with approximately 6 liters of distilled water per rinse. After the rinse the swatches were put on top of paper towels to dry.
- microswatch was pre-washed in deionised water for 20 minutes at ambient temperature. After the pre-washing step, the swatches were put on top of paper towels to dry. The air-dried swatches were then punched using a 1 ⁇ 4′′ circular die on an expulsion press. Finally two microswatches were put into each well of a 96-well MTP vertically to expose the whole surface area (i.e. not flat on the bottom of the well).
- the stain removal performance of reference serine proteases and variants therefrom on microswatches was determined on a MTP scale in commercially available detergent (Calgonit 5 int).
- the reagents used were: 5 mM HEPES, pH 8.0 or 5 mM MOPS, pH 7 buffer, 3:1 Ca:Mg for medium water hardness.
- the incubator was set at the desired temperature (16° C. or 32° C.). 10 ⁇ L samples from the master dilution plate of ⁇ 10 ppm enzyme was added to BMI 2-swatch plates with 190 ⁇ L working detergent solutions listed above. The volume was adjusted to give final concentration of 0.5 ppm for variants in the assay plates. The plates were immediately transferred to iEMS incubators and incubated for 30 minutes with 1400 rpm shaking at given temperature. Following incubation, 100 ⁇ L of supernatant was transferred into a new 96-well plate and the absorbance was measured in MTP Reader at 405 nm and/or 600 nm.
- Control wells containing 1 or 2 microswatches and detergent without the addition of protease samples were also included in the test.
- the measurement at 405 nm provides a higher value and tracks pigment removal, while the measurement at 600 nm tracks turbidity and cleaning.
- the absorbance value obtained was corrected for the blank value (substrate without enzyme), providing a measure of hydrolytic activity.
- the performance index was calculated.
- the performance index compares the performance of the variant (actual value) and the standard enzyme (theoretical value) at the same protein concentration.
- the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard enzyme.
- Samples of reference serine proteases variants thereof were obtained from filtered culture broth of cultures grown in MTP plates.
- the equipment used was a Biomek FX Robot (Beckman Coulter), a SpectraMAX MTP Reader (type 340; Molecular Devices), an iEMS incubator/shaker (Thermo/Labsystems); F-bottom MTPs (Costar type 9017 used for reading reaction plates after incubation); and V-bottom MTPs (Greiner 651101 used for pre-dilution of supernatant).
- the proteases hydrolyze the substrate and liberate pigment and insoluble particles from the substrate.
- the rate of turbidity is a measure of enzyme activity.
- suc-AAPF-pNA suc-AAPF-pNA as a substrate, which enabled the comparison and ranking of the variants versus the wild-type or standard protease.
- the specific activity on the suc-AAPF-pNA substrate was determined by dividing the proteolytic activity by the measured TCA-values of each sample, using the assays described above. Using these values, the relative specific activity was calculated (specific activity of variant/specific activity of reference protease).
- the performance of the protease variants was tested under various automatic dishwashing conditions.
- the compositions of the dish detergents are shown in the following Tables. These detergents are commercially available from wfk Testmaterials (www.testgewebe.de/en/products/detergents/) and are referred to by their wfk Testmaterials designations. These detergents were obtained from the source without the presence of enzymes, to permit analysis of the protease variants.
- each of the stain types egg yolk, minced meat and egg, and egg with milk
- the protocols for preparation of each of the stain types are provided below. Before the individual soil types were applied to the test dishes, the dishes were thoroughly washed. This was particularly necessary, as residues of certain persistent stains may still be present on the dishes from previous tests. New dishes were also subjected to three thorough washes before being used for the first time in a test.
- the stainless steel sheets (10 ⁇ 15 cm; brushed on one side) used in these experiments were thoroughly washed at 95° C. in a laboratory dishwasher with a high-alkalinity commercial detergent (e.g., ECOLAB® detergent; Henkel) to provide sheets that were clean and grease-free. These sheets were deburred prior to their first use.
- the sheets were dried for 30 minutes at 80° C. in a thermal cabinet before being soiled with egg yolk.
- the surfaces to be brushed were not touched prior to soiling. Also, no water stains or fluff on the surfaces were permitted.
- the cooled sheets were weighed before soiling.
- the egg yolks were prepared by separating the yolks of approximately 10-11 eggs (200 g of egg yolk) from the whites. The yolks were stirred with a fork in a glass beaker to homogenize the yolk suspension. The yolks were then strained (approximately 0.5 mm mesh) to remove coarse particles and any egg shell fragments.
- a flat brush (2.5′′) was used to apply 2.0 ⁇ 0.1 g egg yolk suspension as uniformly as possible over an area of 140 cm 2 on the brushed sides of each of the stainless steel sheets, leaving an approximately 1 cm wide unsoiled rim (adhesive tape was used if needed).
- the soiled sheets were dried horizontally (to prevent formation of droplets on the edges of the sheets), at room temperature for 4 hours (max. 24 hr).
- the sheets were immersed for 30 seconds in boiling, demineralized water (using a holding device if necessary). Then the sheets were dried again for 30 min at 80° C. After drying and cooling, the sheets were weighed. After weighing, the sheets were left for at least 24 hrs (20° C., 40-60% relatively humidity) before submitting them to the wash test. In order to meet the testing requirements, only sheets with 1000 ⁇ 100 mg/140 cm 2 (egg yolk after denaturation) were used in the testing. After the wash tests were conducted, the sheets were dried for 30 min at 80° C. in the thermal cabinet and weighed again after cooling. The percent cleaning performance was determined by dividing the mg of egg yolk released upon washing by the mg of denatured egg yolk applied and multiplying by 100.
- dessert plates (Arzberg, 19 cm diameter, white, glazed porcelain) conforming to EN 50242, form 1495, No. 0219, were used.
- a total of 225 g lean pork and beef (50:50 ratio) was finely chopped and maintained cool.
- the mixture was twice run through a mincer. Temperatures above 35° C. were avoided.
- the 225 g of the minced meat was then mixed with 75 g of egg (white and yolk mixed together).
- the preparation was then frozen for up to three months at ⁇ 18° C., prior to use. If pork was not available, 100% beef was used, as these are interchangeable.
- the minced meat and egg mixture (300 g) was brought to room temperature and mixed with 80 ml demineralized water. The mixture was then homogenized for 2 min using a kitchen hand blender. A fork was used to spread 3 g of the minced meat/egg/water mixture on each white porcelain plate, leaving an approximately 2 cm wide unsoiled margin around the rim. The amount applied was 11.8 ⁇ 0.5 mg/cm 2 .
- the plates were dried for 2 hours at 120° C. in a preheated thermal cabinet. As soon as the plates were cooled, they were ready for use.
- the plates were sprayed with ninhydrin solution (prepared to 1% in ethanol) for better identification of the minced meat protein residues.
- ninhydrin solution prepared to 1% in ethanol
- the plates were heated for 10 min at 80° C. in the thermal cabinet. Evaluation of the washing performance was done by visually inspecting the color reactions of the minced meat residue with reference to the IKW photographic catalogue (IKW—The German Cosmetic, Toiletry, Perfumery and Detergent Association).
- the stainless steel sheets (10 ⁇ 15 cm; brushed on one side) used in these experiments were thoroughly washed at 95° C. in a laboratory dishwasher with a high-alkalinity commercial detergent to remove grease and clean the sheets.
- the sheets were polished dry with a cellulose cloth.
- the surfaces to be brushed were not touched prior to soiling. Also, no water stains or fluff on the surfaces were permitted.
- the sheets were placed in a thermal cabinet at 80° C., for 30 min. The cooled sheets were weighed before soiling.
- the egg yolks and whites of whole raw eggs (3-4 eggs; approximately 160 g/egg) were placed in a bowl and beaten with an egg whisk. Then, 50 ml semi-skimmed milk (1.5% fat, ultra-high-temperature, homogenized) were added to the mixture. The milk and egg were mixed without generating froth.
- a flat brush was used to uniformly distribute 1.0 ⁇ 0.1 g of the egg/milk mixture on the brushed side of the stainless steel sheets, using a balance to check the distribution. A margin of approximately 1.0 cm was left around the short sides of the sheets.
- the soiled sheets were dried horizontally (to prevent formation of droplets on the edges of the sheets), at room temperature for 4 hours (max. 24 hr).
- the sheets were then immersed for 30 seconds in boiling, demineralized water (using a holding device if necessary). Then the sheets were dried again for 30 min at 80° C. After drying and cooling the sheets were weighed. After weighing the sheets were left to sit for at least 24 hours (20° C., 40-60% relatively humidity) before submitting them to the wash test. In order to meet the testing requirements, only sheets with 190 ⁇ 10 mg egg yolk/milk were used.
- the sheets were dried for 30 min at 80° C., in the thermal cabinet, and weighed again after cooling.
- the percentage cleaning performance was determined by dividing the mg of egg/milk released upon washing by the mg of egg/milk applied and multiplying by 100.
- the washing tests were performed in an automatic dishwasher (Miele model G690SC), equipped with soiled dishes and stainless steel sheets, prepared as described above. A defined amount of the detergent was used. The temperature tested was 50° C. The water hardness was 21° GH (German hardness). As described above, after washing the plates soiled with minced meat were visually assessed using a photo rating scale of 0 to 10, wherein “0” designated a completely dirty plate and “10” designated a clean plate. These values correspond to the stain or soil removal (SR) capability of the enzyme-containing detergent. The washed stainless steel plates soiled with egg yolk or egg yolk/milk were analyzed gravimetrically to determine the amount of residual stain after washing. The variant proteases were tested at a level of between 0 and 30 mg/active protein per wash.
- Eglin c from the leech Hirudo medicinalis is a tight-binding protein inhibitor of subtilisins and ASP protease (Heinz et al., Biochemistry, 31: 8755-66, 1992), and can therefore be used to measure enzyme concentration, which in turn permits specific activity to be calculated. Briefly, one measures the amount of enzyme inhibition produced by several known concentrations of eglin c. From this information, the concentration of eglin c required for complete inhibition is calculated. This is equivalent to the enzyme concentration in the sample.
- Protease activity was measured using the chromogenic AAPF assay described above.
- the gene for eglin c was synthesized and expressed in E. coli by standard methods. Its properties and inhibitory potency were the same as eglin c purchased from Sigma.
- the concentration of an eglin c stock solution was determined by measuring the inhibition of a sample of Bacillus lentus subtilisin of known specific activity. Then the calibrated eglin c sample was used to determine the concentration and specific activity of subtilisin variants. These values were used to create normalized 96-well enzyme stock plates, where all of the variants were diluted to a common concentration.
- the performance index compares the performance of the variant (actual value) and the standard or reference protease (theoretical value) at the same protein concentration.
- the theoretical values can be calculated, using the parameters of the binding curve (i.e., Langmuir equation) of the standard protease.
- the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.
- Plasmid pJH101 corresponds to pJH101 (Ferrari et al., J Bacteriol, 154:1513-5 [1983]) with the following fragment inserted in the EcoRI/BamHI site:
- This fragment contains the BPN′ gene with a fusion signal sequence (containing the first eight amino acids of the aprE signal sequence of B. subtilis followed by the BPN′ signal sequence of B. amyloliquefaciens starting at the 9th amino acid).
- This fragment was used as a template for a PCR reaction employing the following primers: AK04-14: GtcctctgttaacTTACTGAGCTGCCGCCTGTAC (SEQ ID NO:4) annealing to the 3′ end of the BPN′ mature gene, introducing a HpaI site downstream of the translational stop codon; and AK04-21.1: TTATGCGAGgctagcaaaaggagagggtaaagagtgagaagc (SEQ ID NO:5) annealing to the 5′ end of the BPN′ signal sequence introducing a NheI site at the 5′ end. [01] The PCR fragment obtained from this reaction was cleaned over a Qiagen PCR cleaning kit using standard conditions.
- the pHPLT-BPN′ ligation mixture was transformed into B. subtilis ( ⁇ aprE, ⁇ nprE, oppA, ⁇ spoIIE, degUHy32, ⁇ amyE::(xylR,pxylA-comK) as described in WO 02/14490, incorporated herein by reference.
- Selective growth of B. subtilis transformants harboring the pHPLT-BPN′ vector See, FIG. 3B ) was performed in shake flasks containing 25 ml MBD medium (a MOPS based defined medium), with 20 mg/L neomycin.
- the pHPLT-BPN′ vector containing the BPN′ expression cassette served as template DNA.
- This vector contains a unique BglII restriction site, which was utilized in SEL construction.
- BPN′ SELs To construct BPN′ SELs, three PCR amplifications were performed: two mutagenesis PCRs to introduce the mutated codon of interest in the mature BPN′ DNA sequence and a third PCR to fuse the two mutagenesis PCRs in order to construct the pHPLT-BPN′ expression vector having the desired mutated codon in the mature BPN′ sequence.
- the method of mutagenesis was based on the codon-specific mutation approach.
- NPS specific designed triple DNA sequence
- each SEL began with two primary PCR amplifications using the pHPLT-BglII-FW primer and a specific BPN′ reverse mutagenesis primer, and for the second PCR amplification the pHPLT-BglII-RV primer and a specific BPN′ forward mutagenesis primer (equal BPN′ mature codon positions for the forward and reverse mutagenesis primers).
- the introduction of the mutations in the mature BPN′ sequence was performed using Finnzymes Phusion High-Fidelity DNA Polymerase (Catalog No. F-530L). All PCR amplifications were executed according to Finnzymes protocol supplied with the polymerase. The PCR conditions were as follows:
- PCR was completed using a MJ Research (Location) PTC-200 Peltier thermal cycler with the following PCR program: 30 seconds 98° C., 30 ⁇ (10 seconds 98° C., 20 seconds 55° C., 1 minute 72° C.) and 5 min 72° C.
- the reactions resulted in two fragments of approximately 2 to 3 kB in length, which had about 30 nucleotide base overlap around the BPN′ mature codon of interest. Fragments were fused in a third reaction using the two aforementioned fragments and the forward and reverse BglII primers.
- the fusion PCR reaction was carried out as follows:
- the PCR fusion program was as follows: 30 seconds 98° C., 30 ⁇ (10 seconds 98° C., 20 seconds 55° C., 2:05 minute 72° C.) and 5 min 72° C. using a MJ Research® PTC-200 Peltier thermal cycler.
- the amplified linear 4.8 Kb fragment was purified using the Qiagen® Qiaquick PCR purification kit (Catalog No. 28106) and digested with the BglII restriction enzyme to create cohesive ends on both sides of the fusion fragment.
- the digestion reactions contained:
- the ligation mixture was used to transform B. subtilis ( ⁇ aprE, ⁇ nprE, oppA, ⁇ spoIIE, degUHy32, ⁇ amyE::(xylR,pxylA-comK) as described in WO 02/14490, incorporated herein by reference.
- B. subtilis ⁇ aprE, ⁇ nprE, oppA, ⁇ spoIIE, degUHy32, ⁇ amyE::(xylR,pxylA-comK
- MOPS media containing neomycin and 1.25 g/L yeast extract
- the library numbers ranged from 1 to 275. Each number represents the codon of the mature bpn′ sequence that was randomly mutated.
- Each library contained a maximum of 19 BPN′ variants.
- Synthetic BPN′ multiple mutation libraries were produced by Geneart, Baseclear, and Woke.
- Each synthetic BPN′ multiple mutation library contained a mix of BPN′ genes in which two or more selected codons of the mature sequence were randomly replaced by specific DNA sequences.
- a BPN′ combinatorial library could be designed in such a way that codon 22 of the mature sequence would be randomly substituted for DNA triplets coding for Thr, Gln, Val or Tyr, codon 26 for Val, Gln, Asn or Tyr, codon 31 for Ile, His, Tyr, or Asn and codon 48 for Ala, Glu, His or Asp.
- the combinatorial library would contain a maximum of 256 BPN′ combinatorial variants.
- a typical BPN′ multiple mutation library could contain up to thousands of unique BPN′ variant genes.
- Multiple mutation library fragments having terminal AvaI and HindIII sites See e.g., wild type BPN′) were digested with AvaI and HindIII, gel-purified and cloned into the pHPLT vector by ligase reaction using Invitrogen® T4 DNA Ligase (Catalog No. 15224-025) as recommended by the manufacturer for general cloning of cohesive ends.
- the wild type BPN′AvaI/HindIII fragment See e.g., wild type BPN′.
- the library DNA (BPN′ library fragment mix cloned in pHPLT) was amplified using the TempliPhi kit (Amersham Catalog No. 25-6400).
- TempliPhi kit Amersham Catalog No. 25-6400.
- 1 ⁇ L of the ligation reaction mix was mixed with 5mL of sample buffer from the TempliPhi kit and heated for 3 minutes at 95° C. to denature the DNA.
- the reaction was placed on ice to cool for 2 minutes and then spun down briefly.
- 5mL of reaction buffer and 0.2mL of phi29 polymerase from the TempliPhi kit were added, and the reactions were incubated at 30° C. in an MJ Research PCR machine for 4 hours.
- the phi29 enzyme was heat inactivated by incubation at 65° C. for 10 minutes.
- TempliPhi amplification reaction product 0.1 ⁇ L of the TempliPhi amplification reaction product was mixed with 500 ⁇ L of competent B. subtilis cells ( ⁇ aprE, ⁇ nprE, oppA, ⁇ spoIIE, degUHy32, ⁇ amyE::(xylR,pxylA-comK) followed by vigorous shaking at 37° C. for 1 hour. After this time aliquots of 100 and 500 ⁇ L were plated on HI-agar plates containing 20 ppm neomycin and 0.5% skim milk.
- competent B. subtilis cells ⁇ aprE, ⁇ nprE, oppA, ⁇ spoIIE, degUHy32, ⁇ amyE::(xylR,pxylA-comK
- the BPN′ variant proteins were produced by growing the B. subtilis transformants in 96 well MTP at 37° C. for 68 hours in MBD medium (a MOPS based defined medium).
- MBD medium was made essentially as known in the art (See, Neidhardt et al., J. Bacteriol., 119: 736-747 [1974]), except that NH 4 Cl 2 , FeSO 4 , and CaCl 2 were omitted from the base medium, 3 mM K 2 HPO 4 was used, and the base medium was supplemented with 60 mM urea, 75 g/L glucose, and 1% soytone.
- micronutrients were made up as a 100 ⁇ stock solution containing in one liter, 400 mg FeSO 4 7H 2 O, 100 mg MnSO 4 .H 2 O, 100 mg ZnSO 4 7H 2 O, 50 mg CuCl 2 2H 2 O, 100 mg CoCl 2 6H 2 O, 100 mg NaMoO 4 2H 2 O, 100 mg Na 2 B 4 O 7 10H 2 O, 10 ml of 1M CaCl 2 , and 10 ml of 0.5 M sodium citrate.
- LAS stability was measured by determining the AAPF activity before and after incubation in the presence of LAS at elevated temperature (45° C.), using the methods described in Example 1.
- the results of the following tables are shown as relative stability values in which the stability of the variant BPN′ is compared to the wild-type BPN′ enzyme.
- the relative stability is the ratio of the variant residual activity to the wild-type residual activity. A value of greater than one indicates greater stability in the presence of LAS.
- Table 4-1 contains the relative stability values of BPN′ variants as compared to wild-type BPN′. As determined during development of the present invention, numerous BPN′ variants had demonstrably higher stability in the presence of LAS as compared to the wild-type BPN′. In particular in the LAS stability assay of 92 BPN′ sites examined, 40 sites (43%) were poor (deleterious) and 52 sites (57%) were good (beneficial). Moreover of the 1508 variants tested, 96 (6%) were superior (up), 196 (13%) were neutral (same), and 1216 (81%) were inferior (down).
- PI performance index
- Combinable mutations are mutations that can be combined to deliver proteins with appropriate Performance indices for one or more desired properties. Positions at which mutations occur are classed as follows: Non-restrictive positions have ⁇ 20% neutral mutations for at least one property; and Restrictive positions have ⁇ 20% neutral mutations for activity and stability.
- subtilisin to be engineered has an amino acid different from that of subtilisin BPN′ at a particular position, this data find use in identifying at least one substitution that will alter the desired properties by identifying the best choice substitution, including substitution to the BPN′ wild type amino acid.
- Table 6-1 provides Performance index values (Pi) for 5,004 variants of subtilisin BPN′ at 275 positions. Performance indices less than or equal to 0.05 were fixed to 0.05 and indicated in bold italics in the table. Also, for the stability measure, if the Performance index of activity in the stability assays was less than or equal to 0.05, the associated stability performance index was fixed to 0.05.
- subtilisin BPN′ variants which are Combinable Mutations for the 275 positions (2,907). These variants have Performance index values >0.5 for at least one property, and >0.05 for both properties.
- results of experiments conducted to determine cleaning performance, LAS stability, AAPF activity and protein content (tests of properties of interest) of BPN′, GG36 (GCI-P036) and variant proteases are compared.
- PI performance index
- PI classifications used herein include: Up mutations (PI>1.0); Neutral mutations (PI>0.5); Non-deleterious mutations (PI>0.05); and Deleterious mutations (PI ⁇ 0.05).
- Productive sites are those having at least one Up mutation for any property.
- Productive, non-restrictive sites are those having >20% neutral mutations (PI>0.5) and at least one Up mutation (PI>1.0) for any property tested (besides protein expression).
- PI>0.5 neutral mutations
- PI>1.0 Up mutation
- Table 7-1 the results for variants that meet the definition of a productive, non-restrictive site are shown as a percentage (%) of variants tested that meet the definition of an Up mutation (PI>1).
- Highly productive sites are those having ⁇ 20% Up mutations (PI>1) for at least one property other than protein expression (TCA assay).
- TCA assay TCA assay
- Table 7-2 the results for variants that meet the definition of a highly productive site are shown as a percentage (%) of variants tested that meet the definition of an Up mutation (PI>1).
- Restrictive sites are those having less than 20% neutral mutations for activity and stability.
- Table 7-3 the results for variants that meet the definition of a restrictive site are shown as a percentage (%) of variants evaluated that meet definition of a neutral mutation (PI>0.5).
- GG36 also referred to herein as B. lentos subtilisin
- the expression plasmid pAC-GG36ci was assembled using the GG36 codon-improved gene fused at the eighth codon of the aprE signal sequence under the control of the consensus aprE promoter and the BPN′ transcriptional terminator.
- bold and italicized font indicates the consensus aprE promoter
- standard font indicates the signal sequence
- underlined font indicates the pro sequence
- bold font indicates DNA that encodes the GG36 mature protease
- underlined italicized font indicates the BPN′ terminator.
- the coding region of the GG36 mature protease is flanked by KpnI and XhoI restriction sites for cloning purposes:
- the amino acid sequence of the GG36 precursor protein is provided below. In this sequence, bold indicates the mature GG36 protease:
- amino acid sequence of the mature GG36 protease (SEQ ID NO:562) was used as the basis for making the variant libraries described herein:
- the plasmid features as follows: On for B.
- subtilis origin of replication from pUB110
- CAT chloramphenicol resistance gene from pC194
- pMB1 origin origin of replication from pBR322
- bla beta-lactamase from pBR322
- Short aprE promoter consensus transcriptional promoter
- Signal Peptide signal peptide
- Pro Peptide GG36 pro region
- GG36ci Mature Peptide mature GG36 (replaced by the coding regions for each variant expressed in this study)
- BPN′ Terminator transcriptional terminator from subtilisin BPN′.
- B. amyloliquefaciens subtilisin BPN′-Y217L (commercially available as PURAFECT® PRIME subtilisin; also referred to as “FNA”) in B. subtilis are described.
- the expression plasmid pAC-FNAre was assembled using the BPN′-Y217L gene, fused at the eighth codon of the aprE signal sequence under the control of the consensus aprE promoter and BPN′ transcriptional terminator.
- bold and italicized font indicates the consensus aprE promoter
- standard font indicates the signal sequence
- underlined font indicates the pro sequence
- bold font indicates DNA that encodes the BPN′-Y217L mature protease
- underlined italicized font indicates the BPN′ terminator.
- the coding region of the BPN′-Y217L mature protease contains the KvnI and XhoI restriction sites for cloning purposes:
- subtilis origin of replication from pUB110
- CAT chloramphenicol resistance gene from pC194
- pMB1 origin origin of replication from pBR322
- bla beta-lactamase from pBR322
- Short aprE promoter consensus transcriptional promoter
- Signal Peptide signal peptide
- Pro Peptide BPN′-Y217L pro region
- BPN′ Terminator transcriptional terminator from subtilisin BPN′.
- This Example describes the methods used to express various recombinant enzymes of transformed B. subtilis.
- B. subtilis clones containing BPN′-Y217L or GG36 expression vectors were replicated with a steel 96-well replicator from glycerol stocks into 96-well culture plates (BD, 353075) containing 200 ⁇ l of LB media +25 ⁇ g/ml chloramphenicol, grown overnight at 37° C., 220 rpm in a humidified enclosure. A 200 ⁇ l aliquot from the overnight culture was used to inoculate 2000 ⁇ l defined media +25 ⁇ g/ml chloramphenicol in 5 ml plastic culture tubes.
- the cultivation media was an enriched semi-defined media based on MOPS buffer, with urea as major nitrogen source, glucose as the main carbon source, and supplemented with 1% soytone for robust cell growth.
- Culture tubes were incubated at 37° C., 220 rpm, for 60 hours. Following this incubation, the culture broths were centrifuged at greater than 8000 ⁇ RCF. The supernatant solution was decanted into 15 ml polypropylene conical tubes for storage. No further purification or concentration was performed. Supernatant stocks were formulated to 40% propylene glycol final concentration for long-term stability and stored at 4° C.
- This Example describes the production of enzyme charge ladders and combinatorial charge libraries.
- Exemplary protease charge ladder variants are shown in the following tables and assayed as described herein. In these tables, the charge change is relative to the wild-type enzyme.
- BPN′-Y217L Charge Ladder Variants BPN′-Y217L Variant (BPN′ numbering) Charge Change S87D-N109D-S188D-S248D ⁇ 4 S87D-N109D-S188D ⁇ 3 S87D-N109D ⁇ 2 N109D ⁇ 1 (BPN′-Y217L) 0 N109R +1 S87R-N109R +2 S87R-N109R-S188R +3 S87R-N109R-S188R-S248R +4
- GG36 Charge Ladder Variants GG36 Variant GG36 Variant Charge (GG36 numbering) (BPN′ numbering) Change S85D-Q107D-S182D-N242D S87D-Q109D-S188D-N248D ⁇ 4 S85D-Q107D-S182D S87D-Q109D-S188D ⁇ 3 S85D-Q107D S87D-Q109D ⁇ 2 Q107D Q109D ⁇ 1 (GG36) (GG36) 0 Q107R Q109R +1 S85R-Q107R S87R-Q109R +2 S85R-Q107R-S182R S87R-Q109R-S188R +3 S85R-Q107R-S182R-N242R S87R-Q109R-S188R-N248R +4
- the pAC-GG36ci plasmid containing the codon-improved GG36 gene was sent to DNA 2.0, for the generation of combinatorial charge libraries (CCL). They were also provided with the Bacillus subtilis strain (genotype: ⁇ aprE, ⁇ nprE, ⁇ spoIIE, amyE::xylRPxylAcomK-phleo) for transformations. In addition a request was made to DNA2.0 Inc. for the generation of positional libraries at each of the four sites in GG36 protease that are shown in Table 10-3. Variants were supplied as glycerol stocks in 96-well plates.
- the GG36 CCL was designed by identifying four well-distributed, surface-exposed, uncharged polar amino-acid residues outside the active site. These residues are Ser-85, Gln-107, Ser-182, and Asn-242 (residues 87, 109, 188, and 248 in BPN′ numbering).
- An 81-member combinatorial library (G-1 to G-81) was created by making all combinations of three possibilities at each site: wild-type, arginine, or aspartic acid.
- the pAC-FNAre plasmid containing the BPN′-Y217L gene was sent to DNA 2.0 Inc. (Menlo Park, Calif.) for the generation of CCL. They were also provided with the Bacillus subtilis strain (genotype: ⁇ aprE, ⁇ nprE, ⁇ spoIIE, amyE::xylRPxylAcomK-phleo) for transformations. A request was made to DNA 2.0 Inc. for the generation of positional libraries at each of the four BPN′-Y217L protease sites that are shown in Table 10-4. Variants were supplied as glycerol stocks in 96-well plates.
- subtilisin BPN′-Y217L combinatorial charge library was designed by identifying four well-distributed, surface-exposed, uncharged polar amino-acid residues outside the active site. These residues are Ser-87, Asn-109, Ser-188, and Ser-248.
- An 81-member combinatorial library (F-1 to F-81) was created by making all combinations of three possibilities at each site: wild-type, arginine, or aspartic acid.
- This Example describes the testing of BPN′-Y217L and GG36 variants in BMI microswatch and baked egg assays in detergents representing various market geographies (e.g., differing pH, T, and/or water hardness), in both laundry and automatic dishwashing applications, as described in Example 1.
- FIGS. 2A and 2B shows an optimum charge for BPN′-Y217L and GG36 respectively, in cleaning performance under North American laundry conditions using off-the-shelf, heat-inactivated TIDE® 2 ⁇ detergent.
- the left Y-axes shows microswatch cleaning performance, where a higher number indicates superior BMI stain removal.
- the right Y-axes shows the performance index defined as cleaning performance of variants (filled symbols) relative to the parent molecule (unfilled symbols).
- the horizontal lines indicate a performance index at either 2 or 3 standard deviations above the noise of the assay.
- the BPN′-Y217L charge combinatorial library exhibits a charge optimum at zero charge changes with respect to the parent BPN′-Y217L while the GG36 CCL exhibits an optimum at negative two charges relative to the GG36 parent.
- FIGS. 3A , 3 B, 4 A, 4 B, 5 A and 5 B demonstrate that the location of the charge optimum is a function of the solution environment determined by detergent formulation, pH, temperature and ionic strength due to water hardness and detergent concentration. For instance the charge optimum for BPN′-Y217L CCL shifts dramatically from zero under North American laundry conditions to more positive charges under Western European and Japanese conditions. Moreover the charge optimum is observed for both liquid and granular (powder) laundry detergent formulations.
- protease charge variants e.g., GG36, BPN′-Y217L, etc
- Final conductivity is a measure of ionic strength and is due to water hardness, detergent concentration and composition.
- cleaning performance of charge variants is well correlated provided pH and conductivity are the same.
- This Example describes determining the relationship between protein charge and stability in a reaction medium containing one or both of an anionic surfactant and a chelant.
- an anionic surfactant and a chelant For the determination of protease activity of the stressed and unstressed samples, the suc-AAPF-pNA assay was used.
- the BODIPY-starch assay was used. Residual LAS and EDTA from the stress plates do not affect the suc-AAPF-pNA or BODIPY-starch assays.
- control buffer 50 mM HEPES, 0.005% Tween-80, pH 8.0
- stress buffer 50 mM HEPES, 0.1% (w/v) LAS (dodecylbenzene-sulfonate, sodium salt, Sigma D-2525), 10 mM EDTA, pH 8.0.
- Enzyme variants (20 ppm) were diluted 1:20 into 96-well non-binding flat-bottom plate containing either control or stress buffer and mixed. The control plate was incubated at room temperature while the stress plate was immediately placed at 37° C. for 30-60 min (depending on the stability of the enzyme being tested). Following incubation, enzyme activity was measured using suc-AAPF-pNA assay for proteases. The fraction of remaining or residual activity was equal to the reaction rate of the stressed sample divided by the reaction rate of the control sample. The parent enzymes and variants were found to be stable for 60 min in the control buffer.
- FIG. 6 depicts LAS/EDTA stability as a function of net charge change relative to parent BPN′-Y217L, for a library containing 80 variants.
- This library was designed and constructed according to the methods described in Example 2, to span several net charges relative to the parent BPN′-Y217L molecule.
- accumulation of negative charges (up to ⁇ 4) relative to parent BPN′-Y217L are beneficial for combined LAS/chelant stability. This is an example of optimizing a protein physical property, in this case net charge, for improving protein stability in a complex liquid laundry environment.
- BPN′ multiple mutation libraries were produced by Geneart or DNA 2.0, as described previously herein, using BPN′ as the parent protein.
- the BPN′ variant proteins were also produced as described earlier. Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
- the stain removal performance of the variants was tested in laundry applications on EMPA 116 swatches (BMI stain, CFT) at pH 8/16° C., pH 7/16° C. and pH 8/32° C. using methods described in Example 1, with the following modifications.
- the test detergent used was heat inactivated TIDE® 2 ⁇ Cold detergent (Procter & Gamble).
- Heat inactivation of commercial detergent formulas serves to destroy the endogenous enzymatic activity of any protein components while retaining the properties of nonenzymatic components.
- Heat inactivation of the detergents was performed by placing pre-weighed amounts of liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The detergent was purchased from local supermarket stores. Both unheated and heated detergents were assayed within 5 minutes of dissolving the detergent, in order to accurately determine percentage deactivated. Enzyme activity was tested by AAPF assay. Functionality of BPN′ variants was quantified as a performance index (Pi) (i.e., the ratio of performance of a variant relative to parent BPN′). Results are shown in Table 13-1.
- BPN′ variants showing a Pi value greater than or equal to 0.5 for one or more BMI stain removal performance tests and/or TCA precipitation showed improved cleaning benefits and/or expression.
- Performance indices less than or equal to 0.05 were fixed to 0.05 and indicated in bold italics in the table.
- “ND” indicates “not determined”
- BPN′ As the parent protein, two site variants were generated at positions 217 and 222 (BPN′ numbering) using fusion PCR.
- the BPN′ variant proteins were produced as described before. Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
- the stain removal performance of the variants was tested in laundry applications on EMPA 116 swatches (BMI stain, CFT) at pH 8/16° C. in heat-inactivated TIDE® 2 ⁇ Cold (Procter & Gamble) using methods described in Example 1.
- Functionality of BPN′ variants was quantified as a performance index (Pi) (i.e., the ratio of performance of a variant relative to BPN′-Y217L. Results are shown in Table 14-1.
- Bi performance index
- the mature serine protease enzyme derived from Cellulomonas strain 69B4 (DSM 983316035) is 189 amino acids long, with a catalytic triad consisting of His32, Asp56, and Ser137, as shown below (with the catalytic triad indicated in bold and underline):
- the multiple mutation library was constructed as outlined in the Stratagene QCMS kit, with the exception of the primer concentration used in the reactions. Specifically, 1 ⁇ L, of the methylated, purified pUC18-ASP plasmid (about 70 ng) was mixed with 15 ⁇ L of sterile distilled water, 1.5 ⁇ L of dNTP, 2.5 ⁇ L of 10 ⁇ buffer, 1 ⁇ L of the enzyme blend and 1.0 ⁇ L mutant primer mix (for a total of 100 ⁇ mol of primers).
- the primer mix was prepared using 10 ⁇ L of each of the eighteen mutant primers (100 pmol/ ⁇ L); adding 50 ng of each primer for the library as recommended by Stratagene, resulted in fewer mutations in a previous round of mutagenesis.
- the protocol was modified in the present round of mutagenesis to include a total of 100 pmol of primers in each reaction.
- the cycling conditions were 95° C. for 1 min, followed by 30 cycles of 95° C. for 1 min, 55° C. for 1 min, and 65° C. for 12 min, in an MJ Research PTC2-200 thermocycler using thin-walled 0.2 mL PCR tubes.
- the reaction product was digested with 1 ⁇ L of DpnI from the QCMS kit by incubating at 37° C. overnight. An additional 0.5 ⁇ L of DpnI was added, and the reaction was incubated for 1 hour.
- the library DNA (mutagenized single stranded pUC18-ASP product) was electroporated into electrocompetent E. coli cells (Invitrogen®, Catalog No. C4040-52, One Shot® TOP10 ElectrocompTM E. coli , dam+) and growth of transformed cells was selected on agar plates containing 100 mg/L ampicillin resulting in the ASP multiple mutation library in E. coli cells. Colonies (tens of thousands) were harvested and the Qiagen spin miniprep DNA kit (Catalog No. 27106) was used for preparing the plasmid DNA by the steps outlined by the manufacturer. The miniprep DNA was eluted with 50 ⁇ L of Qiagen buffer EB provided in the kit.
- Miniprep DNA was digested using the PstI and HindIII DNA restriction enzymes.
- the ASP library fragment mix (PstI x HindIII) was gel purified and cloned in the 4154 basepair HindIII x PstI pHPLT vector fragment by a ligase reaction using Invitrogen® T4 DNA Ligase (Catalog No. 15224-025) as recommended by the manufacturer for cloning cohesive ends).
- synthetic ASP library fragments were produced by GeneArt. These ASP library fragments were also digested with PstI and HindIII, purified and cloned in the 4154 basepair HindIII x PstI pHPLT vector fragment by a ligase reaction.
- the library DNA (ASP library fragment mix cloned in pHPLT) was amplified using the TempliPhi kit (Amersham Catalog No. 25-6400).
- 1 ⁇ L of the ligation reaction mix was mixed with 5mL of sample buffer from the TempliPhi kit and heated for 3 minutes at 95° C. to denature the DNA.
- the reaction was placed on ice to cool for 2 minutes and then spun down briefly.
- 5 ⁇ L of reaction buffer and 0.2 ⁇ L of phi29 polymerase from the TempliPhi kit were added, and the reactions were incubated at 30° C. in an MJ Research PCR machine for 4 hours.
- the phi29 enzyme was heat inactivated in the reactions by incubation at 65° C. for 10 min in the PCR machine.
- 0.1 ⁇ L of the TempliPhi amplification reaction product was mixed with 500 ⁇ L of competent B. subtilis cells ( ⁇ aprE, ⁇ nprE, oppA, ⁇ spoIIE, degUHy32, ⁇ amyE::(xylR,pxylA-comK) followed by vigorous shaking at 37° C. for 1 hour and 100 and 500 ⁇ L was plated on HI-agar plates containing 20 ppm neomycin sulfate (Sigma, Catalog No. N-1876; contains 732 ⁇ g neomycin per mg) and 0.5% skim milk Ninety-five clones from the library were picked for sequencing.
- the mutagenesis worked well, in that only 14% of the clones were equal to the backbone (parent) sequence (ASP with R0141-A064K-T086K-T116E-R123F), and about 3% of clones had extra mutations. The remaining sequenced clones (72%) were all mutants and of these about 94% were unique mutants.
- the sequencing results for the library are provided below in Table 16-1.
- the variant enzymes were produced as described herein and within U.S. patent application Ser. Nos. 10/576,331, 10/581,014, 11/581,102, and 11/583,334, all of which are incorporated by reference in their entirety.
- the tables below provide pair-wise comparisons of the numbers of variants with more than 5% wt activity and less than 5% activity for each of two properties, along with correlation coefficients for the two properties.
- the assay systems used in this Example are also provided in the above referenced applications.
- the properties used herein were casein activity (CAS), keratin activity (KER), AAPF activity (AAPF), LAS stability (LAS) and thermal stability for ASP; and peracid formation (PAF) and peracid degradation (PAD) for ACT.
- keratin Prior to the incubations, keratin was sieved on a 100 ⁇ m sieve in small portions at a time. Then, 10 g of the ⁇ 100 ⁇ m keratin was stirred in detergent solution for at least 20 minutes at room temperature with regular adjustment of the pH to 8.2. Finally, the suspension was centrifuged for 20 minutes at room temperature (Sorvall, GSA rotor, 13,000 rpm). This procedure was then repeated. Finally, the wet sediment was suspended in detergent to a total volume of 200 ml, and the suspension was kept stirred during pipetting.
- MTPs Prior to incubation, MTPs were filled with 200 ⁇ l substrate per well with a Biohit multichannel pipette and 1200 ⁇ l tip (6 dispenses of 200 ⁇ l and dispensed as fast as possible to avoid settling of keratin in the tips). Then, 10 ⁇ l of the filtered culture was added to the substrate containing MTPs. The plates were covered with tape, placed in an incubator and incubated at 20° C. for 3 hours at 350 rpm (New Brunswick Innova 4330). Following incubation, the plates were centrifuged for 3 minutes at 3000 rpm (Sigma 6K 15 centrifuge). About 15 minutes before removal of the first plate from the incubator, the TNBS reagent was prepared by mixing 1 ml TNBS solution per 50 ml of reagent A.
- MTPs were filled with 60 ⁇ l TNBS reagent A per well. From the incubated plates, 10 ⁇ l was transferred to the MTPs with TNBS reagent A. The plates were covered with tape and shaken for 20 minutes in a bench shaker (BMG Thermostar) at room temperature and 500 rpm. Finally, 200 ⁇ l of reagent B was added to the wells, mixed for 1 minute on a shaker, and the absorbance at 405 nm was measured with the MTP-reader.
- the obtained absorbance value was corrected for the blank value (substrate without enzyme).
- the resulting absorbance provides a measure for the hydrolytic activity.
- the performance index was calculated.
- the performance index compares the performance of the variant (actual value) and the standard enzyme (theoretical value) at the same protein concentration.
- the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard enzyme.
- the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.
- the TNBS reagent was prepared by mixing 1 ml TNBS solution per 50 ml of reagent A. MTPs were filled with 60 ⁇ l TNBS reagent A per well. The incubated plates were shaken for a few seconds, after which 10 ⁇ l were transferred to the MTPs with TNBS reagent A. The plates were covered with tape and shaken for 20 minutes in a bench shaker (BMG Thermostar) at room temperature and 500 rpm. Finally, 200 ⁇ l of reagent B were added to the wells, mixed for 1 minute on a shaker, and the absorbance at 405 nm was determined using an MTP reader.
- BMG Thermostar bench shaker
- the obtained absorbance value was corrected for the blank value (substrate without enzyme).
- the resulting absorbance is a measure for the hydrolytic activity.
- the (arbitrary) specific activity of a sample was calculated by dividing the absorbance and the determined protein concentration.
- This assay is based on the dimethylcasein (DMC) hydrolysis, before and after heating of the buffered culture supernatant.
- DMC dimethylcasein
- the filtered culture supernatants were diluted to 20 ppm in PIPES buffer (based on the concentration of the controls in the growth plates). Then, 50 ⁇ l of each diluted supernatant were placed in the empty wells of a MTP. The MTP plate was incubated in an iEMS incubator/shaker HT (Thermo Labsystems) for 90 minutes at 60° C. and 400 rpm. The plates were cooled on ice for 5 minutes. Then, 10 ⁇ l of the solution was added to a fresh MTP containing 200 ⁇ l DMC substrate/well. This MTP was covered with tape, shaken for a few seconds and placed in an oven at 37° C. for 2 hours without agitation. The same detection method as used for the DMC hydrolysis assay was employed.
- the residual activity of a sample was expressed as the ratio of the final absorbance and the initial absorbance, both corrected for blanks.
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