US20150056682A1 - Compositions and Methods Comprising Variant Microbial Proteases - Google Patents

Compositions and Methods Comprising Variant Microbial Proteases Download PDF

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US20150056682A1
US20150056682A1 US14/537,776 US201414537776A US2015056682A1 US 20150056682 A1 US20150056682 A1 US 20150056682A1 US 201414537776 A US201414537776 A US 201414537776A US 2015056682 A1 US2015056682 A1 US 2015056682A1
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Prior art keywords
sequence
subtilisin
cleaning
cleaning composition
protease
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US14/537,776
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Viktor Yuryevich Alekseyev
Luis G. Cascao-Pereira
James T. Kellis, Jr.
Alexander Pisarchik
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Procter and Gamble Co
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Danisco US Inc
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Priority to US14/537,776 priority Critical patent/US20150056682A1/en
Assigned to DANISCO US INC. reassignment DANISCO US INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ALEKSEYEV, VIKTOR YURYEVICH, KELLIS, JR., JAMES T., CASCAO-PEREIRA, LUIS G., PISARCHIK, ALEXANDER
Publication of US20150056682A1 publication Critical patent/US20150056682A1/en
Priority to US14/635,832 priority patent/US20150247140A1/en
Assigned to THE PROCTER & GAMBLE COMPANY reassignment THE PROCTER & GAMBLE COMPANY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DANISCO US INC.
Priority to US15/677,239 priority patent/US20180223269A1/en
Priority to US16/707,088 priority patent/US20200190497A1/en
Abandoned legal-status Critical Current

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    • 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 variant subtilisins and compositions comprising at least one variant subtilisin set forth herein, as well as methods for using these variants and compositions.
  • 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.
  • the present invention provides variant subtilisins and compositions comprising at least one variant subtilisin set forth herein, as well as methods for using these variants and compositions.
  • the present invention provides variant proteases suitable for laundry cleaning applications.
  • subtilisin variants comprising at least one modification, wherein the at least one modification is selected from G100C, S101D, S101H, Q103D, M124G, L126G, S132H, S161N, Q275Z, and/or at least one set of modifications selected from A001T/L096V/G097S/A098D/D099I, A001V/G097S/A098R/D099G, V004M/A098D/D099G, V004M/D099R/G100S/Z100.01L, V004M/G127C/G128S/P129R, V004M/M119C/D120S, V008I/A098G/D099R, V008I/A098N, V008I/M119C/D120G, Q010L/G097R/A098C/D099S, K012N/A098R/D099V
  • the present invention also provides subtilisin variants comprising at least one set of modifications, wherein said set of modifications is selected from: V072G/G097A/G128A/Y217Q, G097A/G128S/Y217Q, G097A/G128S/P129D/Y217Q, G097A/G128A/A134T/Y217Q, M124V/L126A/Y217Q, G097A/G128A/P129D/Y217Q/A232P, G097A/G128A/P129D/Y217Q, G097A/G128S/P129V/A134T/Y217Q, G097A/G128S/P129V/Y217Q, V068I/G097A/G128S/P129D/Y217Q, G097A/S101D/G128S/S204F/Y217Q/S236F/T254
  • the present invention further provides subtilisin variants comprising at one set of modifications, wherein said set of modifications is selected from: G097A/G128S/P129T/Y217Q, G097A/G128A/P129S/Y217Q, G097A/G128S/P129C/Y217Q, G097A/G128S/P129Q/Y217Q, G097A/G128S/P129E/Y217Q, G097A/P129S/Y217Q, G097A/G128S/P129A/Y217Q, G097A/G128S/P129K/Y217Q, G097A/P129N/Y217Q, G097A/P129E/Y217Q, G097A/G128S/P129K/G131S/Y217Q, G097A/G128S/P129R/Y217Q, G097A/G128S
  • the present invention also provides subtilisin variants comprising at least one set of modifications, wherein said set of modifications is selected from: G097A/G128A/Y217Q/M222Q, M124V/L126A/P129V/Y217Q, G128A/P129V/Y217Q, G097A/G128A/P129V/Y217Q, N123G/P129V/Y217Q, V30I/Z096.01D/G097A/A098R/G128A/Y217Q, Z096.01D/A098R/G128A/Y217Q, Z096.01D/A098R/M124V/L126A/Y217Q, G097A/Z098.01S/G128A/Y217Q, Z096.01D/A098R/N123G/Y217Q, Z096.01D/G097A/A098R/G128A/Y217Q/S
  • the present invention also provides subtilisin variants comprising the substitution Y217L and further at least one set of any of the modification sets provided above, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
  • the present invention further provides subtilisin variants comprising the substitutions G97A/G128A/Y217Q and further comprising at least one modification of any of the modifications sets provided herein, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
  • the present invention also provides cleaning compositions comprising at least one subtilisin variant as provided herein.
  • the cleaning composition is a laundry detergent.
  • the laundry detergent is a heavy duty liquid laundry detergent.
  • the cleaning composition is a dish detergent.
  • the cleaning compositions further comprise one or more additional enzymes or enzyme derivatives selected from the group consisting of 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.
  • additional enzymes or enzyme derivatives selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectina
  • the cleaning compositions further comprise at least one stabilizing agent.
  • the cleaning compositions comprise at least 0.0001 weight percent of at least one of any of the subtilisin variants provided herein, and optionally, at least one suitable adjunct ingredient.
  • the present invention provides methods of cleaning, the method comprising the steps of: contacting a surface and/or an article comprising a fabric with at least one of the cleaning compositions set forth herein and optionally washing and/or rinsing the surface or article.
  • the present invention also provides animal feeds comprising at least one subtilisin variant provided herein, as well as food processing compositions comprising at least one subtilisin variant provided herein.
  • FIG. 1 shows a diagram illustrating the method used for creating in-frame deletions and insertions. Library was designed to have 33% of clones with substitutions; 33% of clones with insertions and 33% of clones with deletions.
  • FIG. 2A Plasmid map of pAC-FNA10.
  • FIG. 2B provides a map of pHPLT-FNA.
  • FIG. 2C provides a map of pHPLT-BPN.
  • Vector pHPLT contains the LAT promoter (PLAT), a sequence encoding the LAT signal peptide (preLAT).
  • preLAT LAT signal peptide
  • the origin of replication and neoR are derived from plasmid pUB110.
  • the present invention provides variant subtilisins and compositions comprising at least one variant subtilisin set forth herein, as well as methods for using these variants and compositions.
  • the modification of the precursor proteases of the invention to produce these variant subtilisins includes at least one substitution, at least one deletion, or at least one insertion.
  • the modification comprises a combination of mutations.
  • the modification includes a combination of at least one substitution and at least one deletion.
  • the modification includes a combination of at least one substitution and at least one insertion.
  • the modification includes a combination of at least one deletion and at least one insertion.
  • the modification includes a combination of at least one substitution, at least one deletion, and at least one insertion.
  • Suitable methods are known in the art for generating modified polynucleotide sequences of the present invention, including but not limited to site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.
  • the commonly used methods include DNA shuffling (See, Stemmer, Proc Natl Acad Sci USA. 25:10747-51 [1994]), methods based on non-homologous recombination of genes (e.g. ITCHY) (Ostermeier et al., Bioorg Med Chem.
  • the full-length parent polynucleotide is ligated into an appropriate expression plasmid, and the following mutagenesis method is used to facilitate the construction of the modified protease of the present invention, although other methods may be used.
  • the method is based on that described by Pisarchik et al. (Pisarchik et al., Prot. Eng. Des. Select., 20:257-265 [2007]).
  • an added advantage is provided, in that the restriction enzyme used herein cuts outside its recognition sequence, which allows digestion of practically any nucleotide sequence and precludes formation of a restriction site scar..
  • a naturally-occurring gene encoding the full-length protease is obtained and sequenced and scanned for one or more points at which it is desired to make a mutation (deletion, insertion, substitution, or a combination thereof) at one or more amino acids.
  • Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by primer extension in accord with generally known methods. Fragments to the left and to the right of the desired point(s) of mutation are amplified by PCR and to include the Eam1104I restriction site.
  • the left and right fragments are digested with Eam1104I to generate a plurality of fragments having complementary three base overhangs, which are then pooled and ligated to generate a library of modified sequences containing one or more mutations.
  • This method avoids the occurrence of frame-shift mutations.
  • this method simplifies the mutagenesis process because all of the oligonucleotides can be synthesized so as to have the same restriction site, and no synthetic linkers are necessary to create the restriction sites as is required by some other methods
  • the present invention provides variant proteases and compositions comprising at least one of the variant subtilisins set forth herein.
  • the present invention provides means to produce protease with various commercial applications where degradation or synthesis of polypeptides are desired, including cleaning compositions, as well as feed components, textile processing, leather finishing, grain processing, meat processing, food processing, preparation of protein hydrolysates, digestive aids, microbicidal compositions, bacteriostatic compositions, fungistatic compositions, and personal care products (e.g., oral care, hair care, and/or skin care).
  • the present invention further provides enzyme compositions have comparable or improved wash performance, as compared to presently used subtilisin proteases.
  • the cleaning composition of the present invention are advantageously employed for example, in laundry applications. Indeed, due to the unique advantages of increased effectiveness in lower temperature solutions, the enzymes of the present invention are ideally suited for laundry applications. Furthermore, 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 laundry 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 additive 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 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 laundry 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 2000ppm 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 to10 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.
  • Preferred commercially available 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 Verysch, 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. 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 l [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 l [1991]
  • 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.
  • 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 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 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 HC.
  • 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 least
  • 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 catalytic and auxiliary metal cations, particularly ethylenediaminetetraace
  • 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.
  • 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).
  • ortholog and “orthologous genes” refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typically, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.
  • paralogous genes refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paralogs evolve new functions, even though some functions are often related to the original one. Examples of paralogous genes include, but are not limited to genes encoding trypsin, chymotrypsin, elastase, and thrombin, which are all serine proteinases and occur together within the same species.
  • 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 wild-type subtilisin protease. Additionally, analogous genes include at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity with the sequence of the wild-type subtilisin protease. Alternately, analogous sequences have an alignment of between about 70 to about 100% of the genes found in the B. amyloliquefaciens subtilisin protease region. In additional embodiments more than one of the above properties applies to the sequence. 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.
  • mature form of the BPN′ subtilisin protease of the present invention includes at least the amino acid sequence identical to residue positions 1-275 of SEQ ID NO:2, as set forth below:
  • 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 subtilisin 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.
  • multiple sequence alignment refers to the sequences of multiple homologs of a starting gene that are aligned using an algorithm (e.g., Clustal W).
  • 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.
  • 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. However, in some preferred embodiments, 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.
  • 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.
  • 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 (i.e., the “parent” sequence).
  • 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.
  • proteases that are similar to a wild-type subtilisin or a parent protease (e.g., subtilisin) used as a starting point in protein engineering. These variants are similar to the wild-type or other parent in their function, but have mutations in their amino acid sequence that make them different in sequence from the wild-type or parent protease (e.g., subtilisin).
  • 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).
  • 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, MAXAPEMTM, 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).
  • 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.
  • cleaning compositions and “cleaning formulations” refer to compositions that find use in the removal of undesired compounds from items to be cleaned, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes) etc.
  • the term encompasses any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, granule, or spray composition), as long as the composition is compatible with the perhydrolase and other enzyme(s) used in the composition.
  • the specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.
  • the terms further refer to any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any object and/or surface. It is intended that the terms include, but are not limited to detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents).
  • detergent compositions e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents).
  • cleaning composition includes unless otherwise indicated, granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries such as bleach additives and “stain-stick” or pre-treat types.
  • HDL heavy-duty liquid
  • cleaning and disinfecting agents including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and
  • detergent composition and “detergent formulation” are used in reference to mixtures which are intended for use in a wash medium for the cleaning of soiled objects.
  • the term is used in reference to laundering fabrics and/or garments (e.g., “laundry detergents”).
  • laundry detergents e.g., “laundry detergents”.
  • the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e.g., “dishwashing detergents”). It is not intended that the present invention be limited to any particular detergent formulation or composition.
  • the term encompasses detergents that contain surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and solubilizers.
  • 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.
  • subtilisin numbering corresponds to that of the mature BPN′ sequence, as set forth below:
  • BPN′ e.g., reference protease
  • BPN′ variants this assay was started using filtered culture supernatant from microtiter 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
  • 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 ml of diluted protease solution to each well, immediately followed by the addition of 190 ⁇ l 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.
  • 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 g/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) from 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 pl 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 microtiter 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.
  • 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).
  • 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.
  • the stain removal performance of reference serine proteases and variants therefrom on microswatches was determined on a MTP scale in commercially available heat-inactivated detergent.
  • 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 18 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.
  • 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 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 Langmuir equation of the standard protease.
  • the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.
  • a PCR-based method was used to create a library of modified B. amyloliquefaciens subtilisin BPN′-Y217L (commercially available as PURAFECT® PRIME subtilisin) polynucleotides containing in-frame insertions, deletions and/or substitutions in two regions of BPN′-Y217L (positions 63-77 and 92-132 of the mature region) as shown in FIG. 1 (See, Pisarchik et al., Prot. Eng. Des. Select., 20:257-265 [2007]).
  • Two sets of oligonucleotides that evenly cover the targeted regions of the BPN′-Y217L gene of a full-length protein of 392 amino acids, in both forward and reverse direction were used to amplify the 5′ and 3′ segments of the portion of the BPN′-Y217L gene.
  • the coding region of the BPN′-Y217L mature protease contains the Kpnl and Xhol restriction sites for cloning purposes:
  • Each oligonucleotide contained about 20-24 nucleotides necessary for annealing to the template, targeted codons (with or without degeneracy) and the Eam1104I recognition sequence. Every following primer was moved 3 by downstream until the end of the targeted region was reached.
  • Two PCR reactions (5′ and 3′ segments) contained either the 5′ forward or the 3′ reverse gene sequence flanking oligonucleotides each in combination with the corresponding oppositely priming oligonucleotides. The 5′ fragments were amplified from pAC-FNA10 plasmid (See, FIG.
  • Each amplification reaction contained 30 pmol of each oligonucleotide and 100 ng of pAC-FNA10 template DNA. Amplifications were carried out using Vent DNA polymerase (New England Biolabs). The PCR mix (20 ul) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15s, annealing at 55° C. for 15s and extension at 72° C. for 40s. Following amplification, left and right fragments generated by the PCR reactions were gel-purified and mixed (about 200 ng of each fragment). Every mix contained three left fragments targeting three adjacent codons and three right fragments targeting same codons.
  • the polynucleotide sequence of the expression cassette from the pHPLT vector (Plat promoter-pre-pro-BPN′-Y217L -terminator) (SEQ ID NO. 124) is shown below:
  • Ligation mixtures were amplified using rolling circle amplification according to manufacturer's recommendation (Epicentre). One ul of the ligation mixture was mixed with 5 ul of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 ul of the reaction buffer and 0.2 ul of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform Bacillus subtilis.
  • Bacillus subtilis cells made competent by the induction of the comK gene under control of a xylose inducible promoter (See e.g., Hahn et al., Mol Microbiol, 21:763-775 [1996]) were transformed with 45 libraries containing DNA sequences encoding BPN′-Y217L protease with mutated regions. The cells were grown in 1 ml of Luria Broth (LB) at 37° C.
  • LB Luria Broth
  • results of experiments conducted to determine stain removal activity are shown in Table 3-1.
  • the results were obtained using the methods described in Example 1, with the following modifications for the stain removal performance assay.
  • the test detergent used was heat inactivated Tide 2X 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 to accurately determine percentage deactivated. Enzyme activity was tested by AAPF assay.
  • PI performance index
  • BPN′-Y217L variants showing a PI value greater or equal than 0.5 for BMI stain removal performance and/or TCA precipitation showed improved cleaning benefits and/or expression.
  • Mutations are named by the one letter code for the parent amino acid, followed by a three digit position number and then the one letter code for the variant amino acid.
  • mutating glycine (G) at position 87 to serine (S) is represented as “G087S.”
  • Multiple mutations are indicated by inserting a “I” between the mutations.
  • Mutations at positions 87 and 90 are represented as “G087S/A090Y.”
  • the one letter code “Z” is used.
  • the one letter code “Z” is on the left side of the position number.
  • the one letter code “Z” is on the right side of the position number.
  • the position number is the position number before the inserted amino acid(s), plus 0.01 for each amino acid.
  • an insertion of three amino acids alanine (A), serine (S) and tyrosine (Y) between position 87 and 88 is shown as “Z087.01A/Z087.02S/Z087.03Y.”
  • Z087.01A/Z087.02S/Z087.03Y is shown as “Z087.01A/Z087.02S/Z087.03Y.”
  • Table 3-2 provides a list of the insertions that make up the combinations.
  • BPN′3 BPN′G97A-G128A-Y217Q
  • Fragment 1 was amplified by primers P4974 and P4977 (See, Table 4-1).
  • Fragment 4 was amplified by primers 4978 and P4976 (Table 4-1). Both fragment 1 and 4 did not contain any mutations.
  • Fragments 2 and 3 each were created as a set of 12 and 16 sequences respectively that contained the desired mutations, using either BPN′3 as template or in the absence of template using partially overlapping forward and reverse primers.
  • the set of Fragment 2 sequences were amplified by the following primers:
  • the set of Fragment 3 sequences were amplified by the following primers:
  • Each amplification reaction contained 30 pmol of each oligonucleotide and 100 ng of the template DNA. Amplifications were carried out using Vent DNA polymerase (New England Biolabs). The PCR mix (20 ul) was initially heated at 95° C. for 25 min followed by 30 cycles of denaturation at 94° C. for 15s, annealing at 55° C. for 15s and extension at 72° C. for 40s. Following amplification all the fragments were purified by the Gel Band Purification kit (Qiagen) and mixed to serve as templates for a fusion PCR using primers P4975 and P4948.
  • Vent DNA polymerase New England Biolabs
  • the full-length DNA fragment from the fusion PCR reaction was gel-purified by the QIAGEN gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated in the Bacillus expression plasmid pHPLT-BPN partial opt ( FIG. 2C ), which was cut with the same restriction enzymes.
  • Ligation mixtures were amplified using rolling circle amplification according to manufacturer's recommendation (Epicentre).
  • One ul of the ligation mixture was mixed with 5 ul of the sample buffer, heated to 95° C. for 3 min and cooled on ice.
  • 5 ul of the reaction buffer and 0.2 ul of the enzyme was added to each tube, followed by incubation at 30° C. for 10 hours.
  • Products of the rolling circle amplification were diluted 100 times and 1 ul of this dilution was used to transform Bacillus subtilis cells (genotype: AaprE, AnprE, AspoIIE, amyE::xylRPxylAcomK-phleo) as described in Example 2.
  • Transformations were plated on LB plates containing 1.6% skim milk and 10 ppm Neomycin and incubated overnight at 37° C. Only colonies with halos were picked and grown in microtiter plates for further analysis as described in Example 2. Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
  • BPN′ 3 The stain removal activity of combinatorial variants of BPN′ 3 generated as described in Example 4 was tested as described in Example 3.
  • functionality of BPN′ variants was quantified as a performance index (Pi), which is the ratio of performance of a variant to parent protein BPN′3.
  • Bi performance index
  • BPN′3 variants showing a Pi value greater or equal than 0.5 for BMI stain removal performance 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. Results are shown in Table 5-1.
  • Combinatorial libraries focused around positions 128/129/130 in BPN′G97A-G128A-Y217Q (BPN′3) were created using site specific saturation mutagenesis and/or site specific insertion and/or deletion.
  • the variants generated contain substitution, insertion, and deletion mutations.
  • Libraries L1, L2 and L3 are based on WT position S130S and L4 is based on S130A.
  • Four saturation libraries focused around positions 128/129 and 130 were constructed as shown in Table 6-1.
  • Mutagenesis was performed using methods known in the art (See, Amin et al., Biotechniques 35: 1134-1140, [2003]) using a modified version of the QuikChange multi site-directed mutagenesis (QCMS) kit from Stratagene.
  • QCMS QuikChange multi site-directed mutagenesis
  • the mutagenesis reaction contained 60 ng template plasmid, 0.5 ul forward primer 128/129 Stop F (25 uM), 0.5 ul reverse primer 128/129 Stop R (25 uM), 1 ul dNTP's (QCMS kit), 2.5 ul 10 ⁇ QCMS reaction buffer, 18.5 ul deionized water, and 1 ul of enzyme blend (QCMS kit), for a total volume of 25 ul.
  • the thermocycler program used was 1 cycle at 95° for 1 min., followed by 25 cycles of 95° C. for 1 min, 55° C. for 1 min., and 68° C. for 11 minutes (MJ Research thermocycler).
  • the template DNA was digested by DpnI (QCMS kit, Qiagen) and 1.5 uL of the reaction was amplified by rolling circle amplification (RCA) using the Templiphi kit (Amersham) using manufacturer's protocol.
  • One microliter of the amplified DNA was used to transform 100 ⁇ L of competent Bacillus subtilis cells (genotype: AaprE, AnprE, AspoIIE, amyE::xylRPxylAcomK-phleo). Aliquots of 20 uL or 80 uL of the transformation mixture were plated on Luria Agar plates supplemented with 10 ⁇ g/ml neomycin+1.6% skim milk (Teknova). After growth, 4 colonies not forming halos on the skim milk plates were picked to identify the plasmid with the correct sequence. The correct variant with the two stop codons at positions 128/129, called “pHPLT-BPN′ 128/129 Stop” was chosen as the parent plasmid for the construction of combinatorial libraries described in Table 6-1.
  • Plasmid pHPLT-BPN′ 128/129 Stop was methylated and used as template with the degenerate primer pairs shown in Table 6-3.
  • cultures were grown overnight in micro-filter plates (.22um, Millipore) containing 180 ul of a enriched semi-defined media based on MOPs buffer, with urea as major nitrogen source, glucose as the main carbon source, 1% soytone for robust cell growth, and supplemented with 2.5ug/ml neomycin. Cultures were grown for 64 hours at 37 C, 250rpm, and 70% humidity. Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
  • BPN′ 3 The stain removal activity of combinatorial variants of BPN′ 3 at positions 128, 129, and 130 generated as described in Example 6 was tested as described in Example 3.
  • functionality of BPN′ variants was quantified as a performance index (Pi), which is the ratio of performance of a variant to parent protein BPN′3.
  • Bi performance index
  • BPN′3 variants showing a Pi value greater or equal than 0.5 for BMI stain removal performance and/or TCA precipitation showed improved cleaning benefits and/or expression. Results are shown in Table 7-1.
  • BPN′ mutants as parent molecules—BPN′ G97A/G128A/Y217Q, BPN′ M124V/L126A/Y217Q, BPN′ G128A/Y217Q, and BPN′ N123G/Y217Q to create insertions or substitutions at positions 96, 98, 129 and/or 222.
  • the parent DNA molecule, mutations introduced and PCR primers used are listed in the Table 8-1.
  • two fragments 1 and 2 were generated by the PCR primers (Table 8-2) in the 5′ and 3′ regions of the gene using the respective parent molecule listed in Table 8-1. These fragments were mixed and amplified by the flanking primers P4973 and P4950 (Table 8-2) to restore the full-length gene.
  • Each amplification reaction contained 30pmol of each PCR primer and 100 ng of the template DNA. Amplifications were carried out using Vent DNA polymerase (New England Biolabs). The PCR mix (20 ul) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15s, annealing at 55° C. for 15s and extension at 72° C. for 40s. Following amplification, the 5′ and 3′ fragments were gel-purified by the QIAGEN gel-band purification kit, mixed and amplified by the flanking primers P4973 and P4950.
  • the full-length DNA fragment produced was gel-purified by the QIAGEN gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′ vector containing the respective parent molecules, which were also cut with the same restriction enzymes.
  • Ligation mixtures were amplified using rolling circle amplification according to manufacturer's recommendation (Epicentre).
  • One microliter of the ligation mixture was mixed with 5 ul of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 ul of the reaction buffer and 0.2 ul of the enzyme was added to each tube, followed by incubation at 30° C. for 10 hours.
  • Products of the rolling circle amplification were diluted 100 times and used to transform Bacillus subtilis cells (genotype: AaprE, ⁇ nprE, AspoIIE, amyE::xylRPxylAcomK-phleo) as described in Example 2. Transformations were plated on LB plates containing 1.6% skim milk and 10 ppm Neomycin and incubated overnight at 37° C. Only colonies with halos were picked and grown in microtiter plates for further analysis as described in Example 2 Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
  • Example 9 The stain removal activity of combinatorial variants of BPN′ generated as described in Example 8 was tested as described in Example 3.
  • functionality of BPN′ variants was quantified as a performance index (Pi), which is the ratio of performance of a variant to BPN′3 (BPN′ G97A-G128A-Y217Q).
  • Pi performance index
  • Variants showing a Pi value greater or equal than 0.5 for BMI stain removal performance 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. Results are shown in Table 9-1.

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Abstract

The present invention provides variant subtilisins and compositions comprising at least one variant subtilisin set forth herein, as well as methods for using these variants and compositions. In some embodiments, the present invention provides variant proteases suitable for laundry cleaning applications.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • The present application is a divisional application of U.S. patent application Ser. No. 14/470,709, filed Aug. 27, 2014, which is a continuation of U.S. patent application Ser. No. 12/996,576, filed Jun. 14, 2011, which is a U.S. National Phase Application of International Application No. PCT/US2009/046067, filed Jun. 3, 2009, which claims the benefit of U.S. Provisional Application No. 61/059,695, filed Jun. 6, 2008, all of which are hereby incorporated by reference in their entirety.
    • FIELD OF THE INVENTION
  • The present invention provides variant subtilisins and compositions comprising at least one variant subtilisin set forth herein, as well as methods for using these variants and compositions.
    • BACKGROUND OF THE INVENTION
  • 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.
    • SUMMARY OF THE INVENTION
  • The present invention provides variant subtilisins and compositions comprising at least one variant subtilisin set forth herein, as well as methods for using these variants and compositions. In particular, the present invention provides variant proteases suitable for laundry cleaning applications.
  • The present invention provides subtilisin variants comprising at least one modification, wherein the at least one modification is selected from G100C, S101D, S101H, Q103D, M124G, L126G, S132H, S161N, Q275Z, and/or at least one set of modifications selected from A001T/L096V/G097S/A098D/D099I, A001V/G097S/A098R/D099G, V004M/A098D/D099G, V004M/D099R/G100S/Z100.01L, V004M/G127C/G128S/P129R, V004M/M119C/D120S, V008I/A098G/D099R, V008I/A098N, V008I/M119C/D120G, Q010L/G097R/A098C/D099S, K012N/A098R/D099V, K012R/G097D/A098H/D099G, A013T/S101R/Q103S, A013V/G097N/A098H/D099G/G160S, A013V/G097S/Z097.01R/A098R, A013V/G100R/S101G, A013V/N118G/M119I, A013V/P129Z/S130H/S132G, P014L/N062D/S101R/Q103S, P014S/A098S/D099G, A015T/V068N/A069G, L016M/G128R/P129R/Z129.01G, H017Q/G127R/P129G, H017Y/A098R/D099I, H017Y/A098R/D099Z/S101G, H017Y/P129R, H017Y/S130Y/G131S/S132L, Q019H/G128R/Z128.01R/P129D, Q019H/Z128.01G/Z128.02G, G020D/G097R/A098G/D099C, G020D/P129Z/S130G/G131R/S132C, N025Y/S101L/Q103N, A029V/G097R/A098S, S033I/T066G/H067R/V068D/A216E, S038F/A098R/D099S, V044I/G097C/A098D/D099G, V044I/G097R/A098R, V044I/G100S/S101G, A045V/G097C/A098R/D099S, A048V/G097L/A098G/D099S, A048V/G100R/S101G, A048V/G128S/P129L/Z129.01G, M050I/A098G, S053F/S130I/S132G, S053T/D099H/S101Z, E054D/A098R/D099G, T055I/S101R/Q103R, T055S/T066I/Z066.01G/Z066.02G, Q059H/D099S/S101G, Q059H/M119S/D120R, Q059K/M119V/V121C, Q059L/P129G, N061D/G128R/P129V/Z129.01G, S063T/G097D/A098R/D099S, G065R/Z101.01R/Z101.02G, T066S/H067S, V068C/A069G, V068C/A069G/H238Q, V068G/A069G, V068G/A069G/V150I, V068G/A069S, V068I/A069G/A144V, V068I/G097D/A098G/D099Z/G100R, V068L/A069G, V068L/A069S, V068S/A069G, V068S/A069G/A116T, A069G/T071G, A069G/T071I, L075G/N076G/N077Z, L075R/N076G/N077Z, N076D/D099R/Z100.01G, I079F/A098G, V081A/S101R/Q103V, V081I/G097R/A098G/D099C, V084D/G097S/A098G/D099N, V084I/G127C/P129G, V084I/G127L, A085V/A098G, A085V/G097R/A098S/D099N, A085V/G128S/P129R/Z129.01G, A085V/N218I, A085V/S101G, A085V/S204P, A085V/Z099.01H/S101R, P086S/G131D/S132G, P086T/S101D/Q103S/V147I, A088S/S101R/Q103V, A088T/A098R/D099R, A088T/D099G/Z100.01G, A088T/G127N/P129R, A088T/G127S/P129R/Q206R/A273S, A088T/P129S/G146D, A088T/S101G, A088T/S130R/G131H/S132N, A088V/G100Z/S101C/Q103R, A088V/P129Z, A092G/S249R, A092G/V093C, A092G/V093I, A092G/V093L, A092L/V093G/K094Z, A092S/K094V, A092S/V093C, A092S/V093G, A092S/V093S, A092V/V093L, V093I/G128D/P129R, V093I/P129Z, V093R/Z093.01G/K094R, K094E/V095D, K094S/V148I, L096I/N118R/M119C/D120G/A151V, Z096.01D/A098R, Z096.01H/A098G, Z096.01N/A098H,Z096.01R/A098C, Z096.01R/A098G, Z096.01R/A098G/V192I, Z096.01R/A098R, Z096.01R/A098R/V192A, Z096.01R/A098S, Z096.01R/A098S/G157S, Z096.01R/Z096.02G, Z096.01R/Z096.02G/V147I, Z096.01S/A098G, Z096.01S/A098N, G097C/A098D/D099S, G097C/A098G/D099G, G097C/A098G/D099N, G097C/A098G/D099S, G097C/A098G/D099S/G100Z, G097C/A098G/D099V, G097C/A098N/D099S, G097C/A098R, G097C/A098R/D099G, G097C/A098R/D099H, G097C/A098R/D099H, G097C/A098R/D099N, G097C/A098R/D099R, G097C/A098R/D099R/N109D/A114S, G097C/A098S/D099H/A151V, G097C/A098V/D099S, G097C/Z097.01G/A098H/T253I, G097C/Z097.01S/A098G, G097D/A098C, G097D/A098G/D099G, G097D/A098G/D099R, G097D/A098H/D099R, G097D/A098H/D099V, G097D/A098R/D099G, G097D/A098R/D099R, G097D/A098R/D099S, G097D/A098R/D099Z, G097D/G100C/S101R, G097D/G100R/S101G, G097H/A098C/D099G, G097H/A098D/D099N, G097H/A098G, G097H/A098G/D099G, G097H/A098G/D099R, G097H/A098G/D099Z, G097H/A098G/D099Z/A151V, G097H/A098G/D099Z/G100R, G097H/A098G/D099Z/G100V, G097H/A098G/D099Z/S101N/G166S, G097H/A098R, G097H/A098R/D099G, G097H/A098R/D099L, G097H/A098R/D099N, G097H/A098S/D099G, G097H/A098S/D099I, G097H/A098S/D099L, G097H/A098S/D099S/K237E, G097L/A098C/D099S, G097L/A098G/D099S, G097L/A098G/D099S/G100Z, G097L/A098G/D099Z, G097L/A098H, G097L/A098R/D099N, G097L/A098V, G097N/A098G/D099C, G097N/A098G/D099R, G097N/A098G/D099R/V270I, G097N/A098G/D099S, G097N/A098G/D099V, G097N/A098G/D099V/A151V, G097N/A098G/D099V/S101F, G097N/A098H/D099N, G097N/A098R/D099R, G097N/A098R/D099S, G097N/A098R/D099V, G097N/A098S/D099S, G097R/A098C, G097R/A098C/D099S, G097R/A098F/A200V, G097R/A098G, G097R/A098G/D099C, G097R/A098G/D099G, G097R/A098G/D099G/A273T, G097R/A098G/D099S, G097R/A098G/D099S/A133D, G097R/A098G/D099V, G097R/A098H/D099C/S182N, G097R/A098H/D099G, G097R/A098N/D099G, G097R/A098N/D099Z, G097R/A098R, G097R/A098R/D099C, G097R/A098R/D099G, G097R/A098R/D099S, G097R/A098S/D099C, G097R/A098S/D099G, G097R/A098S/D099L, G097R/A098S/D099R, G097R/A098S/D099S, G097R/A098S/D099V, G097R/A098V/D099G, G097R/Z097.01H/A098C, G097R/Z097.01R/A098G, G097R/Z097.01R/A098G/V147I, G097R/Z097.01R/A098S, G097R/Z097.01R, G097R/Z097.01S/A098G, G097R/Z097.01S/A098R, G097S/A098C, G097S/A098C/D099G, G097S/A098C/D099H, G097S/A098C/D099L, G097S/A098C/D099R, G097S/A098C/D099S, G097S/A098C/D099Z, G097S/A098G/D099F, G097S/A098G/D099G, G097S/A098G/D099G, G097S/A098G/D099S, G097S/A098G/D099Z, G097S/A098G/D099Z/G100R, G097S/A098H/D099C, G097S/A098H/D099G, G097S/A098N/D099G, G097S/A098R, G097S/A098R/D099C, G097S/A098R/D099G, G097S/A098R/D099L, G097S/A098R/D099V, G097S/A098R/D099Y, G097S/A098S/D099G, G097S/A098S/D099G/A133V/A153V/S236Y, G097S/A098S/D099G/A153V, G097S/A098S/D099Z, G097S/Z097.01D/A098G/A144T/Q271H, G097S/Z097.01G/A098V, G097S/Z097.01S/A098G, G097S/Z097.01S/A098G/A273T, G097S/Z097.01Y, G097V/A098R, G097V/A098R/D099G, G097V/A098S/D099S, G097Y/A098H/D099N, G097Z/A098S/D099H, Z097.01D/A098R/D099G, Z097.01D/A098S/D099L, Z097.01D/A098V/D099G, Z097.01G, Z097.01G/A098G, Z097.01G/A098G/D099S, Z097.01G/A098R, Z097.01G/A098R/D099C, Z097.01G/A098R/D099G, Z097.01G/A098R/D099S, A098C/D099G, A098C/D099G/A116T, A098C/D099H, A098C/D099L, A098C/D099L/K213R, A098C/D099L, A098C/D099N, A098C/D099R, A098C/D099S, A098C/D099S/P194L, A098D/D099C, A098D/D099G, A098D/D099G/G100L, A098D/D099H/G100S, A098D/D099H/H238Y, A098D/D099R, A098D/D099R/G100D, A098D/D099R/G160S, A098F/D099S/G100L, A098G/D099C, A098G/D099G, A098G/D099G/A228T, A098G/D099G/G100H, A098G/D099G/N243D, A098G/D099G/S101P, A098G/D099G/T244S, A098G/D099G, A098G/D099H, A098G/D099L, A098G/D099R, A098G/D099R/A116T, A098G/D099R/G100D, A098G/D099R/V148I, A098G/D099S, A098G/D099S/L267M, A098G/D099V, A098G/D099Z, A098H/D099C, A098H/D099G, A098H/D099G/A216T, A098H/D099G/G100D, A098H/D099G/G100N/L267M, A098H/D099G/G100S, A098H/D099G/V143I, A098H/D099R, A098H/D099R/G100S, A098H/D099S/G100C, A098H/D099S/G100N, A098H/D099Y, A098I/D099G, A098I/D099S, A098L/D099G, A098L/D099S, A098N/D099G, A098N/D099L, A098N/D099S, A098R/A144T, A098R/A274V, A098R/D099C, A098R/D099G, A098R/D099G/G100C, A098R/D099G/G100D, A098R/D099G/G100L/S145T, A098R/D099G/G100N, A098R/D099G/G100R, A098R/D099G/G100S, A098R/D099G/V150I, A098R/D099H, A098R/D099I, A098R/D099L, A098R/D099N, A098R/D099N/G100L, A098R/D099R, A098R/D099R/G100D, A098R/D099R/S101Z, A098R/D099R/S130F, A098R/D099R, A098R/D099S, A098R/D099S/G100L/A153V, A098R/D099S/, A098R/D099V, A098R/D099Z/S101G, A098R/G100C/A137T, A098R/G100S, A098S/D099C, A098S/D099G, A098S/D099G/G100C, A098S/D099G/G100D, A098S/D099G/G100R/L250I, A098S/D099G/G100S, A098S/D099G/Z099.01R, A098S/D099H, A098S/D099H/M124V, A098S/D099H/Z099.01R/G169A/V180A, A098S/D099R, A098S/D099R/G100L, A098S/D099R/G154S, A098S/D099R/G166S, A098S/D099R/Z100.01G, A098S/D099S, A098S/D099S/Z099.01R, A098S/D099V, A098T/P129Z/S130R/S132G, A098T/S101G, A098V/D099G, A098V/D099G/G100D, A098V/D099G/G100D/A153V, A098V/D099G/G100S, A098Y/D099R/G100L, A098Y/D099R/G100Z/S101Z/G102D, A098Z/S101R, Z098.01N, Z098.01R, Z098.01R/G100R, D099C/K136N, D099C/S101D, D099C/S101R, D099C/S101Z, D099C/Z099.01S, D099C/Z100.01G, D099F/S101Z, D099G/G100C, D099G/G100D/S101R, D099G/G100H, D099G/G100R/S101V, D099G/G100S/S101R, D099G/S101G, D099G/S101H, D099G/S101V, D099G/S101Z, D099G/S101Z/N269D, D099G/Z099.01N, D099G/Z099.01R/G100S, D099G/Z099.01S, D099G/Z100.01G, D099G/Z100.01G/S183N/Q275K, D099H/S101Z, D099H/S101Z/V150I, D099H/Z100.01G/A273V, D099H/Z100.01G/Z100.02G, D099I/Z099.01S, D099L/N118D, D099L/S101G, D099L/S101V, D099L/Z100.01G, D099N/A116T, D099N/G100R/S101V, D099N/S101G, D099N/S101H, D099N/S101Z, D099N/Z099.01S, D099N/Z100.01G, D099R/S101D, D099R/S101G, D099R/S101V, D099R/S101Z, D099R/S101Z/A133T, D099R/Z099.01N, D099R/Z099.01S, D099R/Z100.01G, D099R/Z100.01N, D099S/S101G, D099S/S101I, D099S/S101Z, D099S/Z099.01H, D099S/Z099.01L, D099S/Z099.01V, D099S/Z100.01G, D099S/Z100.01G/Z100.02G, D099V/S101D, D099V/S101V, D099V/S101Z, Z099.01H/S101L, Z099.01H/Z099.02R, Z099.01N/S101R, Z099.01S, Z099.01S/S101V, Z099.01S/Z099.02G/A228T, G100D/S101G, G100D/S101R, G100H/G102V, G100H/S101G, G100H/S101R, G100L/S101G, G100L/S101R, G100N/S101D, G100N/S101L, G100N/S101R, G100R/L267R/I268V, G100R/S101G, G100R/S101G/G160S/S182N, G100R/S101G/Q103Z/Y104Z/S161N, G100R/S101G/S163N, G100R/S101N, G100R/S101R, G100R/S101R/Q103Z/V270I, G100R/S101Z/G102C/Q103D, G100S/S101C, G100S/S101G, G100S/S101R, G100S/S101R, G100S/S101V, G100S/S101V/A231T, Z100.01G, Z100.01L, Z100.01N, Z100.01R, Z100.01R/S101G, S101C/Q103I, S101C/Q103S, S101D/Q103C, S101D/Q103D, S101D/Q103F, S101D/Q103H, S101D/Q103H, S101D/Q103R, S101D/Q103R/H238Y, S101D/Q103S, S101D/Q103V, S101F/Q103G, S101G/A151V, S101G/A179V, S101G/G102S, S101G/Q103G, S101G/Q103L, S101G/Q103N, S101G/Q103R, S101G/Q103R/A179T, S101G/Q103R/G157D, S101G/Q103S, S101G/Q103S/L233S, S101G/Q103S/L233S, S101G/Q245H, S101G/S249N, S101G/V139I, S101G/Z101.01R, S101G/Z101.01R/A200T, S101G/Z102.01G, S101H/G102S, S101H/Q103D, S101H/Q103G, S101H/Q103G/E251Q, S101H/Q103H, S101H/Q103I, S101H/Q103R, S101H/Q103R/A274Z/Q275Z, S101H/Q103S, S101H/Q103V, S101I/Q103R, S101L/G102S/Q103R, S101L/Q103D, S101L/Q103G, S101L/Q103R, S101L/Q103R/A138T, S101L/Q103R/V121I/K213E, S101L/Q103S, S101L/Q103V, S101L/Z101.01R, S101N/Q103R, S101N/Z102.01G, S101R/A151V, S101R/A273V, S101R/E156G, S101R/G102L/Q103G, S101R/G102S, S101R/G102S/Q103R, S101R/G102S/Q103V, S101R/G131D, S101R/Q103C, S101R/Q103D, S101R/Q103D/S248R, S101R/Q103G, S101R/Q103G/A116T, S101R/Q103G/V147I/A153V, S101R/Q103G, S101R/Q103L, S101R/Q103N, S101R/Q103R, S101R/Q103R/S161N, S101R/Q103R/V148I, S101R/Q103S, S101R/Q103S/L126F, S101R/Q103V, S101V/Q103G, S101V/Q103H, S101V/Q103N/Z275.01K, S101V/Q103R, S101V/Q103V, S101Y/G102S/Q103R, S101Y/Q103L, S101Y/Q103S, S101Y/Q103V, S101Z/G102I/Q103H/Y104G/L135F, S101Z/G102V/Q103R/Y104L, S101Z/Q103D, S101Z/Q103F, S101Z/Q103L, S101Z/Q103R, G102C/Q103L/Y104S, G102R/Q103C/Y104C/V192I, G102R/Q103G/Y104R, G102R/Q103R/Z103.01G, G102Z/Q103S, Z102.01G/Q103R, Z102.01G/Q103R/L267V, Z102.01G/Z102.02R/Z102.03G, Q103H/Y104N/Q275Z, Q103N/E156D, Q103R/I122L/N123S/M124V, Q103R/Y104F, Q103Y/S182N, S105G/P129S/S130H/G131H, W106C/A116D/N117S/N118C, N109S/M119C/D120G/V121L, A116G/N117Z/N118R/M119C, A116H/N117G/N118R, N117C/N118G/M119S, N117C/N118G/M119S/A216V, N117G/N118G/M119C, N117G/N118R/M119C, N117H/N118C/M119C, N117H/N118D/M119S, N117H/N118G/M119C, N117H/N118G/M119L, N117H/N118G/M119S, N117H/N118G/M119V, N117H/N118H/M119V, N117H/N118R/M119C, N117H/N118R/M119S, N117H/N118S/M119C, N117H/N118S/M119S, N117H/N118S/M119V, N117I/N118G/M119V, N117L/N118G/M119V, N117R/N118G/M119C, N117R/N118G/M119I/V270I, N117R/N118G/M119S, N117R/N118H/M119V, N117R/N118R/M119C, N117R/N118R/M119I, N117R/N118R/M119S, N117R/N118R/M119V, N117R/N118S/M119I, N117R/N118S/M119L, N117S/N118G/M119C, N117S/N118G/M119S, N117S/N118H/M119V, N117S/N118R/M119V, N117V/N118G/M119C, N117V/N118H/M119C, N117Y/N118G/M119S, N117Y/N118G/M119V, Z117.01G/N118R/M119C, N118C/M119V/D120S, N118C/Z118.01G/M119C, N118D/M119C/D120L, N118D/M119S/D120R, N118G/M119C/D120G, N118G/M119C/D120L, N118G/M119I/D120R, N118G/M119N, N118G/M119S/D120G, N118G/M119S/D120R, N118G/M119V/D120G, N118G/M119V/D120S/S182N, N118H/M119C/D120R, N118H/M119I/D120G, N118H/M119N/V148I, N118H/M119S/D120G, N118H/M119V/D120G, N118R/M119C, N118R/M119C/D120G, N118R/M119C/D120R, N118R/M119S, N118R/M119S/D120G, N118R/M119S/D120R, N118R/M119S/D120S, N118R/M119V/D120C, N118R/M119V/D120G, N118R/M119V/D120G/A232T, N118S/M119C/D120R, M119C/D120G, M119C/D120G/V121I, M119C/D120G/V121L, M119C/D120H, M119C/D120H/A187V, M119C/D120R, M119H/V121I, M119I/D120C, M119I/D120G, M119I/D120R, M119L/D120G, M119S/D120R, M119S/D120S/V121I, M119S/V121I, M119V/D120C, M119V/D120G, M119V/D120R, M119V/D120S/V198L, D120G/I122C, D120G/I122C/L217S, D120G/I122G, D120G/I122V, D120H/I122L, D120H/V121C/I122V, D120N/V121L/I122V, D120R/A151V, D120R/I122V, D120R/V121I, D120R/V121I/I122C, D120R/V121I/I122C/A228T, D120S/I122C/A133S, V121C/I122C/N123C, V121C/I122G/N123V, V121C/I122S/N123V, V121C/I122V/N123S, V121I/I112C/N123C, V121I/I122C/V150I, V121I/I122G/N123C, V121I/I122S/N123C, V121I/I122S/N123I, V121I/I122V/N123S, V121L/I122S/N123V, V121L/N123C, I122C/N123C, I122C/N123S/M124L, I122D/N123V/M124L, I122G/N123C/M124L, I122G/N123G/Z123.01C, I122H/N123V/M124LI122S/N123S, I122V/N123G/M124C/A138V, I122V/N123S/M124I, I122V/S183G, N123C/M124S, N123C/M124V, N123S/M124L, N123S/M124S, M124C/L126S, M124C/L126S/T244I, M124C/L126V, M124I/L126H, L126F/P129Z, L126F/P129Z/S182N, L126I/P129Z, L126R/G127R, Z126.01S, G127C/G128S/P129R, G127C/P129G, G127C/P129G/A200T, G127C/P129R, G127C/P129R/A153T, G127D/P129G, G127D/P129S, G127H/P129G, G127H/P129R, G127L/P129R, G127N/P129C, G127N/P129G, G127N/P129G/A133T, G127N/P129G/A151V/K213N, G127N/P129R, G127N/P129S/A273V, G127N/P129V, G127R/A230V, G127R/P129D, G127R/P129D/L217S, G127R/P129G, G127R/P129R, G127R/P129S, G127S/P129D, G127S/P129F, G127S/P129G, G127S/P129G/G166D, G127S/P129G/I175T, G127S/P129R, G127S/P129S, G127S/P129V, G127V/P129G, G127V/P129R, Z127.01G/Z127.02H/P129D, Z127.01G/Z127.02H/P129G, Z127.01G/Z127.02H/P129R, Z127.01H/G128C/P129C/A230V, Z127.01H/P129G, Z127.01H/P129G/V148I, Z127.01H/P129R, Z127.01N/G128C/P129V, Z127.01N/G128S/P129S, Z127.01N/G128S/P129V, Z127.01N/P129R, Z127.01R/G128S/P129S, Z127.01R/P129G, Z127.01S/P129D, Z127.01S/P129G, Z127.01S/P129R, G128D/P129G, G128D/P129R/Z129.01R, G128D/P129S/G154S, G128H/P129C, G128H/P129H/Z129.01G, G128H/P129H/Z129.01R, G128H/P129R, G128H/P129R/Z129.01G, G128H/P129R/Z129.01R, G128H/P129S/Z129.01G, G128H/P129S/Z129.01R, G128H/P129S/Z129.01R/S248N, G128H/P129Y, G128H/Z128.01R/P129D, G128H/Z128.01R/P129S, G128H/Z128.01R/P129S/A144T, G128H/Z129.01R, 128N/P129R/Z129.01G, G128N/P129R/Z129.01R, G128N/P129S/Z129.01D, G128N/Z128.01R/P129S, G128R/P129G, G128R/P129H/Z129.01G, G128R/P129L/Z130.01S, G128R/P129R, G128R/P129R/Z129.01G, G128R/P129R/Z129.01G/A216V, G128R/P129S, G128R/P129S/Z129.01G, 128R/P129Z/S130R/S132G, G128R/Z128.01R/P129D, G128R/Z128.01R/P129F, G128S/P129C, G128S/P129C/Z129.01R, G128S/P129D, G128S/P129D/S248R, G128S/P129D/T244N, G128S/P129G, G128S/P129G/A187V, G128S/P129H/Z129.01R, G128S/P129R/L209F, G128S/P129R/Z129.01G, G128S/P129S/Z129.01D, G128S/P129S/Z129.01R, G128S/P129V, G128Y/P129R/Z129.01G, Z128.01G, Z128.01G/P129D, Z128.01G/P129R, Z128.01G/P129S/A134T, Z128.01G/Z128.02G, Z128.01R/P129D, Z128.01R/Z128.02G/P129S, Z128.01R/Z128.02H/P129G/G160S, Z128.01R/Z128.02R/P129D, Z128.01Y/Z128.02H/P129R, P129C/S130C/S132Z, P129D/Z129.01G/Z129.02D/G160D, P129D/Z129.01G/Z129.02G, P129G/G131Z, P129G/S130G/Z131.01G, P129G/S130H/S132Z, P129G/S130R/S132Z, P129G/V27I, P129G/Z129.01G, P129G/Z129.01R/Z129.02G, P129H/G131Z, P129H/G131Z/S132C, P129H/G131Z/S132H, P129H/S132Z, P129H/Z130.01D/Z130.02S, P129R/G131Z/S132H, P129R/S130D/S132Z, P129R/S130N/G131Z, P129R/S132T, P129R/S132Z, P129R/Z129.01G, P129R/Z129.01G/Z129.02G, P129R/Z129.01G/Z129.02G/A144T, P129R/Z129.01G/Z129.02G/G146D, P129R/Z129.01G/Z129.02R, P129R/Z129.01G, P129R/Z129.01R/Z129.02G, P129S/G131ZP129S/S130H/S132Z, P129S/Z129.01C, P129S/Z129.01R/Z129.02S, P129S/Z130.01N/Z130.02S, P129V/S132Z, P129Z, P129Z/G131R/S132C, P129Z/S130G, P129Z/S130G/G131H/S132H, P129Z/S130G/G131V, P129Z/S130H, P129Z/S130H/S132G, P129Z/S130H/S132N, P129Z/S130L, P129Z/S130R, P129Z/S130R/G131C, P129Z/S130R/G131C, P129Z/S130R/G131D/S132C, P129Z/S130R/G131H/A144E, P129Z/S130R/G131H/S132D, P129Z/S130R/G131R/S132D, P129Z/S130R/L267M, P129Z/S130R/S132C, P129Z/S130R/S132G, P129Z/S130V/S132G, P129Z/S130Z/G131N/S132G, S130D/G131C/S132D, S130D/G131R, S130D/G131R/S132D, S130D/G131Z, S130D/G131Z/S132H, S130F/G131N/S132V, S130F/G131S/S132V, S130F/S132V, S130G/G131D/S132C, S130G/G131H/S132D, S130G/G131S/S132I, S130G/S132G, S130H/G131C/S132G, S130H/G131H/S132R, S130H/G131R/S132C, S130H/G131S, S130H/G131S/S132C, S130H/S132Z, S130I/G131S/S132H, S130I/S132G, S130N/G131S/S132C, S130R/G131R/S132C, S130R/G131S/S132C/S161N, S130R/S132G, S130R/S132G/S183N, S130R/S132Z, S130V/G131D/S132I, S130V/G131H/S132C, S130V/S132N, S130V/S132Z, S130Y/G131H/S132C, S130Y/S132G, S130Z/G131H/S132H, G131H/S132L/W241R, G131N/S132C, G131S/S132G, G131Y/S132G, and A142V/G211V, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
  • The present invention also provides subtilisin variants comprising at least one set of modifications, wherein said set of modifications is selected from: V072G/G097A/G128A/Y217Q, G097A/G128S/Y217Q, G097A/G128S/P129D/Y217Q, G097A/G128A/A134T/Y217Q, M124V/L126A/Y217Q, G097A/G128A/P129D/Y217Q/A232P, G097A/G128A/P129D/Y217Q, G097A/G128S/P129V/A134T/Y217Q, G097A/G128S/P129V/Y217Q, V068I/G097A/G128S/P129D/Y217Q, G097A/S101D/G128S/S204F/Y217Q/S236F/T254P, G097A/S101D/G128S/Y217Q, G097A/G128A/P129V/Y217Q, G097A/S101D/G128A/Y217Q, G097A/S101D/G128S/P129D/Y217Q, G097A/S101D/G128A/A134T/Y217Q, G097A/Z099.01S/A114S/G128S/Y217Q, G097A/S101D/G128A/P194L/Y217Q, G097A/Z099.01S/G128S/Y217Q, G097A/S101D/G128A/P129D/Y217Q, Q019R/G097A/Z099.01S/G128S/P129D/Y217Q, G097A/G100S/G128S/Y217Q, G097A/S101D/G128S/P129V/Y217Q, G097A/Z099.01S/G128A/A134T/Y217Q, G097A/G100S/1107V/G128S/P129D/Y217Q, G097A/G100S/G128S/Y217Q/A230V, G097A/G100S/G128A/Y217Q/K237N, G097A/Z099.01S/G128S/P129D/Y217Q, G097A/G100S/G128S/P129D/Y217Q, Z096.01D/G097A/A098R/G128A/Y217Q, G097A/Z099.01S/I122S/N123G/G128A/Y217Q,G097A/Z099.01S/I122S/N123G/G128A/Y217Q/T220A/A232T, P005S/G097A/Z099.015/S101D/G128A/Y217Q, G097A/S101D/G128A/P129V/Y217Q, V093Z/K094S/V095C/L096S/G097A/G128A/Y217Q, G097A/G100S/S101D/G128A/Y217Q/A232P, Z096.01D/G097A/A098R/S101D/G128A/Y217Q, and G097A/Z099.015/G100S/G128A/Y217Q, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
  • The present invention further provides subtilisin variants comprising at one set of modifications, wherein said set of modifications is selected from: G097A/G128S/P129T/Y217Q, G097A/G128A/P129S/Y217Q, G097A/G128S/P129C/Y217Q, G097A/G128S/P129Q/Y217Q, G097A/G128S/P129E/Y217Q, G097A/P129S/Y217Q, G097A/G128S/P129A/Y217Q, G097A/G128S/P129K/Y217Q, G097A/P129N/Y217Q, G097A/P129E/Y217Q, G097A/G128S/P129K/G131S/Y217Q, G097A/G128S/P129R/Y217Q, G097A/G128S/P129V/Y217Q, G097A/G128S/P129Y/Y217Q, G097A/G128A/P129R/Y217Q, G097A/G128H/P129S/Y217Q, G097A/G128N/P129S/Y217Q/V270A, G097A/G128S/P129L/Y217Q, G097A/P129R/Y217Q, G097A/P129W/Y217Q, G097A/P129Z/S130A/Y217Q, G097A/G128H/P129R/Y217Q, G097A/G128H/P129T/Y217Q, G097A/P129G/Y217Q, G097A/G128N/P129A/Y217Q, G097A/G128H/Y217Q, G097A/G128S/P129W/Y217Q, G097A/P129Z/Y217Q, G097A/G128N/Z128.01A/P129S/Y217Q, G097A/G128N/Y217Q, G097A/G128A/P129L/Y217Q, G097A/G128T/P129N/Y217Q, G097A/G128H/P129A/Y217Q, G097A/G128M/P129R/Y217Q, G097A/G128Q/P129K/Y217Q, G097A/G128T/P129Q/Y217Q, G097A/G128T/P129H/Y217Q, G097A/G128T/P129R/Y217Q, G097A/G128D/P129E/Y217Q, G097A/G128T/P129T/Y217Q, G097A/G128T/P129A/Y217Q, G097A/G128Z/P129Z/Y217Q, G097A/G128S/Z128.01A/P129S/Y217Q, G097A/G128N/P129M/Y217Q, G097A/G128N/P129V/Y217Q, G097A/G128E/P129K/Y217Q, G097A/G128N/Z128.01A/P129F/Y217Q, G097A/G128C/P129R/Y217Q, G097A/G128T/P129M/Y217Q, G097A/G128R/Z128.01A/P129S/Y217Q, G097A/G128S/Z128.01A/P129A/Y217Q, G097A/G128D/Y217Q, G097A/G128T/P129Y/Y217Q, G097A/G128R/P129S/Y217Q, G097A/G128R/Z128.01A/P129T/Y217Q, G097A/G128R/Z128.01A/P129A/Y217Q, G097A/G128T/P129W/Y217Q, G097A/G128Y/Z128.01A/P129R/Y217Q, G097A/G128C/P129D/Y217Q, V084L/G097A/P129Z/Y217Q, G097A/G128R/P129D/Y217Q, G097A/G128A/Z128.01A/P129H/E195A/Y217Q, G097A/G128S/Z128.01A/P129Y/Y217Q, G097A/G128N/P129L/Y217Q, G097A/G128C/P129H/Y217Q, G097A/G128T/Z128.01A/P129T/Y217Q, G097A/G128C/Z128.01A/P129A/Y217Q, G097A/G128S/Z128.01A/P129I/Y217Q, G097A/G128S/Z128.01A/P129E/Y217Q/Q271H, G097A/G128S/Z128.01A/P129L/Y217Q, G097A/G128Y/Z128.01A/P129S/Y217Q, G097A/G128S/Z128.01A/P129M/R186H/Y217Q, G097A/G128E/Z128.01A/P129D/Y217Q, G097A/G128T/P129L/Y217Q, G097A/G128F/P129D/Y217Q, G097A/G128V/Z128.01A/P129R/Y217Q, G097A/G128L/Z128.01A/P129S/Y217Q, G097A/G128Y/P129T/Y217Q, G097A/G128C/P129Y/Y217Q, G097A/G128C/P129T/Y217Q, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
  • The present invention also provides subtilisin variants comprising at least one set of modifications, wherein said set of modifications is selected from: G097A/G128A/Y217Q/M222Q, M124V/L126A/P129V/Y217Q, G128A/P129V/Y217Q, G097A/G128A/P129V/Y217Q, N123G/P129V/Y217Q, V30I/Z096.01D/G097A/A098R/G128A/Y217Q, Z096.01D/A098R/G128A/Y217Q, Z096.01D/A098R/M124V/L126A/Y217Q, G097A/Z098.01S/G128A/Y217Q, Z096.01D/A098R/N123G/Y217Q, Z096.01D/G097A/A098R/G128A/Y217Q/S236F, Z098.01S/M124V/L126A/Y217Q, Z098.01S/G128A/Y217Q, Z096.01D/G097A/A098R/G128A/Y217Q, V093I/Z098.01S/M124V/L126A/Y217Q, A073V/Z098.01S/N123G/Y217Q, G065D/Z098.01S/G128A/Y217Q, I011L/Z098.01S/G128A/Y217Q, L082F/Z096.01D/A098R/N123G/Y217Q, Z098.01S/N123G/A138V/Y217Q, and Z098.01S/N123G/Y217Q, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
  • The present invention also provides subtilisin variants comprising the substitution Y217L and further at least one set of any of the modification sets provided above, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2. The present invention further provides subtilisin variants comprising the substitutions G97A/G128A/Y217Q and further comprising at least one modification of any of the modifications sets provided herein, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
  • The present invention also provides cleaning compositions comprising at least one subtilisin variant as provided herein. In some preferred embodiments, the cleaning composition is a laundry detergent. In some particularly preferred embodiments, the laundry detergent is a heavy duty liquid laundry detergent. In some alternative embodiments, the cleaning composition is a dish detergent. In some further embodiments, the cleaning compositions further comprise one or more additional enzymes or enzyme derivatives selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. In some further embodiments, the cleaning compositions further comprise at least one stabilizing agent. In still some additional embodiments, the cleaning compositions comprise at least 0.0001 weight percent of at least one of any of the subtilisin variants provided herein, and optionally, at least one suitable adjunct ingredient.
  • In some embodiments, the present invention provides methods of cleaning, the method comprising the steps of: contacting a surface and/or an article comprising a fabric with at least one of the cleaning compositions set forth herein and optionally washing and/or rinsing the surface or article.
  • The present invention also provides animal feeds comprising at least one subtilisin variant provided herein, as well as food processing compositions comprising at least one subtilisin variant provided herein.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows a diagram illustrating the method used for creating in-frame deletions and insertions. Library was designed to have 33% of clones with substitutions; 33% of clones with insertions and 33% of clones with deletions.
  • FIG. 2A Plasmid map of pAC-FNA10. The plasmid elements are as follows: pUB110=DNA fragment from plasmid pUB110 (McKenzie et al., Plasmid 15:93-103 [1986], pBR322=DNA fragment from plasmid pBR322 (Bolivar et al., Gene 2:95-113 [1977]), pC194=DNA fragment from plasmid pC194 (Horinouchi and Weisblum, J. Bacteriol 150:815-825 [1982]).
  • FIG. 2B provides a map of pHPLT-FNA.
  • FIG. 2C provides a map of pHPLT-BPN. Vector pHPLT contains the LAT promoter (PLAT), a sequence encoding the LAT signal peptide (preLAT). The origin of replication and neoR are derived from plasmid pUB110.
    •  DESCRIPTION OF THE INVENTION
  • The present invention provides variant subtilisins and compositions comprising at least one variant subtilisin set forth herein, as well as methods for using these variants and compositions. The modification of the precursor proteases of the invention to produce these variant subtilisins includes at least one substitution, at least one deletion, or at least one insertion. In some embodiments, the modification comprises a combination of mutations. For example, in some embodiments, the modification includes a combination of at least one substitution and at least one deletion. In some other embodiments, the modification includes a combination of at least one substitution and at least one insertion. In some further embodiments, the modification includes a combination of at least one deletion and at least one insertion. In yet other embodiments, the modification includes a combination of at least one substitution, at least one deletion, and at least one insertion.
  • Several suitable methods are known in the art for generating modified polynucleotide sequences of the present invention, including but not limited to site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches. The commonly used methods include DNA shuffling (See, Stemmer, Proc Natl Acad Sci USA. 25:10747-51 [1994]), methods based on non-homologous recombination of genes (e.g. ITCHY) (Ostermeier et al., Bioorg Med Chem. 7:2139-44 [1999]), SCRACHY (Lutz et al. Proc Nati Acad Sci USA. 98:11248-53 [2001]), SHIPREC (Sieber et al., Nat Biotechnol., 19:456-60 [2001 ]),and NRR (Bittker et al., Nat Biotechnol., 20:1024-9 [2001]; Bittker et al., Proc Nati Acad Sci. USA. 101:7011-6 [2004]), and methods that rely on the use of oligonucleotides to insert random and targeted mutations, deletions and/or insertions (Ness et al., Nat Biotechnol. 20:1251-5 [2002]; Coco et al., Nat Biotechnol., 20:1246-50 [2002]; Zha et al., Chembiochem. 3:34-9 [2003], Glaser et al., J Immunol , 149:3903-13 [1992], Sondek and Shortie, Proc Nati Acad Sci USA 89:3581-5 [1992], Yáñez et al., Nucleic Acids Res., 32:e158 [2004], Osuna et al., Nucleic Acids Res., 32:e136 [2004], Gaytán et al., Nucleic Acids Res., 29:E9 [2001], and Gaytán et al., Nucleic Acids Res., 30:e84 [2002]).
  • In some embodiments, the full-length parent polynucleotide is ligated into an appropriate expression plasmid, and the following mutagenesis method is used to facilitate the construction of the modified protease of the present invention, although other methods may be used. The method is based on that described by Pisarchik et al. (Pisarchik et al., Prot. Eng. Des. Select., 20:257-265 [2007]). In some embodiments, an added advantage is provided, in that the restriction enzyme used herein cuts outside its recognition sequence, which allows digestion of practically any nucleotide sequence and precludes formation of a restriction site scar.. First, as described herein, a naturally-occurring gene encoding the full-length protease is obtained and sequenced and scanned for one or more points at which it is desired to make a mutation (deletion, insertion, substitution, or a combination thereof) at one or more amino acids. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by primer extension in accord with generally known methods. Fragments to the left and to the right of the desired point(s) of mutation are amplified by PCR and to include the Eam1104I restriction site. The left and right fragments are digested with Eam1104I to generate a plurality of fragments having complementary three base overhangs, which are then pooled and ligated to generate a library of modified sequences containing one or more mutations. This method avoids the occurrence of frame-shift mutations. In addition, this method simplifies the mutagenesis process because all of the oligonucleotides can be synthesized so as to have the same restriction site, and no synthetic linkers are necessary to create the restriction sites as is required by some other methods
    • Compositions
  • The present invention provides variant proteases and compositions comprising at least one of the variant subtilisins set forth herein. In some embodiments, the present invention provides means to produce protease with various commercial applications where degradation or synthesis of polypeptides are desired, including cleaning compositions, as well as feed components, textile processing, leather finishing, grain processing, meat processing, food processing, preparation of protein hydrolysates, digestive aids, microbicidal compositions, bacteriostatic compositions, fungistatic compositions, and personal care products (e.g., oral care, hair care, and/or skin care).
  • The present invention further provides enzyme compositions have comparable or improved wash performance, as compared to presently used subtilisin proteases.
    • Cleaning Compositions
  • The cleaning composition of the present invention are advantageously employed for example, in laundry applications. Indeed, due to the unique advantages of increased effectiveness in lower temperature solutions, the enzymes of the present invention are ideally suited for laundry applications. Furthermore, 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 laundry cleaning additive products. In some embodiments, low temperature solution cleaning applications find use. The additive product may be, in its simplest form, one or more proteases. In some embodiments, 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. In some embodiments, 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 additive 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. Typically the present cleaning compositions 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. Such 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. In some embodiments, 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.
  • In some embodiments, 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. In addition, encapsulation is also a means of controlling the availability of the variant protease during the cleaning process. In some embodiments, encapsulation enhances the performance of the variant protease(s) and/or additional enzymes. In this regard, the variant proteases of the present invention are encapsulated with any suitable encapsulating material known in the art. In some embodiments, the encapsulating material typically encapsulates at least part of the catalyst for the variant protease(s) of the present invention. Typically, the encapsulating material is water-soluble and/or water-dispersible. In some embodiments, 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. 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. In some embodiments, 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.)>
  • As described herein, the variant proteases of the present invention find particular use in laundry 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.
  • Indeed, there are a variety of 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. In addition, detergent formulations used in different geographical areas have different concentrations of their relevant components present in the wash water. For example, a European detergent typically has about 4500-5000 ppm of detergent components in the wash water, while a Japanese detergent typically has approximately 667 ppm of detergent components in the wash water. In North America, particularly the United States, 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 2000ppm 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.
  • In light of the foregoing, it is evident that 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.
  • The 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.
  • As a further example, different geographies use different wash temperatures. The temperature of the wash water in Japan is typically less than that used in Europe. For example, 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.).
  • As a further example, different geographies typically have different water hardness. Water hardness is usually described in terms of the grains per gallon mixed Ca2+/Mg2+. Hardness is a measure of the amount of calcium (Ca2+) and magnesium (Mg2+) 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.
  • Water Grains per gallon Parts per million
    Soft less than 1.0 less than 17
    Slightly hard 1.0 to 3.5 17 to 60
    Moderately hard 3.5 to 7.0 60 to 120
    Hard 7.0 to 10.5 120 to 180
    Very hard Greater than 10.5 greater than 180
  • European water hardness is typically greater than 10.5 (for example 10.5-20.0) grains per gallon mixed Ca2±/Mg2+ (e.g., about 15 grains per gallon mixed Ca2+/Mg2+). North American water hardness is typically greater than Japanese water hardness, but less than European water hardness. For example, North American water hardness can be between 3 to10 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 Ca2+/Mg2+.
  • Accordingly, in some embodiments, 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). In some embodiments, the variant proteases of the present invention are comparable in wash performance to other subtilisin proteases. In some embodiments, the variant proteases of the present invention exhibit enhanced wash performance as compared to subtilisin proteases currently commercially available. Thus, in some preferred embodiments of the present invention, the variant proteases provided herein exhibit enhanced oxidative stability, enhanced thermal stability, and/or enhanced chelator stability. In addition, 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.
  • In some embodiments of the present invention, 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. In other aspects of the present invention, 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.
  • In some embodiments, 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. Such enzymes include, but are not limited to other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g., laccases), and/or mannanases.
  • Any other suitable protease finds use in the compositions of the present invention. 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. In some embodiments, 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. Preferred commercially available protease enzymes include MAXATASE®, MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT® and PURAFECT® OXP (Genencor); ALCALASE®, SAVINASE®, PRIMASE®, DURAZYM™, RELASE® and ESPERASE® (Novozymes); and BLAP™ (Henkel Kommanditgesellschaft 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. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, and various other patents.
  • In addition, 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. Examples of 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. 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]).
  • Furthermore, a number of 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 l [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.
  • 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).
  • Additional suitable lipases include commercially available lipases such as M1 LIPASE™, LUMA FAST™, and LIPOMAX™ (Genencor); LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE P™ “Amano” (Amano Pharmaceutical Co. Ltd., Japan).
  • In some embodiments of the present invention, 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. In other aspects of the present invention, 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.
  • Any amylase (alpha and/or beta) suitable for use in alkaline solutions also find use in some embodiments of the present invention. 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 α-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 BAN™ (Novozymes) and RAPIDASE® and MAXAMYL® P (Genencor).
  • In some embodiments of the present invention, 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. In other aspects of the present invention, 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.
  • In some further embodiments, 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)™ (Kao Corporation). In some embodiments, 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). In some embodiments, 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. In other aspects of the present invention, 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.
  • Any 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). In some embodiments, 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. In other aspects of the present invention, 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.
  • In some embodiments, 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. In some alternative embodiments, 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. Chemically or genetically modified mutants are included in some embodiments. In some embodiments, 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. In other aspects of the present invention, 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.
  • In some embodiments, additional enzymes find use, including but not limited to perhydrolases (See e.g., WO 05/056782). In addition, in some particularly preferred embodiments, 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. It is also contemplated that 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).
  • Examples of suitable 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. Pat. Nos. 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of which are incorporated herein by reference). Embodiments of specific cleaning composition materials are exemplified in detail below. In embodiments in which the cleaning adjunct materials are not compatible with the variant proteases of the present invention in the cleaning compositions, then suitable methods of keeping the cleaning adjunct materials and the protease(s) separated (i.e., not in contact with each other) until combination of the two components is appropriate are used. Such separation methods include any suitable method known in the art (e.g., gelcaps, encapulation, tablets, physical separation, etc.).
  • By way of example, several cleaning compositions wherein the variant proteases of the present invention find use are described in greater detail below. In embodiments in which the cleaning compositions of the present invention are formulated as compositions suitable for use in laundry machine washing method(s), the 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. In some embodiments, 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. Such additive products are intended to supplement and/or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process. In some embodiments, 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.
  • In some embodiments, various cleaning compositions such as those provided in U.S. Pat. No. 6,605,458 find use with the variant proteases of the present invention. Thus, in some embodiments, the compositions comprising at least one variant protease of the present invention is 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. In some embodiments, 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. In addition, 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 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. When a low pH cleaning composition is desired, the pH of such composition is adjusted via the addition of a material such as monoethanolamine or an acidic material such as HC.
  • While not essential for the purposes of the present invention, the non-limiting list of adjuncts illustrated hereinafter are suitable for use in the instant cleaning compositions. In some embodiments, 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. In addition to the disclosure below, 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.
  • In some embodiments, 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.
  • In some additional embodiments, 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.
  • In some further embodiments, the cleaning compositions herein contain a chelating agent. Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof.
  • In some still further embodiments, 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.
  • In some additional embodiments, 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.
  • In some still additional embodiments, 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.
  • In some particularly preferred embodiments, the cleaning compositions comprise one or more detergent enzymes which provide cleaning performance and/or fabric care benefits. Examples of 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,β-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 least one amylase.
  • In some further embodiments, the enzymes used in the cleaning compositions are stabilized any suitable technique. In some embodiments, 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. In some still further embodiments, 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. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.
  • In some embodiments, the compositions herein are catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art and include, for example, the manganese-based catalysts disclosed in U.S. Pat. No. 5,576,282. In addition, 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. In some embodiments, the compositions provided herein also suitably include a transition metal complex of a macropolycyclic rigid ligand (i.e., “MRL”). As a practical matter, and not by way of limitation, 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.
  • As indicated above, 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). Typically, at least a portion of the situs is contacted with an embodiment of the present cleaning composition, in neat form or diluted in a wash liquor, and then the situs is optionally washed and/or rinsed. For purposes of the present invention, “washing” includes but is not limited to, scrubbing, and mechanical agitation. In some embodiments, the cleaning compositions are typically employed at concentrations of from about 500 ppm to about 15,000 ppm in solution. When the wash solvent is water, the water temperature typically ranges from about 5° C. to about 90° C. and, when the situs comprises a fabric, the water to fabric mass ratio is typically from about 1:1 to about 30:1.
    • Definitions
  • Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, protein engineering, microbiology, and recombinant DNA, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Second Edition (Cold Spring Harbor), [1989]); and Ausubel et al., “Current Protocols in Molecular Biology” [1987]). All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.
  • Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. There are various dictionaries available and known to those in the art that provide definitions of these terms. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular “a”, “an” and “the” includes the plural reference unless the context clearly indicates otherwise. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not 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.
  • The practice of the present invention employs, unless otherwise indicated, conventional techniques of protein purification, molecular biology, microbiology, recombinant DNA techniques and protein sequencing, all of which are within the skill of those in the art.
  • Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.
  • As used herein, the terms “protease,” and “proteolytic activity” refer 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]). For example, 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). 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. In addition, absorbance measurements at 280 nm can be used to determine the total protein concentration. The active enzyme/total-protein ratio gives the enzyme purity.
  • As used herein, “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. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “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.
  • The terms “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. The following are non-limiting examples of 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. In some embodiments, 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. In alternative embodiments, the sequence of nucleotides is interrupted by non-nucleotide components.
  • As used herein, the terms “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. In particularly preferred embodiments, the DNA construct comprises a sequence of interest (e.g., as an incoming sequence). In some embodiments, 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. In a further embodiment, the transforming DNA comprises other non-homologous sequences, added to the ends (e.g., stuffer sequences or flanks). In some embodiments, 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. In some embodiments, 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.
  • As used herein, the terms “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. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In preferred embodiments, expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those of skill in the art. The term “expression cassette” is used interchangeably herein with “DNA construct,” and their grammatical equivalents. Selection of appropriate expression vectors is within the knowledge of those of skill in the art.
  • As used herein, 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. In some embodiments, 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.
  • As used herein, the term “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.
  • As used herein in the context of introducing a nucleic acid sequence into a cell, 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]).
  • As used herein, 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. For example, DNA encoding a secretory leader (i.e., a signal peptide), is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “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.
  • As used herein the term “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).
  • As used herein, “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).
  • As used herein, “ortholog” and “orthologous genes” refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typically, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.
  • As used herein, “paralog” and “paralogous genes” refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paralogs evolve new functions, even though some functions are often related to the original one. Examples of paralogous genes include, but are not limited to genes encoding trypsin, chymotrypsin, elastase, and thrombin, which are all serine proteinases and occur together within the same species.
  • As used herein, 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).
  • As used herein, “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]).
  • As used herein, an “analogous sequence” is one wherein the function of the gene is essentially the same as the gene based on the wild-type subtilisin protease. Additionally, analogous genes include at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity with the sequence of the wild-type subtilisin protease. Alternately, analogous sequences have an alignment of between about 70 to about 100% of the genes found in the B. amyloliquefaciens subtilisin protease region. In additional embodiments more than one of the above properties applies to the sequence. 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.
  • One example of a useful algorithm is PILEUP. 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.
  • Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al., (Altschul et al., J. Mol. Biol., 215:403-410, [1990]; and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). A particularly useful BLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth. Enzymol., 266:460-480 [1996]). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. 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).
  • Thus, “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.
  • As used herein, “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. Thus, for example, 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.
  • In some preferred embodiments, 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.
  • As used herein, the terms “amplification” and “gene amplification” refer to a process by which specific DNA sequences are disproportionately replicated such that the amplified gene becomes present in a higher copy number than was initially present in the genome. In some embodiments, selection of cells by growth in the presence of a drug (e.g., an inhibitor of an inhibitable enzyme) results in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the 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.
  • As used herein, 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. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • As used herein, the term “probe” refers to an oligonucleotide (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. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
  • As used herein, the term “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.
  • As used herein, the term “polymerase chain reaction” (“PCR”) refers to the methods of U.S. Pat. Nos. 4,683,195 4,683,202, and 4,965,188, hereby incorporated by reference, which include methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified”.
  • As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
  • As used herein, the term “RT-PCR” refers to the replication and amplification of RNA sequences. In this method, 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. In RT-PCR, 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).
  • As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • A “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. In certain embodiments of the invention 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. In a preferred embodiment, chromosomal integration is homologous recombination.
  • As used herein “amino acid” refers to peptide or protein sequences or portions thereof. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably.
  • As used herein, “protein of interest” and “polypeptide of interest” refer to a protein/polypeptide that is desired and/or being assessed. In some embodiments, the protein of interest is expressed intracellularly, while in other embodiments, it is a secreted polypeptide. In particularly preferred embodiments, these enzymes include the serine proteases of the present invention. In some embodiments, 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. As is known in the art, 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. Thus 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.
  • The terms “protein” 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.
  • The term “signal sequence” or “signal peptide” 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).
  • The term “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.
  • The term “mature” form of a protein or peptide refers to the final functional form of the protein or peptide. For example, the mature form of the BPN′ subtilisin protease of the present invention includes at least the amino acid sequence identical to residue positions 1-275 of SEQ ID NO:2, as set forth below:
  • (SEQ ID NO: 2)
    AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGAS
    MVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGA
    DGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGV
    VVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPEL
    DVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQ
    VRSSLENTTTKLGDSFYYGKGLINVQAAAQ
  • The term “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.
  • The term “identical” in the context of two 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.
  • The term “optimal alignment” refers to the alignment giving the highest percent identity score.
  • “Percent sequence identity,” “percent amino acid sequence identity,” “percent gene sequence identity,” and/or “percent nucleic acid/polynucloetide sequence identity,” with respect to two amino acid, polynucleotide and/or gene sequences (as appropriate), refer 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.
  • The phrase “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. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative 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).
  • The phrase “equivalent,” in this context, 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. For example, being equivalent means that 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 subtilisin protease having the amino acid sequence of SEQ ID NO:2.
  • The term “isolated” or “purified” refers to a material that is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, 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. For example, 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. In some embodiments, 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. In preferred embodiments, 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.
  • The term “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”.
  • The term “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.
  • As used herein, the term, “combinatorial mutagenesis” refers to methods in which libraries of variants of a starting sequence are generated. In these libraries, the variants contain one or several mutations chosen from a predefined set of mutations. In addition, the methods provide means to introduce random mutations which were not members of the predefined set of mutations. In some embodiments, the methods include those set forth in U.S. patent application Ser. No. 09/699,250, filed Oct. 26, 2000, hereby incorporated by reference. In alternative embodiments, combinatorial mutagenesis methods encompass commercially available kits (e.g., QuikChange® Multisite, Stratagene, San Diego, Calif.).
  • As used herein, the term “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.
  • As used herein, the term “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. As used herein, the term “multiple sequence alignment” (“MSA”) refers to the sequences of multiple homologs of a starting gene that are aligned using an algorithm (e.g., Clustal W).
  • As used herein, 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.
  • As used herein, the term “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.
  • As used herein, 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. In some embodiments, the enhanced CCM library is based on the sequence of an initial hit resulting from an earlier round of CCM. In additional embodiments, 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.
  • As used herein, the term “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. In preferred embodiments, the screening process removes and/or reduces the abundance of variants that contain “performance-reducing mutations.”
  • As used herein, the term “functional assay” refers to an assay that provides an indication of a protein's activity. In particularly preferred embodiments, the term refers to assay systems in which a protein is analyzed for its ability to function in its usual capacity. For example, in the case of enzymes, a functional assay involves determining the effectiveness of the enzyme in catalyzing a reaction.
  • As used herein, the term “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. However, in some preferred embodiments, 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.
  • The term “property” or grammatical equivalents thereof in the context of a nucleic acid, as used herein, refer 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). For example, 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.
  • The term “property” 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, KM, kcat, kcat/kM 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 terms “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. In some preferred embodiments, the expression product of the modified sequence is a truncated protein (e.g., if the modification is a deletion or interruption of the sequence). In some particularly preferred embodiments, the truncated protein retains biological activity. In alternative embodiments, the expression product of the modified sequence is an elongated protein (e.g., modifications comprising an insertion into the nucleic acid sequence). In some embodiments, an insertion leads to a truncated protein (e.g., when the insertion results in the formation of a stop codon). Thus, an insertion may result in either a truncated protein or an elongated protein as an expression product.
  • As used herein, the terms “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).
  • The terms “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. In another preferred embodiment of the invention, 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. By adding 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. For example, if it is desired that 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. Alternatively, 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.
  • As used herein, the phrase “contiguous mutations” refers to mutations which are presented within the same oligonucleotide primer. For example, 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.
  • The terms “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. In some embodiments, the wild-type sequence refers to a sequence of interest that is the starting point of a protein engineering project (i.e., the “parent” sequence). 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.
  • As used herein, the terms “protease variant,” “subtilisin variant,” “subtilisin protease variant,” are used in reference to proteases that are similar to a wild-type subtilisin or a parent protease (e.g., subtilisin) used as a starting point in protein engineering. These variants are similar to the wild-type or other parent in their function, but have mutations in their amino acid sequence that make them different in sequence from the wild-type or parent protease (e.g., subtilisin).
  • Unless otherwise indicated, the amino acid position numbers refer to those assigned to the mature Bacillus amyloliquefaciens subtilisin sequence of SEQ ID NO:2. The invention, however, 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.
  • As used herein, the terms “modification” and “mutation” 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).
  • The term “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. In some embodiments, 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. In some embodiments, the stability is measured as described in the Examples.
  • The term “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. In some embodiments, 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. In some embodiments, the chelator stability is measured as described in the Examples.
  • The terms “thermally stable” and “thermostable” refer 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. In some embodiments, 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. In some embodiments, the thermostability is determined as described in the Examples.
  • The term “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.
  • The term “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.
  • The term “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. In some embodiments, 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.
  • The term “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.
  • The term “cleaning adjunct materials,” as used herein, 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. In some embodiments, granular compositions are in “compact” form, while in other embodiments, the liquid compositions are in a “concentrated” form.
  • The term “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.
  • The term “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.
  • The term “comparative performance” in the context of cleaning activity refers to at least about 60%, at least about 70%, at least about 80% at least about 90%, or at least about 95% of the cleaning activity of a comparative subtilisin protease (e.g., commercially available proteases), including but not limited to OPTIMASE™ protease (Genencor), PURAFECT™ protease products (Genencor), SAVINASE™ protease (Novozymes), BPN′-variants (See e.g., U.S. Pat. No. Re 34,606), RELASE™ DURAZYME™, EVERLASE™, KANNASE™ protease (Novozymes), MAXACAL™, MAXAPEM™, PROPERASE™ 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).
  • 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.
  • As used herein, “cleaning compositions” and “cleaning formulations” refer to compositions that find use in the removal of undesired compounds from items to be cleaned, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes) etc. The term encompasses any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, granule, or spray composition), as long as the composition is compatible with the perhydrolase and other enzyme(s) used in the composition. The specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.
  • The terms further refer to any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any object and/or surface. It is intended that the terms include, but are not limited to detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents).
  • Indeed, the term “cleaning composition” as used herein, includes unless otherwise indicated, granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries such as bleach additives and “stain-stick” or pre-treat types.
  • As used herein, the terms “detergent composition” and “detergent formulation” are used in reference to mixtures which are intended for use in a wash medium for the cleaning of soiled objects. In some preferred embodiments, the term is used in reference to laundering fabrics and/or garments (e.g., “laundry detergents”). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e.g., “dishwashing detergents”). It is not intended that the present invention be limited to any particular detergent formulation or composition. Indeed, it is intended that in addition to perhydrolase, the term encompasses detergents that contain surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and solubilizers.
  • As used herein, “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).
  • As used herein, “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.
  • The “compact” form of the cleaning compositions herein is best reflected by density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically about 17 to about 35% by weight of the total composition. In contrast, in compact compositions, the filler salt is present in amounts not exceeding about 15% of the total composition. In some embodiments, the filler salt is present in amounts that do not exceed about 10%, or more preferably, about 5%, by weight of the composition. In some embodiments, 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.
  • As used herein, the term “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.
    • Experimental
  • The present invention is described in further detail in the following Examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. The following Examples are offered to illustrate, but not to limit the claimed invention
  • In the experimental disclosure which follows, the following abbreviations apply: PI or Pi (Performance Index), ppm (parts per million); M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); μg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); H2O (water); dH2O (deionized water); (HCl (hydrochloric acid); aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl2 (magnesium chloride); NaCl (sodium chloride); w/v (weight to volume); v/v (volume to volume); g (gravity); OD (optical density); Dulbecco's phosphate buffered solution (DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto Tryptone, 24 g/l glycerol, 2.31 g/l KH2PO4, and 12.54 g/l K2HPO4); OD280 (optical density at 280 nm); OD600 (optical density at 600 nm); A405 (absorbance at 405 nm); Vmax (the maximum initial velocity of an enzyme catalyzed reaction); PAGE (polyacrylamide gel electrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST (PBS+0.25% TWEEN® 20); PEG (polyethylene glycol); PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); HEPES (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPES buffered saline); Tris-HCl (tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine (N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino) ethane-sulfonic acid); TAPS (3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS (3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide); DTT (1,4-dithio-DL-threitol); SA (sinapinic acid (s,5-dimethoxy-4-hydroxy cinnamic acid); TCA (trichloroacetic acid); Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP (Tris[2-carboxyethyl] phosphine); Ci (Curies); mCi (milliCuries); μCi (microCuries); HPLC (high pressure liquid chromatography); RP-HPLC (reverse phase high pressure liquid chromatography); TLC (thin layer chromatography); MALDI-TOF (matrix-assisted laser desorption/ionization—time of flight); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (Therms aquaticus DNA polymerase); Klenow (DNA polymerase I large (Klenow) fragment); EGTA (ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid); EDTA (ethylenediaminetetracetic acid); bla (β-lactamase or ampicillin-resistance gene); HDL (high density liquid); MJ Research (MJ Research, Reno, Nev.); Baseclear (Baseclear BV, Inc., Leiden, the Netherlands); PerSeptive (PerSeptive Biosystems, Framingham, Mass.); ThermoFinnigan (ThermoFinnigan, San Jose, Calif.); Argo (Argo BioAnalytica, Morris Plains, N.J.);Seitz EKS (SeitzSchenk Filtersystems GmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East Hills, N.Y.); Spectrum (Spectrum Laboratories, Dominguez Rancho, Calif.); Molecular Structure (Molecular Structure Corp., Woodlands, Tex.); Accelrys (Accelrys, Inc., San Diego, Calif.); Chemical Computing (Chemical Computing Corp., Montreal, Canada); New Brunswick (New Brunswick Scientific, Co., Edison, N.J.); CFT (Center for Test Materials, Vlaardingen, the Netherlands); Test Fabrics (Test Fabrics, Inc., West Pittiston, Pa.), Procter & Gamble (Procter & Gamble, Inc., Cincinnati, Ohio); Epicentre (Epicentre Biotechnologies, Madison, Wis.); GE Healthcare (GE Healthcare, Chalfont St. Giles, United Kingdom); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (Megazyme International Ireland Ltd., Bray Business Park, Bray, Co., Wicklow, Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP Kelco, Wilmington, Del.); Corning (Corning Life Sciences, Corning, N.Y.); (NEN (NEN Life Science Products, Boston, Mass.); Pharma AS (Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis, Lewisville, Tex.); ATCC (American Type Culture Collection, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island , N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Piscataway, N.J.); NCBI (National Center for Biotechnology Information); Applied Biosystems (Applied Biosystems, Foster City, Calif.); BD Biosciences and/or Clontech (BD Biosciences CLONTECH Laboratories, Palo Alto, Calif.); Operon Technologies (Operon Technologies, Inc., Alameda, Calif.); MWG Biotech (MWG Biotech, High Point, N.C.); Oligos Etc (Oligos Etc. Inc, Wilsonville, Oreg.); Bachem (Bachem Bioscience, Inc., King of Prussia, Pa.); Difco (Difco Laboratories, Detroit, Mich.); Mediatech (Mediatech, Herndon, Va.; Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); Oxoid (Oxoid Inc., Ogdensburg, N.Y.); Worthington (Worthington Biochemical Corp., Freehold, N.J.); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp., San Diego, Calif.); New England Biolabs and NEB (New England Biolabs, Ipswich, Mass.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pierce (Pierce Biotechnology, Rockford, Ill.); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.); Biodesign (Biodesign Intl., Saco, Me.); Aptagen (Aptagen, Inc., Herndon, Va.); Sorvall (Sorvall brand, from Kendro Laboratory Products, Asheville, N.C.); United States Testing (United States Testing Co., Hoboken, N.J.);Molecular Devices (Molecular Devices, Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems, Minneapolis, Minn.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Marsh (Marsh Biosciences, Rochester, N.Y.); Geneart (Geneart GmbH, Regensburg, Germany); DNA2.0 (DNA2.0, Menlo Park, Calif.); Gene Oracle (Gene Oracle, Mountain View, Calif.); Zymo Research (Zymo Research, Orange, Calif.); Bio-Tek (Bio-Tek Instruments, Winooski, Vt.); Biacore (Biacore, Inc., Piscataway, N.J.); PeproTech (PeproTech, Rocky Hill, N.J.); SynPep (SynPep, Dublin, Calif.); New Objective (New Objective brand; Scientific Instrument Services, Inc., Ringoes, N.J.); Waters (Waters, Inc., Milford, Mass.); Matrix Science (Matrix Science, Boston, Mass.); Dionex (Dionex, Corp., Sunnyvale, Calif.); Monsanto (Monsanto Co., St. Louis, Mo.); Wintershall (Wintershall AG, Kassel, Germany); BASF (BASF Co., Florham Park, N.J.); Huntsman (Huntsman Petrochemical Corp., Salt Lake City, Utah); Enichem (Enichem Iberica, Barcelona, Spain); Fluka Chemie AG (Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV, Delft, the Netherlands); Dow Corning (Dow Corning Corp., Midland, Mich.); and Microsoft (Microsoft, Inc., Redmond, Wash.).
    • EXAMPLE 1 Assays
  • In the following Examples, various assays were used as set forth below for ease in reading. Any deviations from the protocols provided below are indicated in the Examples. In the present invention, the subtilisin numbering corresponds to that of the mature BPN′ sequence, as set forth below:
  • (SEQ ID NO: 1)
    AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGAS
    MVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGA
    DGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGV
    VVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPEL
    DVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQ
    VRSSLENTTTKLGDSFYYGKGLINVQAAAQ 

    A. TCA Assay for Protein Content Determination in 96-well Microtiter Plates
  • For BPN′ (e.g., reference protease) and BPN′ variants, this assay was started using filtered culture supernatant from microtiter 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 μL/well of 0.25 N HCl was placed in each well. Then, 50 μL 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. For the test, 100 μL/well of 15% (w/v) trichloroacetic acid (TCA) was placed in the plates and incubated between 5 and 30 min at room temperature. The light scattering/absorbance at 405 rim (use 5 sec mixing mode in the plate reader) was then determined
  • 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).
  • The calculations were performed by subtracting the blank (no TCA) from the test reading with TCA to provide a relative measure of the protein content in the samples. If desired, a standard curve can be created by calibrating the TCA readings with AAPF assays of clones with known conversion factors. However, 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 turbidity/light scatter increase in the samples correlates to the total amount of precipitable protein in the culture supernatant.
  • B. AAPF Protease Assay in 96-well Microtiter Plates
  • In order to determine the protease activity of the proteases and variants thereof of the present invention, the hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide (suc-AAPF-pNA) was measured. 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 CaCl2 and 0.005% TWEEN®-80 (Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare a 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 ml of diluted protease solution to each well, immediately followed by the addition of 190 μl 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. The protease activity was expressed as AU (activity=ΔOD·min−1 ml−1).
  • C. Surfactant and Chelant Stability Assays
  • 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.
    • LAS Stability Method Reagents:
    • Dodecylbenzenesulfonate, Sodium salt (=LAS): Sigma D-2525
    • TWEEN®-80: Sigma P-8074
    • 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.
    • LAS stock solution: Prepare a 10.5% LAS solution in MQ water (=10.5 g per 100 ml MQ)
    • TRIS buffer-100 mM/pH 8.6 (100 mM Tris/0.005% Tween®-80)
    • TRIS-Ca buffer, pH 8.6 (100 mM Tris/10 mM CaCl2/0.005% Tween-®80)
    Hardware:
  • Flat bottom MTPs (Costar No. 9017)
    • Biomek FX
    • ASYS Multipipettor
    • Spectramax MTP Reader
    • iEMS Incubator/Shaker
    • Innova 4330 Incubator/Shaker
    • Biohit multichannel pipette
    • BMG Thermostar Shaker
  • 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.
  • Ten μl of the diluted supernatant were added to 190 μl 0.063% LAS buffer/well. The MTP was covered with tape, shaken for a few seconds and placed in an incubator (Innova 4230) at 45° C. for for 60 minutes at 200 rpm agitation. The initial activity (t=10 minutes) was determined after 10 minutes of incubation by transferring 10 μl of the mixture in each well to a fresh MTP containing 190μl suc-AAPF-pNA work solution. These solutions were mixed well and the AAPF activity was measured using a MTP Reader (20 readings in 5 minutes and 25° C.).
  • The final activity (t=60 minutes) was determined by removing another 10 μl of solution from the incubating plate after 60 minutes of incubation. 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:

  • Residual Activity (%)=[t−60 value]*100/[t−10 value].
    • LAS/EDTA Stability Method
  • The stability of protease variants in the presence of a representative anionic surfactant (LAS=linear alkylbene sulfonate, sodium dodecylbenzenesulfonate-DOBS) and di-sodium EDTA was measured after incubation under defined conditions and the residual activity was determined using the AAPF assay. 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 g/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 μg/ml protein. The equipment used was 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) from 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 μl of sample from the master dilution plate was added to plates containing 180 pl 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. 20 μl of sample from the master dilution plate was also added to plates containing 180 μl 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). The solutions were mixed and immediately placed in 29° C. iEMS shaker for 30 min at 400 rpm. Following 30 minutes of incubation, a AAPF assay was performed on the stress plate. The stability of the samples was determined by calculating the ration of the residual and initial AAPF activity as follows: Residual Activity (%)=[mOD.min-1 stressed]*100/[mOD. min-1 unstressed].
    • D. Cleaning Performance Assays
  • 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:
  • Microswatches of ¼″ 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).
    • BMI Microswatch Assay
  • 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 microtiter 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 μl of detergent solution was added to each well of the MTP, containing microswatches. To this mixture, 10 μl of the diluted enzyme solution was added so that the final enzyme concentration was 1 μg/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 μl of the solution from each well was transferred into a fresh MTP. The new MTP containing 100 μl 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.
    • “Pre-washed” Swatch
  • This type of 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 ¼″ 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).
    • Detergents
  • For North American (NA) and Western European (WE) heavy duty liquid laundry (HDL) detergents, heat inactivation was performed by placing pre-weighed liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The detergents were purchased from local supermarket stores. Both un-heated and heated detergents were assayed within 5 minutes of dissolving the detergent to accurately determine percentage deactivated. Enzyme activity was tested by the AAPF assay.
  • For testing of enzyme activity in heat-inactivated detergents, working solutions of detergents were made from the heat inactivated stocks. Appropriate amounts of water hardness (6 gpg or 12 gpg) and buffer were added to the detergent solutions to match the desired conditions. The solutions were mixed by vortexing or inverting the bottles.
    • Enzymes and Equipment
  • 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). In this assay, the proteases hydrolyze the substrate and liberate pigment and insoluble particles from the substrate. Thus the rate of turbidity is a measure of enzyme activity.
  • The stain removal performance of reference serine proteases and variants therefrom on microswatches was determined on a MTP scale in commercially available heat-inactivated detergent. 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. (CaCl2: MgCl2.6H2O); 15000 grains per gallon (gpg) stock diluted to 6 gpg, 2 BMI (blood/milk/ink) swatches per plate: EMPA-116 BMI cotton swatches processed by CFT: pre-rinsed and punched 2 swatches per well, and heat inactivated TIDE® 2X Cold off-the-shelf detergent in which lack of protease activity was confirmed.
  • TABLE 1-2
    Working Detergent Solutions
    Temp Detergent
    Detergent (C.) g/L pH Buffer Gpg Protease
    TIDE ® 2X Cold 16 0.98 8 5 mM 6 BPN′
    HEPES
    TIDE ® 2X Cold 32 0.98 8 5 mM 6 BPN′
    HEPES
    TIDE ® 2X Cold 16 0.98 7 5 mM 6 BPN′
    MOPS
  • The incubator was set at the desired temperature (16° C. or 32° C.). 10 μL samples from the master dilution plate of 18 10 ppm enzyme was added to BMI 2-swatch plates with 190 μL 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 μL 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.
    • Calculation of the Stain Removal Activity for All Microswatch Assay Methods:
  • The absorbance value obtained was corrected for the blank value (substrate without enzyme), providing a measure of hydrolytic activity. For each sample (variant) 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. In addition, the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard enzyme.
    • E. Relative Specific Activity of Proteases and Variants Thereof
  • In order to discriminate the protease variants, the relative specific activity was calculated using 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).
    • F. Performance Index
  • The performance index compares the performance of the variant (actual value) and the standard or reference protease (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard protease. A performance index (PI) that is greater than 1 (PI>1) identifies a better variant as compared to the standard (e.g., wild-type), while a PI of 1 (PI=1) identifies a variant that performs the same as the standard, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the standard. Thus, the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.
    • EXAMPLE 2 Targeted ISD (Insertion Substitution Deletion) Library Construction
  • A PCR-based method was used to create a library of modified B. amyloliquefaciens subtilisin BPN′-Y217L (commercially available as PURAFECT® PRIME subtilisin) polynucleotides containing in-frame insertions, deletions and/or substitutions in two regions of BPN′-Y217L (positions 63-77 and 92-132 of the mature region) as shown in FIG. 1 (See, Pisarchik et al., Prot. Eng. Des. Select., 20:257-265 [2007]). Two sets of oligonucleotides that evenly cover the targeted regions of the BPN′-Y217L gene of a full-length protein of 392 amino acids, in both forward and reverse direction were used to amplify the 5′ and 3′ segments of the portion of the BPN′-Y217L gene. The coding region of the BPN′-Y217L mature protease contains the Kpnl and Xhol restriction sites for cloning purposes:
  • (SEQ ID NO: 3)
    Figure US20150056682A1-20150226-P00001
    Figure US20150056682A1-20150226-P00002
    Figure US20150056682A1-20150226-P00003
    Figure US20150056682A1-20150226-P00004
    Figure US20150056682A1-20150226-P00005
    Figure US20150056682A1-20150226-P00006
    gtgagaagcaaaaaattgtggatcagtttgctgtttgctttagcgttaatctttacgatggcgttcggc
    agcacatccagcgcgcaggctgcagggaaatcaaacggggaaaagaaatatattgtcgggtttaaacagacaatgagcacgatgagcgccgctaaga
    agaaagacgtcatttctgaaaaaggcgggaaagtgcaaaagcaattcaaatatgtagacgcaptagcgctacattaaacgaaaaagagtaaaagaatt
    gaaaaaagacccgagcgtcgcttacgttgaagaagatcacgtagcacacgcgtac gcgcagtccgtgccatatggcgtatcacaaattaaagcccc
    tgctctgcactctcaaggctacaccggttcaaatgttaaagtagcggttatcgacagcggtatcgattcttctcatccagatcttaaagtagcagg
    cggagccagcatggttccttctgaaacaaatcattccaagacaacaactctcacggaacacacgttgctggtaccgttgcggctcttaataactc
    aatcggtgtattaggcgttgcgccaagcgcatcactttacgctgtaaaagttctcggcgccgacggttccggccaatacagctggatcattaacg
    gaatcgagtgggcgatcgcaaacaatatggacgttattaacatgagcctcggcggaccgtccggttctgctgctttaaaagcggcagttgataa
    agccgttgcatccggcgtcgtagtcgttgcggcagccggcaacgaaggcacttccggcagctcaagcacagtgggctaccctggtaaataccct
    tctgtcattgcagtaggcgctgtcgacagcagcaaccaaagagcatctttctcaagcgtaggacctgagctcgatgtcatggcacctggcgtatc
    tatccaaagcacgcttcctggaaacaaatacggcgcgttgaacggtacatcaatggcatctccgcacgttgccggagccgcggctttgattcttt
    ctaagcacccgaactggacaaacactcaagtccgcagctctctagaaaacaccactacaaaacttggtgattctttctactatggaaaagggct
    gatcaatgtacaggcggcagctcagtaa aactcgagataaaaaaccggccttggccccgccggttttttat .
  • The amino acid sequence of the BPN′-Y217L precursor protein is provided below. In this sequence, bold indicates the mature BPN′-Y217L protease:
  • (SEQ ID NO: 4)
    MRSKKLWISLLFALALIFTMAFGSTSSAQAAGKSNGEKKYIVGFKQTMS
    TMSAAKKKDVISEKGGKVQKQFKYVDAASATLNEKAVKELKKDPSVAYV
    EEDHVAHAYAQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHP
    DLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSAS
    LYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAA
    VDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRA
    SFSSVGPELDVMAPGVSIQSTLPGNKYGALNGTSMASPHVAGAAALILS
    KHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ.
  • The amino acid sequence of the mature BPN′-Y217L protease was used as the basis for making the variant libraries described herein:
  • (SEQ ID NO: 5)
    AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGAS
    MVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGA
    DGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGV
    VVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPEL
    DVMAPGVSIQSTLPGNKYGALNGTSMASPHVAGAAALILSKHPNWTNTQ
    VRSSLENTTTKLGDSFYYGKGLINVQAAAQ.
  • Each oligonucleotide contained about 20-24 nucleotides necessary for annealing to the template, targeted codons (with or without degeneracy) and the Eam1104I recognition sequence. Every following primer was moved 3 by downstream until the end of the targeted region was reached. Two PCR reactions (5′ and 3′ segments) contained either the 5′ forward or the 3′ reverse gene sequence flanking oligonucleotides each in combination with the corresponding oppositely priming oligonucleotides. The 5′ fragments were amplified from pAC-FNA10 plasmid (See, FIG. 2A) using a single forward primer (P3203, TGGATCAGTTTGCTGTTTGCT; SEQ ID NO:6) and reverse primers P4385-P4441 (See, Table 2-1) each containing the Eam11041 restriction site. The 3′ fragments were amplified using a single reverse primer (P3435, ATGTATCAAGATAAGAAAGAACAAG; SEQ ID NO:7) and forward primers P4328-P4384 (See, Table 2-1) each containing an Eam1104I restriction site.
  • TABLE 2-1 
    Primers Used for BPN′-Y217L Library Construction
    PRIMER
    SEQ ID NO. NAME PRIMER SEQUENCE
    8 P4328 AAACTCTTCANDTNDTCACGGAACTCACGTTGCCGGCACA
    9 P4329 AAACTCTTCANDTNDTGGAACTCACGTTGCCGGCACAGTT
    10 P4330 AAACTCTTCANDTNDTACTCACGTTGCCGGCACAGTTG
    11 P4331 AAACTCTTCANDTNDTCACGTTGCCGGCACAGTTGCGGCT
    12 P4332 AAACTCTTCANDTNDTGTTGCCGGCACAGTTGCGGCTCTT
    13 P4333 AAACTCTTCANDTNDTGCCGGCACAGTTGCGGCTCTTAAT
    14 P4334 AAACTCTTCANDTNDTCGGCACAGTTGCGGCTCTTAATAAC
    15 P4335 AAACTCTTCANDTNDTACAGTTGCGGCTCTTAATAACTCA
    16 P4336 AAACTCTTCANDTNDTGTTGCGGCTCTTAATAACTCAATC
    17 P4337 AAACTCTTCANDTNDTGCGGCTCTTAATAACTCAATCGGT
    18 P4338 AAACTCTTCANDTNDTGCTCTTAATAACTCAATCGGTGTA
    19 P4339 AAACTCTTCANDTNDTCTTAATAACTCAATCGGTGTATTA
    20 P4340 AAACTCTTCANDTNDTAATAACTCAATCGGTGTATTAG
    21 P4341 AAACTCTTCANDTNDTAACTCAATCGGTGTATTAGGCGTT
    22 P4342 AAACTCTTCANDTNDTTCAATCGGTGTATTAGGCGTTG
    23 P4343 AAACTCTTCANDTTCAATCGGTGTATTAGGCGTTG
    24 P4344 AAACTCTTCANDTNDTAAAGTTCTCGGTGCTGACGGTTC
    25 P4345 AAACTCTTCANDTNDTGTTCTCGGTGCTGACGGTTCC
    26 P4346 AAACTCTTCANDTNDTCTCGGTGCTGACGGTTCCGGCCAA
    27 P4347 AAACTCTTCANDTNDTGGTGCTGACGGTTCCGGCCAATAC
    28 P4348 AAACTCTTCANDTNDTGCTGACGGTTCCGGCCAATACA
    29 P4349 AAACTCTTCANDTNDTGACGGTTCCGGCCAATACAGCTG
    30 P4350 AAACTCTTCANDTNDTGGTTCCGGCCAATACAGCTGGATC
    31 P4351 AAACTCTTCANDTNDTTCCGGCCAATACAGCTGGATCATT
    32 P4352 AAACTCTTCANDTNDTGGCCAATACAGCTGGATCATTAAC
    33 P4353 AAACTCTTCANDTNDTCAATACAGCTGGATCATTAACGGA
    34 P4354 AAACTCTTCANDTNDTTACAGCTGGATCATTAACGGAATC
    35 P4355 AAACTCTTCANDTNDTAGCTGGATCATTAACGGAATCGAG
    36 P4356 AAACTCTTCANDTNDTTGGATCATTAACGGAATCGAGTG
    37 P4357 AAACTCTTCANDTNDTATCATTAACGGAATCGAGTG
    38 P4358 AAACTCTTCANDTNDTATTAACGGAATCGAGTGGGCGATC
    39 P4359 AAACTCTTCANDTNDTAACGGAATCGAGTGGGCGATCGCA
    40 P4360 AAACTCTTCANDTNDTGGAATCGAGTGGGCGATCGCAAAC
    41 P4361 AAACTCTTCANDTNDTATCGAGTGGGCGATCGCAAACAAT
    42 P4362 AAACTCTTCANDTNDTGAGTGGGCGATCGCAAACAATATG
    43 P4363 AAACTCTTCANDTNDTTGGGCGATCGCAAACAATATGGAC
    44 P4364 AAACTCTTCANDTNDTGCGATCGCAAACAATATGGACGTT
    45 P4365 AAACTCTTCANDTNDTATCGCAAACAATATGGACGTTATT
    46 P4366 AAACTCTTCANDTNDTGCAAACAATATGGACGTTATTAAC
    47 P4367 AAACTCTTCANDTNDTAACAATATGGACGTTATTAACATG
    48 P4368 AAACTCTTCANDTNDTAATATGGACGTTATTAACATGAG
    49 P4369 AAACTCTTCANDTNDTATGGACGTTATTAACATGAGCCTC
    50 P4370 AAACTCTTCANDTNDTGACGTTATTAACATGAGCCTC
    51 P4371 AAACTCTTCANDTNDTGTTATTAACATGAGCCTCGGCGGA
    52 P4372 AAACTCTTCANDTNDTATTAACATGAGCCTCGGCGGACCT
    53 P4373 AAACTCTTCANDTNDTAACATGAGCCTCGGCGGACCTTCT
    54 P4374 AAACTCTTCANDTNDTATGAGCCTCGGCGGACCTTCTGGT
    55 P4375 AAACTCTTCANDTNDTAGCCTCGGCGGACCTTCTGGTTCT
    56 P4376 AAACTCTTCANDTNDTCTCGGCGGACCTTCTGGTTCTGCT
    57 P4377 AAACTCTTCANDTNDTGGCGGACCTTCTGGTTCTGCTGCT
    58 P4378 AAACTCTTCANDTNDTGGACCTTCTGGTTCTGCTGCTTTA
    59 P4379 AAACTCTTCANDTNDTCCTTCTGGTTCTGCTGCTTTAAAA
    60 P4380 AAACTCTTCANDTNDTTCTGGTTCTGCTGCTTTAAAAG
    61 P4381 AAACTCTTCANDTNDTGGTTCTGCTGCTTTAAAAGCGGCA
    62 P4382 AAACTCTTCANDTNDTTCTGCTGCTTTAAAAGCGGCAGTT
    63 P4383 AAACTCTTCANDTNDTGCTGCTTTAAAAGCGGCAGTTGAT
    64 P4384 AAACTCTTCANDTGCTGCTTTAAAAGCGGCAGTTGAT
    65 P4385 AAACTCTTCAAHNGTTGTCTTGGAAAGGATTTGTTTC
    66 P4386 AAACTCTTCAAHNAHNGTTGTCTTGGAAAGGATTTGTTTC
    67 P4387 AAACTCTTCAAHNAHNGTTGTTGTCTTGGAAAGGATTTGT
    68 P4388 AAACTCTTCAAHNAHNAGAGTTGTTGTCTTGGAAAGGATT
    69 P4389 AAACTCTTCAAHNAHNGTGAGAGTTGTTGTCTTGGAAAGG
    70 P4390 AAACTCTTCAAHNAHNTCCGTGAGAGTTGTTGTCTTGGAA
    71 P4391 AAACTCTTCAAHNAHNAGTTCCGTGAGAGTTGTTGTCTTG
    72 P4392 AAACTCTTCAAHNAHNGTGAGTTCCGTGAGAGTTGTTGTC
    73 P4393 AAACTCTTCAAHNAHNAACGTGAGTTCCGTGAGAGTTGTT
    74 P4394 AAACTCTTCAAHNAHNGGCAACGTGAGTTCCGTGAGAGTT
    75 P4395 AAACTCTTCAAHNAHNGCCGGCAACGTGAGTTCCGTGAGA
    76 P4396 AAACTCTTCAAHNAHNTGTGCCGGCAACGTGAGTTCCGTG
    77 P4397 AAACTCTTCAAHNAHNAACTGTGCCGGCAACGTGAGTTCC
    78 P4398 AAACTCTTCAAHNAHNCGCAACTGTGCCGGCAACGTGAGT
    79 P4399 AAACTCTTCAAHNAHNAGCCGCAACTGTGCCGGCAACGTG
    80 P4400 AAACTCTTCAAHNAHNAAGAGCCGCAACTGTGCCGGCAAC
    81 P4401 AAACTCTTCAAHNGTAAAGTGATGCGCTTGGCGCAAC
    82 P4402 AAACTCTTCAAHNAHNGTAAAGTGATGCGCTTGGCGCAAC
    83 P4403 AAACTCTTCAAHNAHNAGCGTAAAGTGATGCGCTTG
    84 P4404 AAACTCTTCAAHNAHNTACAGCGTAAAGTGATGCGCTTG
    85 P4405 AAACTCTTCAAHNAHNTTTTACAGCGTAAAGTGATGCGCT
    86 P4406 AAACTCTTCAAHNAHNAACTTTTACAGCGTAAAGTGATG
    87 P4407 AAACTCTTCAAHNAHNGAGAACTTTTACAGCGTAAAGTGA
    88 P4408 AAACTCTTCAAHNAHNACCGAGAACTTTTACAGCGTAAAG
    89 P4409 AAACTCTTCAAHNAHNAGCACCGAGAACTTTTACAGCGTA
    90 P4410 AAACTCTTCAAHNAHNGTCAGCACCGAGAACTTTTACAG
    91 P4411 AAACTCTTCAAHNAHNACCGTCAGCACCGAGAACTTTTAC
    92 P4412 AAACTCTTCAAHNAHNGGAACCGTCAGCACCGAGAACTTT
    93 P4413 AAACTCTTCAAHNAHNGCCGGAACCGTCAGCACCGAGAAC
    94 P4414 AAACTCTTCAAHNAHNTTGGCCGGAACCGTCAGCACCGAG
    95 P4415 AAACTCTTCAAHNAHNGTATTGGCCGGAACCGTCAGCAC
    96 P4416 AAACTCTTCAAHNAHNGCTGTATTGGCCGGAACCGTCAG
    97 P4417 AAACTCTTCAAHNAHNCCAGCTGTATTGGCCGGAACCGTC
    98 P4418 AAACTCTTCAAHNAHNGATCCAGCTGTATTGGCCGGAAC
    99 P4419 AAACTCTTCAAHNAHNAATGATCCAGCTGTATTGGCCGGA
    100 P4420 AAACTCTTCAAHNAHNGTTAATGATCCAGCTGTATTG
    101 P4421 AAACTCTTCAAHNAHNTCCGTTAATGATCCAGCTGTATTG
    102 P4422 AAACTCTTCAAHNAHNGATTCCGTTAATGATCCAGCTGTA
    103 P4423 AAACTCTTCAAHNAHNCTCGATTCCGTTAATGATCCAGCT
    104 P4424 AAACTCTTCAAHNAHNCCACTCGATTCCGTTAATGATCCA
    105 P4425 AAACTCTTCAAHNAHNCGCCCACTCGATTCCGTTAATGAT
    106 P4426 AAACTCTTCAAHNAHNGATCGCCCACTCGATTCCGTTAAT
    107 P4427 AAACTCTTCAAHNAHNTGCGATCGCCCACTCGATTCCGTT
    108 P4428 AAACTCTTCAAHNAHNGTTTGCGATCGCCCACTCGATTCC
    109 P4429 AAACTCTTCAAHNAHNATTGTTTGCGATCGCCCACTCGAT
    110 P4430 AAACTCTTCAAHNAHNCATATTGTTTGCGATCGCCCACTC
    111 P4431 AAACTCTTCAAHNAHNGTCCATATTGTTTGCGATCGCCCA
    112 P4432 AAACTCTTCAAHNAHNAACGTCCATATTGTTTGCGATCGC
    113 P4433 AAACTCTTCAAHNAHNAATAACGTCCATATTGTTTGCGAT
    114 P4434 AAACTCTTCAAHNAHNGTTAATAACGTCCATATTGTTTGC
    115 P4435 AAACTCTTCAAHNAHNCATGTTAATAACGTCCATATTGTT
    116 P4436 AAACTCTTCAAHNAHNGCTCATGTTAATAACGTCCATATT
    117 P4437 AAACTCTTCAAHNAHNGAGGCTCATGTTAATAACGTCCAT
    118 P4438 AAACTCTTCAAHNAHNGCCGAGGCTCATGTTAATAACGTC
    119 P4439 AAACTCTTCAAHNAHNTCCGCCGAGGCTCATGTTAATAAC
    120 P4440 AAACTCTTCAAHNAHNAGGTCCGCCGAGGCTCATGTTAAT
    121 P4441 AAACTCTTCAAHNAHNAGAAGGTCCGCCGAGGCTCATGTT
  • Each amplification reaction contained 30 pmol of each oligonucleotide and 100 ng of pAC-FNA10 template DNA. Amplifications were carried out using Vent DNA polymerase (New England Biolabs). The PCR mix (20 ul) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15s, annealing at 55° C. for 15s and extension at 72° C. for 40s. Following amplification, left and right fragments generated by the PCR reactions were gel-purified and mixed (about 200 ng of each fragment). Every mix contained three left fragments targeting three adjacent codons and three right fragments targeting same codons. These mixes were digested with Earn11041, ligated with T4 DNA ligase and amplified by flanking primers (P4299 CGTTGAAGAAGATCACGTAGCA; SEQ ID NO:122, and P3246 TTTATTTTATAAACTCATTCCCTGAT; SEQ ID NO:123). The resulting fragments were digested with MluI and XhoI, and cloned into the MluII XhoI sites in the Bacillus subtilis expression plasmid pHPLT- FNA (FIG. 2B). The BPN′-Y217L expression cassette from the pHPLT vector used has the polynucleotide sequence shown below.
  • The polynucleotide sequence of the expression cassette from the pHPLT vector (Plat promoter-pre-pro-BPN′-Y217L -terminator) (SEQ ID NO. 124) is shown below:
  • (SEQ ID NO: 124)
    GCTTTTCTTTTGGAAGAAAATATAGGGAAAATGGTACTTGTTAAAAATTCGGAATATTTATA
    CAATATCATATGTTTCACATTGAAAGGGGAGGAAAATCGTGAAACAACAAAAACGGCTTTA
    GTCTAGCAAAAGGAGAGGGTAAAGAGTGAGAAGCAAAAAATTGTGGATCAGTTTGCTGTTT
    GCTTTAGCGTTAATCTTTACGATGGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAA
    ATCAAACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCACGATGAGCGCC
    GCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATG
    TAGACGCAGCTTCAGCTACATTAAACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAG
    CGTCGCTTACGTTGAAGAAGATCACGTAGCACACGCGTACGCGCAGTCCGTGCCTTACGGCG
    TATCACAAATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAGTA
    GCGGTTATCGACAGCGGTATCGATTCTTCTCATCCTGATTTAAAGGTAGCAGGCGGAGCCAG
    CATGGTTCCTTCTGAAACAAATCCTTTCCAAGACAACAACTCTCACGGAACTCACGTTGCCG
    GCACAGTTGCGGCTCTTAATAACTCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTT
    TACGCTGTAAAAGTTCTCGGTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAAT
    CGAGTGGGCGATCGCAAACAATATGGACGTTATTAACATGAGCCTCGGCGGACCTTCTGGTT
    CTGCTGCTTTAAAAGCGGCAGTTGATAAAGCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCA
    GCCGGTAACGAAGGCACTTCCGGCAGCTCAAGCACAGTGGGCTACCCTGGTAAATACCCTTC
    TGTCATTGCAGTAGGCGCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTCAAGCGTAGGAC
    CTGAGCTTGATGTCATGGCACCTGGCGTATCTATCCAAAGCACGCTTCCTGGAAACAAATAC
    GGCGCGTTGAACGGTACATCAATGGCATCTCCGCACGTTGCCGGAGCGGCTGCTTTGATTCT
    TTCTAAGCACCCGAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACA
    AAACTTGGTGATTCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGTA
    AACTCGAGAGAGGACGGATTTCCTGAAGGAAATCCGTTTTTTTATTTTAAGCTTG 
  • Ligation mixtures were amplified using rolling circle amplification according to manufacturer's recommendation (Epicentre). One ul of the ligation mixture was mixed with 5 ul of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 ul of the reaction buffer and 0.2 ul of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform Bacillus subtilis. Bacillus subtilis cells (genotype: AaprE, AnprE, AspolIE, amyE::xylRPxylAcomK-phleo) made competent by the induction of the comK gene under control of a xylose inducible promoter (See e.g., Hahn et al., Mol Microbiol, 21:763-775 [1996]) were transformed with 45 libraries containing DNA sequences encoding BPN′-Y217L protease with mutated regions. The cells were grown in 1 ml of Luria Broth (LB) at 37° C. for 1 hour and then plated on LB plates containing 1.6% skim milk and 10 mg/l neomycin, and were incubated overnight at 37° C. At least 2000 clones from each of the 45 libraries were screened for producing halos. When plated on skim milk plates, 0.05% to 20% of clones generated halos. A limited number of the clones generating halos were sequenced. These clones varied in the ratio of insertions and deletions, but none of them had frame shift mutations. Clones generating halos were further screened for AAPF activity using a 96-well plate assay as described in Example 1. The BPN′-Y217L variant proteins were produced by growing the B. subtilis transformants in 96 well microtiter plates as described earlier. Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
    • EXAMPLE 3 Stain Removal Performance of BPN′-Y217L Variants Generated by Targeted ISD
  • Results of experiments conducted to determine stain removal activity (microswatch assay to determine stain removal performance in laundry applications using EMPA 116 swatches (BMI stain, CFT) at pH 8/16° C. and protein determination by TCA precipitation (tests of properties of interest) of BPN′-Y217L variants generated as described above are shown in Table 3-1. The results were obtained using the methods described in Example 1, with the following modifications for the stain removal performance assay. The test detergent used was heat inactivated Tide 2X 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 to accurately determine percentage deactivated. Enzyme activity was tested by AAPF assay. As described throughout functionality of BPN′-Y217L variants was quantified as a performance index (“Pi” or “PI”), which is the ratio of performance of a variant to parent protein BPN′-Y217L. BPN′-Y217L variants showing a PI value greater or equal than 0.5 for BMI stain removal performance and/or TCA precipitation showed improved cleaning benefits and/or expression.
  • Mutations are named by the one letter code for the parent amino acid, followed by a three digit position number and then the one letter code for the variant amino acid. For example, mutating glycine (G) at position 87 to serine (S) is represented as “G087S.” Multiple mutations are indicated by inserting a “I” between the mutations. Mutations at positions 87 and 90 are represented as “G087S/A090Y.” For deletions, the one letter code “Z” is used. For an insertion relative to the parent sequence, the one letter code “Z” is on the left side of the position number. For a deletion, the one letter code “Z” is on the right side of the position number. For insertions, the position number is the position number before the inserted amino acid(s), plus 0.01 for each amino acid. For example, an insertion of three amino acids alanine (A), serine (S) and tyrosine (Y) between position 87 and 88 is shown as “Z087.01A/Z087.02S/Z087.03Y.” Thus, combining all the mutations above plus a deletion at position 100 is: “G087S/ Z087.01A/Z087.02S/Z087.03Y/A090Y/A100Z.”
  • TABLE 3-1
    Mutations and Performance Index for TCA protein
    and BMI Activity for BPN'-Y217L Variants
    PI on BMI
    TCA at pH 8
    Mutations PI 16° C.
    A001T/L096V/G097S/A098D/D099I 0.45 1.1
    A001V/G097S/A098R/D099G 0.66 0.74
    S003F 1.08 0.89
    V004M/A098D/D099G 0.69 0.97
    V004M/D099R/G100S/Z100.01L 0.8 0.75
    V004M/G127C/G128S/P129R 0.92 0.52
    V004M/M119C/D120S 0.68 1
    V008I 0.97 0.985
    V008I/A098G/D099R 0.67 0.66
    V008I/A098N 0.52 1.14
    V008I/M119C/D120G 0.53 0.99
    Q010L/G097R/A098C/D099S 0.27 0.52
    Q010R 0.91 0.86
    K012N/A098R/D099V 0.46 0.8
    K012R/G097D/A098H/D099G 0.86 0.96
    A013T/S101R/Q103S 0.52 0.68
    A013V/G097N/A098H/D099G/G160S 0.46 0.74
    A013V/G097S/Z097.01R/A098R 0.46 0.64
    A013V/G100R/S101G 0.43 0.79
    A013V/N118G/M119I 0.5 0.85
    A013V/P129Z/S130H/S132G 0.47 0.58
    P014L/N062D/S101R/Q103S 0.41 1.02
    P014S/A098S/D099G 0.56 0.99
    A015T/V068N/A069G 0.52 0.38
    L016M/G128R/P129R/Z129.01G 0.5 0.28
    H017Q/G127R/P129G 0.54 0.43
    H017R 0.5 1.025
    H017Y/A098R/D099I 0.42 0.56
    H017Y/A098R/D099Z/S101G 0.6 0.47
    H017Y/P129R 0.685 0.85
    H017Y/S130Y/G131S/S132L 0.38 0.58
    Q019H/G128R/Z128.01R/P129D 0.4 0.78
    Q019H/Z128.01G/Z128.02G 0.56 0.29
    G020D/G097R/A098G/D099C 0.39 0.8
    G020D/P129Z/S130G/G131R/S132C 0.37 0.51
    N025Y/S101L/Q103N 1.05 1.13
    A029V/G097R/A098S 0.44 0.94
    S033G 0.9 1.05
    S033I/T066G/H067R/V068D/A216E 0.88 1.01
    S038F/A098R/D099S 0.98 0.6
    V044I/G097C/A098D/D099G 0.42 0.9
    V044I/G097R/A098R 0.83 0.87
    V044I/G100S/S101G 0.36 1.09
    A045V/G097C/A098R/D099S 0.41 0.88
    A048V/G097L/A098G/D099S 0.42 1
    A048V/G100R/S101G 0.65 1.085
    A048V/G128S/P129L/Z129.01G 0.45 0.68
    M050I/A098G 0.2 0.54
    S053F/S130I/S132G 0.65 0.54
    S053T/D099H/S101Z 0.66 0.74
    E054D/A098R/D099G 1.05 0.76
    T055I/S101R/Q103R 0.58 0.51
    T055S/T066I/Z066.01G/Z066.02G 0.33 0.7
    Q059H/D099S/S101G 0.36 0.79
    Q059H/M119S/D120R 0.37 0.52
    Q059K/M119V/V121C 0.23 0.58
    Q059L/P129G 0.69 0.87
    D060N 0.47 0.9
    N061D/G128R/P129V/Z129.01G 0.59 0.43
    S063T/G097D/A098R/D099S 0.97 0.95
    G065R/Z101.01R/Z101.02G 0.57 1.035
    T066S/H067S 0.51 0.61
    V068C/A069G 0.42 1.22
    V068C/A069G/H238Q 0.32 0.62
    V068G/A069G 0.36 0.71
    V068G/A069G/V150I 0.33 0.59
    V068G/A069S 0.34 0.63
    V068I/A069G/A144V 0.46 0.98
    V068I/G097D/A098G/D099Z/G100R 0.41 0.58
    V068L/A069G 0.445 0.905
    V068L/A069S 0.47 0.83
    V068S/A069G 0.48 1.04
    V068S/A069G/A116T 0.4 1.05
    A069G 0.7 1.02
    A069G/T071G 0.23 0.84
    A069G/T071I 0.79 1
    L075G/N076G/N077Z 0.29 0.77
    L075I 0.78 0.94
    L075R/N076G/N077Z 0.34 0.99
    N076D/D099R/Z100.01G 0.28 0.63
    I079F/A098G 1.16 1.16
    V081A/S101R/Q103V 0.4 0.81
    V081I/G097R/A098G/D099C 0.36 0.54
    V084D/G097S/A098G/D099N 0.42 0.63
    V084I/G127C/P129G 1.28 1.01
    V084I/G127L 0.88 0.72
    A085V/A098G 0.75 1.09
    A085V/G097R/A098S/D099N 0.65 0.75
    A085V/G128S/P129R/Z129.01G 0.61 0.47
    A085V/N218I 0.435 0.845
    A085V/S101G 0.59 0.84
    A085V/S204P 0.43 0.78
    A085V/Z099.01H/S101R 0.46 0.6
    P086S/G131D/S132G 0.25 0.64
    P086T/S101D/Q103S/V147I 0.35 1.27
    A088S/S101R/Q103V 0.63 0.88
    A088T 0.855 1.01
    A088T/A098R/D099R 0.73 0.36
    A088T/D099G/Z100.01G 0.21 0.75
    A088T/G127N/P129R 0.99 0.8
    A088T/G127S/P129R/Q206R/A273S 1.22 0.67
    A088T/P129S/G146D 0.74 1.16
    A088T/S101G 1.13 1.08
    A088T/S 130R/G 131 H/S 132N 1.24 0.78
    A088V/G100Z/S101C/Q103R 0.52 0.46
    A088V/P129Z 0.61 1.05
    A092G 0.61 1.15
    A092G/S249R 0.69 1.07
    A092G/V093C 0.2 0.855
    A092G/V093I 0.5 1.15
    A092G/V093L 0.275 0.75
    A092L/V093G/K094Z 0.54 0.96
    A092S/K094V 0.47 1.13
    A092S/V093C 0.72 1.06
    A092S/V093G 0.21 0.79
    A092S/V093S 0.33 0.67
    A092V/V093L 0.34 0.695
    V093I/G128D/P129R 0.6 1.22
    V093I/P129Z 0.6 0.92
    V093R/Z093.01G/K094R 0.28 0.88
    K094E/V095D 0.87 0.95
    K094G 0.33 0.56
    K094S/V148I 0.3 0.85
    L096I/N118R/M119C/D120G/A151V 0.29 0.58
    Z096.01D/A098R 0.66 1.2
    Z096.01H/A098G 0.32 1.16
    Z096.01N/A098H 0.65 1.05
    Z096.01R/A098C 0.47 0.95
    Z096.01R/A098G 0.39 0.82
    Z096.01R/A098G/V192I 0.4 0.77
    Z096.01R/A098R 0.60 0.70
    Z096.01R/A098R/V192A 0.61 0.68
    Z096.01R/A098S 0.47 0.92
    Z096.01R/A098S/G157S 0.3 0.88
    Z096.01R/Z096.02G 0.52 0.67
    Z096.01R/Z096.02G/V147I 0.41 0.96
    Z096.01S/A098G 0.44 1.12
    Z096.01S/A098N 0.47 1.04
    G097C/A098D/D099S 0.675 1.09
    G097C/A098G/D099G 0.42 0.86
    G097C/A098G/D099N 0.82 1.07
    G097C/A098G/D099S 0.45 0.95
    G097C/A098G/D099S/G100Z 0.32 0.64
    G097C/A098G/D099V 0.43 0.88
    G097C/A098N/D099S 0.45 0.94
    G097C/A098R 1.24 1.11
    G097C/A098R/D099G 0.40 0.84
    G097C/A098R/D099H 0.45 0.64
    G097C/A098R/D099H 0.46 0.78
    G097C/A098R/D099N 0.79 0.87
    G097C/A098R/D099R 0.4 0.55
    G097C/A098R/D099R/N109D/A114S 0.56 0.5
    G097C/A098S/D099H/A151V 0.32 0.65
    G097C/A098V/D099S 0.2 0.81
    G097C/Z097.01G/A098H/T253I 0.32 0.58
    G097C/Z097.01S/A098G 0.45 1.04
    G097D/A098C 1 1.03
    G097D/A098G/D099G 0.89 0.985
    G097D/A098G/D099R 1.00 0.83
    G097D/A098H/D099R 1.24 0.92
    G097D/A098H/D099V 0.95 0.86
    G097D/A098R/D099G 0.955 0.96
    G097D/A098R/D099R 1.29 0.79
    G097D/A098R/D099S 1.11 0.87
    G097D/A098R/D099Z 0.255 1.09
    G097D/G100C/S101R 1.15 0.86
    G097D/G100R/S101G 0.47 0.85
    G097H/A098C/D099G 0.34 0.76
    G097H/A098D/D099N 0.5 1.15
    G097H/A098G 0.67 1.1
    G097H/A098G/D099G 0.38 0.78
    G097H/A098G/D099R 0.42 0.73
    G097H/A098G/D099Z 0.39 1.02
    G097H/A098G/D099Z/A151V 0.37 0.52
    G097H/A098G/D099Z/G100R 0.55 0.49
    G097H/A098G/D099Z/G100V 0.2 0.87
    G097H/A098G/D099Z/S101N/G166S 0.41 0.68
    G097H/A098R 0.60 0.97
    G097H/A098R/D099G 0.46 0.67
    G097H/A098R/D099L 0.31 0.5
    G097H/A098R/D099N 0.48 0.74
    G097H/A098S/D099G 0.76 0.84
    G097H/A098S/D099I 0.35 0.55
    G097H/A098S/D099L 0.36 0.83
    G097H/A098S/D099S/K237E 0.53 0.88
    G097L/A098C/D099S 0.26 0.68
    G097L/A098G/D099S 0.33 0.77
    G097L/A098G/D099S/G100Z 0.28 0.53
    G097L/A098G/D099Z 0.29 0.56
    G097L/A098H 0.71 1.1
    G097L/A098R/D099N 0.5 0.78
    G097L/A098V 0.42 1.11
    G097N/A098G/D099C 0.74 0.99
    G097N/A098G/D099R 0.82 0.88
    G097N/A098G/DO99R/V270I 0.74 0.88
    G097N/A098G/D099S 0.45 0.7
    G097N/A098G/D099V 0.47 0.9
    G097N/A098G/D099V/A151V 0.3 1.07
    G097N/A098G/D099V/S101F 0.54 0.82
    G097N/A098H/D099N 0.79 0.75
    G097N/A098R/D099R 0.69 0.25
    G097N/A098R/D099S 0.94 0.73
    G097N/A098R/D099V 0.59 0.675
    G097N/A098S/D099S 0.72 0.91
    G097R/A098C 1.04 0.98
    G097R/A098C/D099S 0.28 0.735
    G097R/A098F/A200V 0.305 0.74
    G097R/A098G 0.97 0.985
    G097R/A098G/D099C 0.31 0.68
    G097R/A098G/D099G 0.33 0.60
    G097R/A098G/D099G/A273T 0.48 0.85
    G097R/A098G/D099N 0.64 0.82
    G097R/A098G/D099S 0.31 0.66
    G097R/A098G/D099S/A133D 0.26 0.61
    G097R/A098G/D099V 0.275 0.55
    G097R/A098H/D099C/S182N 0.38 0.75
    G097R/A098H/D099G 0.47 0.695
    G097R/A098N/D099G 0.32 1.05
    G097R/A098N/D099Z 0.52 0.72
    G097R/A098R 1.06 0.83
    G097R/A098R/D099C 0.50 0.53
    G097R/A098R/D099G 0.54 0.51
    G097R/A098R/D099S 0.45 0.55
    G097R/A098S/D099C 0.39 0.66
    G097R/A098S/D099G 0.51 0.59
    G097R/A098S/D099L 0.2 0.51
    G097R/A098S/D099R 0.45 0.52
    G097R/A098S/D099S 0.33 0.81
    G097R/A098S/D099V 0.32 0.61
    G097R/A098V/D099G 0.45 0.68
    G097R/Z097.01H/A098C 0.56 0.81
    G097R/Z097.1R/A098G 0.5 0.74
    G097R/Z097.01 R/A098G/V147I 0.56 0.74
    G097R/Z097.01R/A098S 0.4 0.59
    G097R/Z097.01R 0.39 0.6
    G097R/Z097.01S/A098G 0.57 0.95
    G097R/Z097.01S/A098R 0.53 0.7
    G097S/A098C 0.87 1.06
    G097S/A098C/D099G 0.47 1.09
    G097S/A098C/D099H 0.62 1.02
    G097S/A098C/D099L 0.45 0.96
    G097S/A098C/D099R 0.575 0.79
    G097S/A098C/D099S 0.71 1.15
    G097S/A098C/D099Z 0.2 0.81
    G097S/A098G/D099F 0.28 0.63
    G097S/A098G/D099G 0.43 0.90
    G097S/A098G/D099G 0.43 0.91
    G097S/A098G/D099S 0.56 0.95
    G097S/A098G/D099Z 0.3 0.94
    G097S/A098G/D099Z/G100R 0.3 0.52
    G097S/A098H/D099C 0.46 0.84
    G097S/A098H/D099G 0.42 0.97
    G097S/A098N/D099G 0.53 1.14
    G097S/A098R 1.06 0.94
    G097S/A098R/D099C 0.45 0.7
    G097S/A098R/D099G 0.63 0.76
    G097S/A098R/D099L 0.55 0.715
    G097S/A098R/D099V 0.27 0.63
    G097S/A098R/D099Y 0.44 0.74
    G097S/A098S/D099G 0.65 0.945
    G097S/A098S/D099G/A133V/A153V/S236Y 0.48 0.69
    G097S/A098S/D099G/A153V 0.46 0.82
    G097S/A098S/D099Z 0.875 0.76
    G097S/Z097.01D/A098G/A144T/Q271H 0.46 1.04
    G097S/Z097.01G/A098V 0.6 1.02
    G097S/Z097.01S/A098G 0.6 1.12
    G097S/Z097.01S/A098G/A273T 0.41 1.2
    G097S/Z097.01Y 0.33 1.04
    G097V/A098R 0.41 0.74
    G097/V/A098R/D099G 0.46 0.56
    G097V/A098S/D099S 0.24 0.84
    G097Y/A098H/D099N 0.3 0.56
    G097Z/A098 S/D099H 0.31 0.77
    Z097.01D/A098R/D099G 0.39 1
    Z097.01D/A098S/D099L 0.36 0.59
    Z097.01D/A098V/D099G 0.345 0.805
    Z097.01G 0.54 1.12
    Z097.01G/A098G 0.47 1.02
    Z097.01G/A098G/D099S 0.21 1.03
    Z097.01G/A098R 0.73 0.86
    Z097.01G/A098R/D099C 0.43 0.73
    Z097.01G/A098R/D099G 0.39 0.64
    Z097.01G/A098R/D099S 0.48 0.63
    A098C 0.97 0.91
    A098C/D099G 0.57 0.98
    A098C/D099G/A116T 0.48 1.03
    A098C/D099H 0.68 0.78
    A098C/D099L 0.4 0.64
    A098C/D099L/K213R 0.36 0.85
    A098C/D099L 0.39 0.95
    A098C/D099N 0.66 0.96
    A098C/D099R 0.62 0.80
    A098C/D099S 0.69 0.98
    A098C/D099S/P194L 0.47 0.79
    A098D/D099C 0.59 0.91
    A098D/D099G 0.59 1.09
    A098D/D099G/G100L 0.65 1.06
    A098D/D099H/G100S 0.3 0.91
    A098D/D099H/H238Y 0.38 1.07
    A098D/D099R 0.69 0.72
    A098D/D099R/G100D 0.28 0.91
    A098D/D099R/G160S 0.6 0.78
    A098F/D099S/G100L 0.3 0.52
    A098G 0.91 1.01
    A098G/D099C 0.67 0.77
    A098G/D099G 0.55 0.85
    A098G/D099G/A228T 0.58 0.9
    A098G/D099G/G100H 0.58 0.38
    A098G/D099G/N243D 0.27 0.71
    A098G/D099G/S 101 P 0.55 0.57
    A098G/D099G/T244S 0.51 0.895
    A098G/D099G 0.49 0.85
    A098G/D099H 0.44 0.89
    A098G/D099L 0.4 0.75
    A098G/D099R 0.56 0.70
    A098G/D099R/A116T 0.6 0.68
    A098G/D099R/G100D 0.28 0.62
    A098G/D099R/V148I 0.79 0.61
    A098G/D099S 0.58 1.01
    A098G/D099S/L267M 0.48 0.89
    A098G/D099V 0.32 0.93
    A098G/D099Z 0.25 0.69
    A098H 1.04 0.96
    A098H/D099C 0.46 0.79
    A098H/D099G 0.82 0.89
    A098H/D099G/A216T 0.72 0.76
    A098H/D099G/G100D 0.52 1.09
    A098H/D099G/G100N/L267M 0.35 1.02
    A098H/D099G/G100S 0.355 0.9
    A098H/D099G/V143I 0.41 0.84
    A098H/D099R 1.07 0.62
    A098H/D099R/G100S 0.37 0.54
    A098H/D099S/G100C 0.33 0.67
    A098H/D099S/G100N 0.43 1.05
    A098H/D099Y 0.32 0.81
    A098I/D099G 0.54 0.97
    A098I/D099S 0.47 0.91
    A098L/D099G 0.44 1.15
    A098L/D099S 0.5 0.91
    A098N 1.05 1
    A098N/D099G 1.14 1.01
    A098N/D099L 0.32 0.71
    A098N/D099S 0.74 1
    A098R/A144T 0.81 0.91
    A098R/A274V 1.11 0.88
    A098R/D099C 0.56 0.82
    A098R/D099G 0.94 0.70
    A098R/D099G/G 100C 0.35 0.68
    A098R/D099G/G 100D 0.83 0.92
    A098R/D099G/G 100L/S 145T 0.68 0.84
    A098R/D099G/G100N 0.66 0.74
    A098R/D099G/G 100R 0.57 0.25
    A098R/D099G/G100S 0.55 0.82
    A098R/D099G/V150I 0.84 0.68
    A098R/D099H 0.85 0.59
    A098R/D099I 0.39 0.665
    A098R/D099L 0.54 0.62
    A098R/D099N 1 0.895
    A098R/D099N/G 100L 0.57 0.79
    A098R/D099R 0.72 0.32
    A098R/D099R/G100D 0.41 0.53
    A098R/D099R/S101Z 0.61 0.23
    A098R/D099R/S 130F 0.23 0.51
    A098R/D099R 0.64 0.51
    A098R/D099S 0.75 0.80
    A098R/D099S/G100L/A153V 0.55 0.79
    A098R/D099S 0.87 0.81
    A098R/D099V 0.505 0.775
    A098R/D099Z/S101G 0.395 0.61
    A098R/G100C/A137T 0.64 0.96
    A098R/G100S 0.95 1
    A098S/D099C 0.655 0.865
    A098S/D099G 0.89 0.91
    A098S/D099G/G100C 0.35 0.86
    A098S/D099G/G100D 0.26 1.26
    A098S/D099G/G100R/L250I 0.58 0.55
    A098S/D099G/G100S 0.48 0.98
    A098S/D099G/Z099.01R 0.25 0.52
    A098S/D099H 0.6 0.95
    A098S/D099H/M124V 0.53 0.95
    A098S/D099H/Z099.01R/G169A/V180A 0.63 0.62
    A098S/D099R 0.80 0.78
    A098S/D099R/G100L 0.71 0.67
    A098S/D099R/G154S 0.41 0.61
    A098S/D099R/G166S 1.16 0.75
    A098S/D099R/Z100.01G 0.47 0.66
    A098S/D099S 0.82 0.97
    A098S/D099S/Z099.01R 0.38 0.66
    A098S/D099V 0.41 1.03
    A098T/P129Z/S130R/S132G 0.33 0.53
    A098T/S101G 0.785 0.925
    A098V/D099G 0.965 0.96
    A098V/D099G/G100D 0.5 0.98
    A098V/D099G/G100D/A153V 0.41 0.62
    A098V/D099G/G100S 0.66 0.83
    A098Y/D099R/G100L 0.31 0.54
    A098Y/D099R/G100Z/S101Z/G102D 0.98 0.66
    A098Z/S101R 0.56 0.62
    Z098.01N 0.5 1.31
    Z098.01R 0.54 0.97
    Z098.01R/G100R 0.55 0.51
    D099C/K136N 0.3 0.8
    D099C/S101D 0.73 1.08
    D099C/S101R 0.655 0.67
    D099C/S101Z 0.40 0.61
    D099C/Z099.01S 0.3 0.88
    D099C/Z100.01G 0.36 0.56
    D099F/S101Z 0.35 0.51
    D099G/G100C 0.5 0.58
    D099G/G100D/S101R 0.54 0.93
    D099G/G100H 0.62 0.71
    D099G/G100R/S101V 0.45 0.58
    D099G/G100S/S101R 0.55 0.79
    D099G/S 101 G 0.67 0.83
    D099G/S 101 H 0.79 0.95
    D099G/S 101 V 0.85 0.95
    D099G/S101Z 0.56 0.81
    D099G/S101Z/N269D 0.45 0.81
    D099G/Z099.01N 0.33 1.21
    D099G/Z099.01R/G100S 0.26 0.59
    D099G/Z099.01S 0.21 1.17
    D099G/Z100.01G 0.26 0.71
    D099G/Z100.01G/S183N/Q275K 0.35 0.71
    D099H/S101Z 0.49 0.74
    D099H/S101Z/V150I 0.56 0.59
    D099H/Z100.01G/A273V 1.91 0.38
    D099H/Z100.01G/Z100.02G 0.2 0.84
    D099I/Z099.01S 0.48 0.805
    D099L/N118D 0.5 0.82
    D099L/S101G 0.23 0.91
    D099L/S101V 0.46 0.88
    D099L/Z100.01G 0.26 0.51
    D099N/A116T 1 0.92
    D099N/G100R/S101V 0.525 0.725
    D099N/S101G 0.57 0.95
    D099N/S101H 0.98 0.89
    D099N/S101Z 0.48 0.94
    D099N/Z099.01S 0.69 1
    D099N/Z100.01G 0.42 0.94
    D099R 0.7 0.71
    D099R/S101D 0.78 1
    D099R/S101G 0.595 0.58
    D099R/S101V 0.75 0.66
    D099R/S101Z 0.48 0.52
    D099R/S101Z/A133T 0.35 0.52
    D099R/Z099.01N 0.28 1.06
    D099R/Z099.01S 0.42 0.71
    D099R/Z100.01G 0.43 0.66
    D099R/Z100.01N 0.44 0.5
    D099S/S101G 0.53 0.98
    D099S/S101I 0.69 0.925
    D099S/S101Z 0.71 0.77
    D099S/Z099.01H 0.62 0.67
    D099S/Z099.01L 0.35 0.59
    D099S/Z099.01V 0.5 0.35
    D099S/Z100.01G 0.47 0.85
    D099S/Z100.01G/Z100.02G 0.21 1.07
    D099V/S101D 0.54 1.24
    D099V/S101V 0.33 0.825
    D099V/S101Z 0.36 0.51
    Z099.01H/S101L 0.56 0.58
    Z099.01H/Z099.02R 0.5 0.44
    Z099.01N/S101R 0.46 1.09
    Z099.01S 0.48 1.17
    Z099.01S/S101V 0.51 0.91
    Z099.01S/Z099.02G/A228T 0.61 0.24
    G 100C 0.85 0.98
    G 100D 0.54 1.18
    G100D/S101G 0.54 0.68
    G100D/S101R 0.74 1.13
    G100H/G102V 0.26 1.04
    G100H/S101G 0.35 0.91
    G100H/S101R 0.74 0.945
    G100L/S101G 0.775 1.135
    G100L/S101R 1.24 1.01
    G100N/S101D 0.5 0.86
    G100N/S101L 0.93 1.075
    G100N/S101R 1.27 0.99
    G100R 0.64 0.84
    G100R/L267R/I268V 0.7 1.01
    G100R/S101G 0.46 0.78
    G100R/S101G/G160S/S182N 0.5 0.69
    G100R/S101G/Q103Z/Y104Z/S161N 0.33 0.73
    G100R/S101G/S163N 0.55 0.54
    G100R/S101N 0.78 0.89
    G100R/S101R 0.60 0.87
    G100R/S101R/Q103Z/V270I 0.58 1
    G100R/S101Z/G102C/Q103D 0.5 0.26
    G100S 0.59 1.14
    G100S/S101C 0.69 0.96
    G100S/S101G 0.47 1.02
    G100S/S101R 0.75 0.99
    G100S/S101R 0.94 1.01
    G100S/S101V 0.53 1.13
    G100S/S101V/A231T 0.51 0.46
    Z100.01G 0.43 0.76
    Z100.01L 0.43 1.05
    Z100.01N 0.43 0.67
    Z100.01R 0.47 0.76
    Z100.01R/S101G 0.45 0.95
    S101C 1.48 0.89
    S101C/Q103I 0.68 0.91
    S101C/Q103S 1.34 0.97
    S101D 1.055 1.125
    S101D/Q103C 0.85 1.15
    S101D/Q103D 0.94 1.1
    S101D/Q103F 1.04 1.1
    S101D/Q103H 0.89 1.24
    S101D/Q103H 0.68 1.09
    S101D/Q103R 1.06 0.87
    S101D/Q103R/H238Y 0.69 1.11
    S101D/Q103S 0.63 1.14
    S101D/Q103V 0.41 1.09
    S101F/Q103G 0.32 0.75
    S101G 0.83 0.99
    S101G/A151V 0.41 1.23
    S101G/A179V 0.29 0.58
    S101G/G102S 0.42 1.13
    S101G/Q103G 0.38 0.59
    S101G/Q103L 0.74 1.01
    S101G/Q103N 0.75 1.14
    S101G/Q103R 0.71 0.83
    S101G/Q103R/A179T 0.32 0.52
    S101G/Q103R/G157D 0.59 0.81
    S101G/Q103S 0.57 1.12
    S101G/Q103S/L233S 0.8 0.97
    S101G/Q103S/L233S 0.45 1.13
    S101G/Q245H 0.56 0.93
    S101G/S249N 0.77 1.115
    S101G/V139I 0.85 1.01
    S101G/Z101.01R 0.435 0.965
    S101G/Z101.01R/A200T 0.52 0.26
    S101G/Z102.01G 0.465 0.69
    S101H 0.92 1.03
    S101H/G102S 0.68 1.23
    S101H/Q103D 0.985 1.185
    S101H/Q103G 0.38 0.84
    S101H/Q103G/E251Q 0.37 0.7
    S101H/Q103H 1.07 0.93
    S101H/Q103I 0.715 1.035
    S101H/Q103R 0.73 0.82
    S101H/Q103R/A274Z/Q275Z 0.67 0.68
    S101H/Q103S 0.76 1.07
    S101H/Q103V 0.67 1.09
    S101I 0.78 0.95
    S101I/Q103R 0.81 0.91
    S101L 1.09 1.04
    S101L/G1025/Q103R 0.87 1.07
    S101L/Q103D 1.03 1.07
    S101L/Q103G 0.33 0.9
    S101L/Q103R 0.61 0.75
    S101L/Q103R/A138T 0.43 0.73
    S101L/Q103R/V121I/K213E 0.81 0.95
    S101L/Q103S 1.04 1.04
    S101L/Q103V 0.66 1.05
    S101L/Z101.01R 0.5 0.99
    S101N/Q103R 0.94 0.925
    S101N/Z102.01G 0.49 0.65
    S101R 0.85 0.90
    S101R/A151V 0.4 0.66
    S101R/A273V 0.78 0.71
    S101R/E156G 1.1 0.66
    S101R/G102L/Q103G 0.95 1.04
    S101R/G102S 1.58 1.1
    S101R/G102S/Q103R 0.81 0.91
    S101R/G102S/Q103V 0.54 0.89
    S101R/G131D 1.45 0.79
    S101R/Q103C 1.05 0.89
    S101R/Q103D 0.75 1.01
    S101R/Q103D/S248R 0.89 0.75
    S101R/Q103G 0.49 0.84
    S101R/Q103G/A116T 0.52 0.97
    S101R/Q103G/V1471/A153V 0.445 1.075
    S101R/Q103G 0.49 0.7
    S101R/Q103L 0.80 0.63
    S101R/Q103N 1.00 0.89
    S101R/Q103R 0.74 0.56
    S101R/Q103R/S161N 1.35 0.63
    S101R/Q103R/V148I 0.58 0.44
    S101R/Q103S 0.93 0.85
    S101R/Q103S/L126F 1.23 0.84
    S101R/Q103V 0.74 0.83
    S101V 0.915 1.05
    S101V/Q103G 0.46 0.99
    S101V/Q103H 0.75 0.98
    S101V/Q103N/Z275.01K 1.17 1.13
    S101V/Q103R 0.62 1.03
    S101V/Q103V 0.58 1.09
    S101Y/G102S/Q103R 0.83 0.94
    S101Y/Q103L 0.54 0.75
    S101Y/Q103S 0.54 0.93
    S101Y/Q103V 0.58 0.95
    S101Z 0.34 0.95
    S101Z/G102I/Q103H/Y104G/L135F 0.32 0.7
    S101Z/G102V/Q103R/Y104L 0.37 0.61
    S101Z/Q103D 0.52 0.29
    S101Z/Q103F 0.45 1.07
    S101Z/Q103L 0.41 1
    S101Z/Q103R 0.49 0.76
    G102C/Q103L/Y104S 0.96 1.15
    G102R/Q103C/Y104C/V192I 0.33 1.26
    G102R/Q103G/Y104R 0.25 0.69
    G102R/QI03R/Z103.01G 0.655 0.885
    G102Z/Q103S 0.33 0.61
    Z102.01G/Q103R 0.41 1.02
    Z102.01G/Q103R/L267V 0.31 0.64
    Z102.01G/Z102.02R/Z102.03G 0.53 0.67
    Q103D 1.04 1.21
    Q103G 0.335 1.04
    Q103H/Y104N/Q275Z 0.56 0.81
    Q103N/E156D 0.92 1.07
    Q103R 0.75 0.82
    Q103R/I122L/N123S/M124V 0.68 0.65
    Q103R/Y104F 0.7 0.795
    Q103S 1.35 0.86
    Q103V 0.61 0.955
    Q103Y/S182N 0.87 0.77
    S105G/P129S/S130H/G131H 1.09 0.78
    W106C/A116D/N117S/N118C 0.47 0.73
    N109S/M119C/D120G/V121L 0.28 0.97
    A116G/N117Z/N118R/M119C 0.37 0.88
    A116H/N117G/N118R 0.39 0.94
    N117C/N118G/M119S 0.44 1.05
    N117C/N118G/M119S/A216V 0.33 0.87
    N117G/N118G/M119C 0.43 0.94
    N117G/N118R/M119C 0.29 1.03
    N117H/N118C/M119C 0.45 0.57
    N117H/N118D/M119S 0.63 0.88
    N117H/N118G/M119C 0.97 0.83
    N117H/N118G/M119L 0.59 0.86
    N117H/N118G/M119S 0.72 0.89
    N117H/N118G/M119V 0.62 0.95
    N117H/N118H/M119V 1.07 0.96
    N117H/NI18R/M119C 0.98 0.75
    N117H/N118R/M119S 0.53 0.77
    N117H/N118S/M119C 0.71 0.84
    N117H/N118S/M119S 0.4 0.985
    N117H/N118S/M119V 0.64 0.89
    N117I/N118G/M119V 0.38 1.01
    N117L/N118G/M119V 0.38 0.77
    N117R/N118G/M119C 0.63 0.80
    N117R/N118G/M119I/V270I 0.46 0.88
    N117R/N118G/M119S 0.375 0.79
    N117R/N118H/M119V 0.66 0.91
    N117R/N118R/M119C 0.755 0.755
    N117R/N118R/M119I 0.67 0.71
    N117R/N118R/M119S 0.28 0.605
    N117R/N118R/M119V 0.80 0.73
    N117R/N118S/M119I 0.38 0.75
    N117R/N118S/M119L 0.23 0.72
    N117S/N118G/M119C 0.595 1.085
    N117S/N118G/M119S 0.3 1.1
    N117S/N118H/M119V 0.49 1.01
    N117S/N118R/M119V 0.58 0.93
    N117V/N118G/M119C 0.47 0.99
    N117V/N118H/M119C 0.39 0.71
    N117Y/N118G/M119S 0.26 0.74
    N117Y/N118G/M119V 0.475 1
    Z117.01G/N118R/M119C 0.29 0.65
    N118C/M119V/D120S 0.27 0.82
    N118C/Z118.01G/M119C 0.485 0.86
    N118D/M119C/D120L 0.43 0.98
    N118D/M119S/D120R 0.24 0.88
    N118G/M119C/D120G 0.63 1.02
    N118G/M119C/D120L 0.5 0.99
    N118G/M119I/D120R 0.65 0.94
    N118G/M119N 0.28 0.73
    N118G/M119S/D120G 0.29 0.69
    N118G/M119S/D120R 0.38 0.83
    N118G/M119V/D120G 0.49 0.94
    N118G/M119V/D120S/S182N 0.52 0.99
    N118H/M119C/D120R 0.88 0.87
    N118H/M119I/D120G 0.54 1.05
    N118H/M119N/V148I 0.38 0.63
    N118H/M119S/D120G 0.28 0.62
    N118H/M119V/D120G 0.54 1
    N118R/M119C 0.97 0.99
    N118R/M119C/D120G 0.63 0.85
    N118R/M119C/D120R 0.8 0.85
    N118R/M119S 0.45 0.99
    N118R/M119S/D120G 0.32 0.53
    N118R/M119S/D120R 0.29 0.57
    N118R/M119S/D120S 0.28 0.82
    N118R/M119V/D120C 0.27 0.97
    N118R/M119V/D120G 0.56 0.76
    N118R/M119V/D120G/A232T 0.32 0.825
    N118S/M119C/D120R 0.56 0.91
    M119C/D120G 0.58 0.98
    M119C/D120G/V121I 0.48 0.95
    M119C/D120G/V121L 0.39 0.52
    M119C/D120H 0.91 1.01
    M119C/D120H/A187V 0.44 0.93
    M119C/D120R 0.76 0.95
    M119H/V121I 0.42 0.58
    M119I/D120C 0.44 0.88
    M119I/D120G 0.47 0.96
    M119I/D120R 0.67 0.86
    M119L/D120G 0.39 0.51
    M119S/D120R 0.28 0.97
    M119S/D120S/V121I 0.46 0.72
    M119S/V121I 0.44 1.15
    M119V/D120C 0.27 0.93
    M119V/D120G 0.43 1.06
    M119V/D120R 0.63 0.86
    M119V/D120S/V198L 0.5 0.93
    D120G/I122C 0.27 0.83
    D120G/1122C/L217S 0.34 0.56
    D120G/I122G 0.54 0.23
    D120G/I122V 0.5 1
    D120H/I122L 0.66 1.03
    D120H/V121C/I122V 0.31 0.74
    D120N/VI21L/1122V 0.32 0.93
    D120R/A151V 0.37 0.71
    D120R/I122V 0.63 0.97
    D120R/V121I 0.72 1
    D120R/V121I/I122C 0.39 0.74
    D120R/V121I/I122C/A228T 0.29 0.98
    D120S/I122C/A133S 0.31 1.1
    V121C 0.63 0.86
    V121C/I122C/N123C 0.54 0.2
    V121C/I122G/N123V 0.56 0.6
    V121C/I122S/N123V 0.56 0.57
    V121C/I122V/N123S 0.52 0.59
    V1211/I122C/N123C 0.53 0.6
    V121I/I122C/V150I 0.8 1.13
    V1211/I122G/N123C 0.65 1
    V121I/I122S/N123C 0.64 1.36
    V121I/I122S/N123I 0.95 0.83
    V1211/I122V/N123S 0.82 0.98
    V121L/I122S/N123V 0.53 0.9
    V121L/N123C 1.02 1.16
    I122C/N123C 0.6 0.6
    I122C/N123S/M124L 0.98 1.015
    I122D/N123V/M124L 0.55 0.63
    I122G/N123C/M124L 0.96 0.96
    I122G/N123G/Z123.01C 0.63 0.45
    I122H/N123V/M124L 0.65 0.76
    I122S/N123S 0.63 1.27
    I122V/N123G/M124C/A138V 0.51 0.25
    I122V/N123S/M124I 0.81 0.75
    I122V/S183G 0.91 0.97
    N123C/M124S 0.98 0.785
    N123C/M124V 1.875 0.745
    N123D 0.7 0.89
    N123S/M124L 1.55 1.13
    N123S/M124S 0.71 0.26
    M124C 0.92 1.05
    M124C/L126S 0.7 0.51
    M124C/L126S/T244I 0.72 0.455
    M124C/L126V 1.535 1.085
    M124G 0.53 0.3
    M124I 1.72 1.16
    M124I/L126H 0.87 0.54
    M124S 0.6 0.58
    M124V 1.08 1.20
    L126C 1.8 0.35
    L126F/P129Z 1.07 1
    L126F/P129Z/S182N 1.47 1.24
    L126G 0.67 0.58
    L126I/P129Z 0.53 0.71
    L126R/G127R 0.54 0.47
    L126S 1.08 0.94
    L126Y 1.04 1.33
    Z126.01S 0.67 0.59
    G127C/G128S/P129R 0.77 0.48
    G127C/P129G 1.53 0.72
    G127C/P129G/A200T 0.56 0.4
    G127C/P129R 1.785 0.825
    G127C/P129R/A153T 0.76 0.48
    G127D/P129G 0.91 0.73
    G127D/P129S 0.71 0.69
    G127H/P129G 1.65 0.71
    G127H/P129R 1.1 0.77
    G127L 1.14 0.83
    G127L/P129R 1.02 0.80
    G127N/P129C 1.05 0.88
    G127N/P129G 1.1 0.91
    G127N/P129G/A133T 0.87 1.04
    G127N/P129G/A151V/K213N 0.37 0.72
    G127N/P129R 1.10 0.77
    G127N/P129S/A273V 1.07 0.9
    G127N/P129V 1.16 0.92
    G127R/A230V 0.5 0.58
    G127R/P129D 1.01 0.8
    G127R/P129D/L217S 0.76 0.78
    G127R/P129G 0.57 0.48
    G127R/P129R 0.85 0.56
    G127R/P1295 0.725 0.725
    G127S/P129D 0.67 1.255
    G127S/P129F 0.94 0.93
    G127S/P129G 1.29 0.99
    G127S/P129G/G166D 0.81 0.98
    G127S/P129G/I175T 0.33 0.77
    G127S/P129R 1.47 0.86
    G127S/P129S 1.06 1.09
    G127S/P129V 1.16 1.15
    G127V/P129G 1.59 0.84
    G127V/P129R 1.01 0.89
    Z127.01G/Z127.02H/P129D 0.58 0.65
    Z127.01G/Z127.02H/P129G 0.55 0.57
    Z127.01G/Z127.02H/P129R 0.52 0.61
    Z127.01H/G128C/P129C/A230V 0.57 0.57
    Z127.01H/P129G 0.59 0.36
    Z127.01H/P129G/V148I 0.61 0.27
    Z127.01H/P129R 0.57 0.44
    Z127.01N/G128C/P129V 0.5 0.2
    Z127.01N/G128S/P129S 0.585 1.19
    Z127.01N/G128S/P129V 0.57 0.67
    Z127.01N/P129R 0.59 1.04
    Z127.01R/G128S/P129S 0.67 0.5
    Z127.01R/P129G 0.485 0.475
    Z127.01S/P129D 0.58 0.5
    Z127.01S/P129G 0.65 0.67
    Z127.01S/P129R 0.42 0.94
    G128D/P129G 0.53 1.00
    G128D/P129R/Z129.01R 0.7 0.33
    G128D/P129S/G154S 0.5 0.54
    G128H/P129C 0.58 0.89
    G128H/P129H/Z129.01G 0.65 0.53
    G128H/P129H/Z129.01R 0.595 0.44
    G128H/P129R 0.78 0.97
    G128H/P129R/Z129.01G 0.52 0.54
    G128H/P129R/Z129.01R 0.57 0.36
    G128H/P129S/Z129.01G 0.66 0.54
    G128H/P129S/Z129.01R 0.47 0.88
    G128H/P129S/Z129.01R/S248N 0.54 0.62
    G128H/P129Y 0.44 1.22
    G128H/Z128.01R/P129D 0.66 0.63
    G128H/Z128.01R/P129S 0.46 0.755
    G128H/Z128.01R/P129S/A144T 0.555 0.705
    G128H/Z129.01R 0.46 0.76
    G128N/P129R/Z129.01G 0.55 0.83
    G128N/P129R/Z129.01R 0.5 0.93
    G128N/P129S/Z129.01D 0.67 0.93
    G128N/Z128.01R/P129S 0.58 1.01
    G128R/P129G 0.54 0.57
    G128R/P129H/Z129.01G 0.495 0.755
    G128R/P129L/Z130.01S 0.58 0.25
    G128R/P129R 0.45 0.53
    G128R/P129R/Z129.01G 0.55 0.32
    G128R/P129R/Z129.01G/A216V 0.64 0.26
    G128R/P129S 0.46 0.78
    G128R/P129S/Z129.01G 0.605 0.7
    G128R/P129Z/S130R/S132G 0.43 0.51
    G128R/Z128.01R/P129D 0.46 0.57
    G128R/Z128.01R/P129F 0.52 0.28
    G128S/P129C 0.845 1.09
    G128S/P129C/Z129.01R 0.575 0.435
    G128S/P129D 0.87 1.37
    G128S/P129D/S248R 0.9 1.28
    G128S/P129D/T244N 1.3 1.13
    G128S/P129G 1.05 1.38
    G128S/P129G/A187V 0.75 0.89
    G128S/P129H/Z129.01R 0.44 0.92
    G128S/P129R/L209F 1.2 1.01
    G128S/P129R/Z129.01G 0.8 0.76
    G128S/P129S/Z129.01D 0.6 0.55
    G128S/P129S/Z129.01R 0.59 0.84
    G128S/P129V 0.735 1.145
    G128Y/P129R/Z129.01G 0.55 0.4
    Z128.01G 0.57 0.515
    Z128.01G/P129D 0.39 1.04
    Z128.01G/P129R 0.46 0.87
    Z128.01G/P129S/A134T 0.55 0.62
    Z128.01G/Z128.02G 0.59 0.27
    Z128.01R/P129D 0.35 0.6
    Z128.01R/Z128.02G/P129S 0.5 0.58
    Z128.01R/Z128.02H/P129G/G160S 0.59 0.44
    Z128.01R/Z128.02R/P129D 0.58 0.74
    Z128.01Y/Z128.02H/P129R 0.57 0.34
    P129C/S130C/S132Z 0.42 0.6
    P129D/Z129.01G/Z129.02D/G160D 0.54 0.61
    P129D/Z129.01G/Z129.02G 0.69 0.3
    P129G 0.64 0.90
    P129G/G131Z 0.57 1.19
    P129G/S130G/Z131.01G 0.51 1.02
    P129G/S130H/S132Z 0.37 1.17
    P129G/S130R/S132Z 0.71 0.3
    P129G/V270I 0.7 0.9
    P129G/Z129.01G 0.45 0.84
    P129G/Z129.01R/Z129.02G 0.69 0.82
    P129H 0.79 1
    P129H/G131Z 0.6 1.16
    P129H/G131Z/S132C 0.52 0.59
    P129H/G131Z/S132H 0.43 1.04
    P129H/S132Z 0.41 0.51
    P129H/Z130.01D/Z130.02S 0.44 1.02
    P129L 1 1.22
    P129R 0.80 0.87
    P129R/G131Z/S132H 0.58 0.78
    P129R/S130D/S132Z 0.745 1.04
    P129R/S130N/G131Z 1.23 0.95
    P129R/S132T 0.37 0.69
    P129R/S132Z 0.395 0.745
    P129R/Z129.01G 0.42 0.60
    P129R/Z129.01G/Z129.02G 0.49 0.57
    P129R/Z129.01G/Z129.02G/A144T 0.64 0.49
    P129R/Z129.01G/Z129.02G/G146D 0.53 0.31
    P129R/Z129.01G/Z129.02R 0.52 0.42
    P129R/Z129.01G 0.46 0.65
    P129R/Z129.01R/Z129.02G 0.58 0.28
    P129S 0.92 1.00
    P129S/G131Z 0.75 1.15
    P129S/S130H/S132Z 0.52 1.19
    P129S/Z129.01C 0.55 0.45
    P129S/Z129.01R/Z129.02S 0.59 0.82
    P129S/Z130.01N/Z130.02S 0.45 1.1
    P129V 1.32 1.09
    P129V/S132Z 0.37 0.59
    P129Z 0.57 1.16
    P129Z/G131R/S132C 0.38 0.51
    P129Z/S130G 0.47 1.29
    P129Z/S130G/G131H/S132H 1.08 1.18
    P129Z/S130G/G131V 0.52 0.82
    P129Z/S130H 0.56 1.17
    P129Z/S130H/S132G 0.42 0.61
    P129Z/S130H/S132N 1.91 0.62
    P129Z/S130L 0.49 1.14
    P129Z/S130R 0.93 1.00
    P129Z/S130R/G131C 0.53 0.75
    P129Z/S130R/G131C 0.61 0.89
    P129Z/S130R/G131D/S132C 0.7 0.75
    P129Z/S130R/G131H/A144E 0.86 0.88
    P129Z/S130R/G131H/S132D 0.33 0.52
    P129Z/S130R/G131R/S132D 0.37 0.64
    P129Z/S130R/L267M 0.67 0.99
    P129Z/S130R/S132C 0.72 0.46
    P129Z/S130R/S132G 0.42 0.64
    P129Z/S130V/S132G 0.37 0.81
    P129Z/S130Z/G131N/S132G 0.5 0.9
    S130C 1.25 0.86
    S130D/G131C/S132D 0.71 0.81
    S130D/G131R 0.77 0.87
    S130D/G131R/S132D 0.52 0.9
    S130D/G131Z 1.53 0.91
    S130D/G131Z/S132H 0.68 0.82
    S130F/G131N/S132V 0.59 0.64
    S130F/G131S/S132V 0.48 0.59
    S130F/S132V 0.29 0.53
    S130G 0.8 1.1
    S130G/G131D/S132C 1.13 0.81
    S130G/G131H/S132D 0.51 0.88
    S130G/G131S/S132I 0.4 0.67
    S130G/S132G 0.635 0.845
    S130H 1.01 0.92
    S130H/G131C/S132G 0.94 0.98
    S130H/G131H/S132R 0.53 0.41
    S130H/G131R/S132C 0.74 0.86
    S130H/G131S 1.08 0.75
    S130H/G131S/S132C 1.11 0.78
    S130H/S132Z 0.81 0.94
    S130I/G131S/S132H 0.69 0.77
    S130I/S132G 0.94 0.76
    S130L 1.06 0.87
    S130N/G131S/S132C 1.04 0.91
    S130R/G131R/S132C 0.7 0.625
    S130R/G131S/S132C/S161N 1.29 0.84
    S130R/S132G 1.05 0.36
    S130R/S132G/S183N 0.98 0.36
    S130R/S132Z 0.48 0.69
    S130V/G131D/S132I 0.98 1.23
    S130V/G131H/S132C 1.34 0.91
    S130V/S132N 0.74 0.91
    S130V/S132Z 0.35 0.52
    S130Y/G131H/S132C 1.14 0.87
    S130Y/S132G 0.97 0.62
    S130Z/G131H/S132H 0.78 0.88
    G131H/S132L/W241R 0.35 0.61
    G131N/S132C 0.94 0.93
    G131S/S132G 0.42 0.63
    G131Y/S132G 0.33 0.615
    S132H 0.65 0.88
    S132Z 0.795 1.045
    A134T 0.66 1.1
    A142V 0.55 1.05
    A142V/G211V 0.33 1.15
    A144T 0.89 0.96
    S161N 0.92 1.04
    A200T 0.5 0.9
    L217Y 1.14 0.95
    A231T 0.69 0.95
    W241R 0.54 0.95
    S248G 0.96 0.95
    S260F 0.81 1.04
    F261L 0.58 0.86
    L267G 1.06 1.08
    A272S 1.21 0.92
    Q275Z 0.79 0.98
  • Table 3-2 provides a list of the insertions that make up the combinations.
  • TABLE 3-2
    Mutations
    POS Variant
    66.01 Z066.01G
    93.01 Z093.01G
    96.01 Z096.01D
    96.01 Z096.01H
    96.01 Z096.01N
    96.01 Z096.01R
    96.01 Z096.01S
    96.02 Z096.02G
    97.01 Z097.01D
    97.01 Z097.01G
    97.01 Z097.01H
    97.01 Z097.01R
    97.01 Z097.01S
    97.01 Z097.01Y
    98.01 Z098.01N
    98.01 Z098.01R
    99.01 Z099.01H
    99.01 Z099.01L
    99.01 Z099.01N
    99.01 Z099.01R
    99.01 Z099.01S
    99.01 Z099.01V
    99.02 Z099.02G
    99.02 Z099.02R
    100.01 Z100.01G
    100.01 Z100.01L
    100.01 Z100.01N
    100.01 Z100.01R
    100.02 Z100.02G
    101.01 Z101.01R
    101.02 Z101.02G
    102.01 Z102.01G
    102.02 Z102.02R
    102.03 Z102.03G
    103.01 Z103.01G
    117.01 Z117.01G
    118.01 Z118.01G
    123.01 Z123.01C
    126.01 Z126.01S
    127.01 Z127.01G
    127.01 Z127.01H
    127.01 Z127.01N
    127.01 Z127.01R
    127.01 Z127.01S
    127.02 Z127.02H
    128.01 Z128.01G
    128.01 Z128.01R
    128.01 Z128.01Y
    128.02 Z128.02G
    128.02 Z128.02H
    128.02 Z128.02R
    129.01 Z129.01C
    129.01 Z129.01D
    129.01 Z129.01G
    129.01 Z129.01R
    129.02 Z129.02D
    129.02 Z129.02G
    129.02 Z129.02R
    129.02 Z129.02S
    130.01 Z130.01D
    130.01 Z130.01N
    130.01 Z130.01S
    131.01 Z131.01G
    275 Q275Z
    • EXAMPLE 4 Construction of Combinatorial Variants of BPN′ 3
  • Using BPN′G97A-G128A-Y217Q (“BPN′3”) as a parent, combinatorial variants were created by a strategy that involved extension (fusion) PCR using four fragments of the gene. Fragment 1 was amplified by primers P4974 and P4977 (See, Table 4-1). Fragment 4 was amplified by primers 4978 and P4976 (Table 4-1). Both fragment 1 and 4 did not contain any mutations. Fragments 2 and 3 each were created as a set of 12 and 16 sequences respectively that contained the desired mutations, using either BPN′3 as template or in the absence of template using partially overlapping forward and reverse primers.
  • The set of Fragment 2 sequences were amplified by the following primers:
    • 01: P4979 and P4980 (BPN′3 as a template)
    • 02: P4981 and P4982 (no template)
    • 03: P4983 and P4984 (no template)
    • 04: P4985 and P4986 (no template)
    • 05: P4987 and P4988 (no template)
    • 06: P4989 and P4990 (no template)
    • 07: P4991 and P4992 (no template)
    • 08: P4993 and P4994 (no template)
    • 09: P4995 and P4996 (no template)
    • 10: P4997 and P4998 (no template)
    • 11: P4999 and P5000 (no template)
    • 12: P5001 and P5002 (no template)
  • The set of Fragment 3 sequences were amplified by the following primers:
    • 01: P5003 and P5004 (BPN′3 as a template)
    • 02: P5005 and P5006 (no template)
    • 03: P5007 and P5008 (no template)
    • 04: P5009 and P5010 (no template)
    • 05: P5011 and P5012 (no template)
    • 06: P5013 and P5014 (no template)
    • 07: P5015 and P5016 (no template)
    • 08: P5017 and P5018 (no template)
    • 09: P5019 and P5020 (no template)
    • 10: P5021 and P5022 (no template)
    • 11: P5023 and P5024 (no template)
    • 12: P5025 and P5026 (no template)
    • 13: P5027 and P5028 (no template)
    • 14: P5029 and P5030 (no template)
    • 15: P5031 and P5032 (no template)
    • 16: P5033 and P5034 (no template)
  • The primer sequences used in the construction of the combinatorial variants are listed in Table 4-1.
  • TABLE 4-1 
    Primer Sequences and Mutations Introduced in
    Combinatorial Variants of BPN′3
    SEQ ID Primer
    NO: Name Sequence
    125 P4948 GTGTTTTTCTTGGAATTGTGCTG
    126 P4974 GCCTCACATTTGTGCCACCTA
    127 P4975 CATATGAGTTATGCAGTTTGTAG
    128 P4976 CCTCTCGGTTATGAGTTAGTTC
    129 P4977 GTAGAGCGAAGCAGACGGGGCTA
    130 P4978 CTTAAAGCAGCAGTTGATAAAGCT
    131 P4979 CTTGGTGTAGCCCCGTCTGCTT
    132 P4980 ATCCATGTTATTCGCGATGGCCCATT
    133 P4981 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCTCTTGCTCTGC
    AGCAGACGGATCAGGCCAATACTCATGGATTATCAA
    134 P4982 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCTGATCCGTCTGCTGCAGAGCAAG
    135 P4983 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGATGCACGTGACGGATCAGGCCAATACTCATGGAT
    136 P4984 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCTGATCCGTCACGTGCATCAAGAA
    137 P4985 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGCAGCAGACTCTGGATCAGGCCAATACTCATGGAT
    138 P4986 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCTGATCCAGAGTCTGCTGCAAGAA
    139 P4987 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGCAGCAGACTCTTCAGGCCAATACTCATGGATTAT
    140 P4988 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCTGAAGAGTCTGCTGCAAGAACTT
    141 P4989 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGCAGCAGACGGAGATGGCCAATACTCATGGATTAT
    142 P4990 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCATCTCCGTCTGCTGCAAGAACTT
    143 P4991 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGATGCACGTGACTCTGGATCAGGCCAATACTCATG
    144 P4992 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCTGATCCAGAGTCACGTGCATCAA
    145 P4993 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGATGCACGTGACTCTTCAGGCCAATACTCATGGAT
    146 P4994 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCTGAAGAGTCACGTGCATCAAGAA
    147 P4995 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGATGCACGTGACGGAGATGGCCAATACTCATGGAT
    148 P4996 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCATCTCCGTCACGTGCATCAAGAA
    149 P4997 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGCAGCAGACTCTTCTTCAGGCCAATACTCATGGAT
    150 P4998 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCTGAAGAAGAGTCTGCTGCAAGAA
    151 P4999 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGCAGCAGACTCTGGAGATGGCCAATACTCATGGAT
    152 P5000 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCATCTCCAGAGTCTGCTGCAAGAA
    153 P5001 CTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTTAAAGTTCT
    TGCAGCAGACTCTGATGGCCAATACTCATGGATTAT
    154 P5002 ATCCATGTTATTCGCGATGGCCCATTCGATGCCGTTGATAATCC
    ATGAGTATTGGCCATCAGAGTCTGCTGCAAGAACTT
    155 P5003 ATCGAATGGGCCATCGCGAAT
    156 P5004 TGCAACAGCTTTATCAACTGCT
    157 P5005 ATCGAATGGGCCATCGCGAATAACATGGATGTATCTGGAATGA
    GCCTGGGAGCACCAAGCGGCAGT
    158 P5006 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTTGGTGCTCCCAGGCTCATTCCAGAT
    159 P5007 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGATCTCCAAGCGGCAGT
    160 P5008 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTTGGAGATCCCAGGCTCATGTTGATT
    161 P5009 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGAGCAGTTAGCGGCAGT
    162 P5010 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTAACTGCTCCCAGGCTCATGTTGATT
    163 P5011 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGAGCAGATAGCGGCAGT
    164 P5012 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTATCTGCTCCCAGGCTCATGTTGATT
    165 P5013 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGAGCAAGCGGCAGTGCGGCA
    166 P5014 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTTGCTCCCAGGCTCATGTTGATTACA
    167 P5015 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGAGCAGGAGGCAGTGCGGCA
    168 P5016 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCTC
    CTGCTCCCAGGCTCATGTTGATTACA
    169 P5017 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGAGCACCAGTTGATAT
    170 P5018 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCGATATCA
    ACTGGTGCTCCCAGGCTCATGTTGATT
    171 P5019 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGAGCACCAAGCGGCAGT
    172 P5020 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGTCGCACTGCCGC
    TTGGTGCTCCCAGGCTCATGTTGATT
    173 P5021 ATCGAATGGGCCATCGCGAATAACATGGATGTATCTGGAATGA
    GCCTGGGATCTCCAAGCGGCAGT
    174 P5022 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTTGGAGATCCCAGGCTCATTCCAGAT
    175 P5023 ATCGAATGGGCCATCGCGAATAACATGGATGTATCTGGAATGA
    GCCTGGGAGCAGTAAGCGGCAGT
    176 P5024 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTTACTGCTCCCAGGCTCATTCCAGAT
    177 P5025 ATCGAATGGGCCATCGCGAATAACATGGATGTATCTGGAATGA
    GCCTGGGAGCAGATAGCGGCAGT
    178 P5026 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTATCTGCTCCCAGGCTCATTCCAGAT
    179 P5027 ATCGAATGGGCCATCGCGAATAACATGGATGTATCTGGAATGA
    GCCTGGGAGCAAGCGGCAGTGCGGCA
    180 P5028 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTTGCTCCCAGGCTCATTCCAGATACATCCATGTT
    181 P5029 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGATCTGTTAGCGGCAGT
    182 P5030 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTAACAGATCCCAGGCTCATGTTGATT
    183 P5031 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGATCTGATAGCGGCAGT
    184 P5032 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTATCAGATCCCAGGCTCATGTTGATT
    185 P5033 ATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGA
    GCCTGGGATCTAGCGGCAGTGCGGCA
    186 P5034 TGCAACAGCTTTATCAACTGCTGCTTTAAGTGCCGCACTGCCG
    CTAGATCCCAGGCTCATGTTGATT
    187 P5035 TGATCCGTCTGAGCAGGGGCTTTAATTTGTGA
    188 P5036 ATTAAAGCCCCTGCTCAGACGGATCAGGCCAATACTC
  • Each amplification reaction contained 30 pmol of each oligonucleotide and 100 ng of the template DNA. Amplifications were carried out using Vent DNA polymerase (New England Biolabs). The PCR mix (20 ul) was initially heated at 95° C. for 25 min followed by 30 cycles of denaturation at 94° C. for 15s, annealing at 55° C. for 15s and extension at 72° C. for 40s. Following amplification all the fragments were purified by the Gel Band Purification kit (Qiagen) and mixed to serve as templates for a fusion PCR using primers P4975 and P4948. The full-length DNA fragment from the fusion PCR reaction was gel-purified by the QIAGEN gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated in the Bacillus expression plasmid pHPLT-BPN partial opt (FIG. 2C), which was cut with the same restriction enzymes.
  • Ligation mixtures were amplified using rolling circle amplification according to manufacturer's recommendation (Epicentre). One ul of the ligation mixture was mixed with 5 ul of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 ul of the reaction buffer and 0.2 ul of the enzyme was added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and 1 ul of this dilution was used to transform Bacillus subtilis cells (genotype: AaprE, AnprE, AspoIIE, amyE::xylRPxylAcomK-phleo) as described in Example 2. Transformations were plated on LB plates containing 1.6% skim milk and 10 ppm Neomycin and incubated overnight at 37° C. Only colonies with halos were picked and grown in microtiter plates for further analysis as described in Example 2. Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
  • TABLE 4.5
    Combinatorial Libraries of
    BPN': G97A-G128A-Y217Q Parent
    Variant Name
    1 V093Z/K094S/V095C/L096S/G097A/G128A/Y217Q
    2 Z096.01D/G097G/A098R/G128A/Y217Q
    3 G097A/Z099.01S/G128A/Y217Q
    4 G097A/G100S/G128A/Y217Q
    5 G097A/S101D/G128A/Y217Q
    6 Z096.01D/G097G/A098R/Z099.01S/G128A/Y217Q
    7 Z096.01D/G097G/A098R/G100S/G128A/Y217Q
    8 Z096.01D/G097G/A098R/S101D/G128A/Y217Q
    9 G097A/Z099.01S/G100S/G128A/Y217Q
    10 G097A/Z099.01S/S101D/G128A/Y217Q
    11 G097A/G100S/S101D/G128A/Y217Q
    12 G097A/I122S/N123G/G128A/Y217Q
    13 V093Z/K094S/V095C/L096S/G097A/I122S/N123G/G128A/
    Y217Q
    14 Z096.01D/G097G/A098R/I122S/N123G/G128A/Y217Q
    15 G097A/Z099.01S/I122S/N123G/G128A/Y217Q
    16 G097A/G100S/I122S/N123G/G128A/Y217Q
    17 G097A/S101D/I122S/N123G/G128A/Y217Q
    18 Z096.01D/G097G/A098R/Z099.01S/I122S/N123G/G128A/Y217Q
    19 Z096.01D/G097G/A098R/G100S/I122S/N123G/G128A/Y217Q
    20 Z096.01D/G097G/A098R/S101D/I122S/N123G/G128A/Y217Q
    21 G097A/Z099.01S/G100S/I122S/N123G/G128A/Y217Q
    22 G097A/Z099.01S/S101D/I122S/N123G/G128A/Y217Q
    23 G097A/G100S/S101D/I122S/N123G/G128A/Y217Q
    24 G097A/G128S/Y217Q
    25 V093Z/K094S/V095C/L096S/G097A/G128S/Y217Q
    26 Z096.01D/G097G/A098R/G128S/Y217Q
    27 G097A/Z099.01S/G128S/Y217Q
    28 G097A/G100S/G128S/Y217Q
    29 G097A/S101D/G128S/Y217Q
    30 Z096.01D/G097G/A098R/Z099.01S/G128S/Y217Q
    31 Z096.01D/G097G/A098R/G100S/G128S/Y217Q
    32 Z096.01D/G097G/A098R/S101D/G128S/Y217Q
    33 G097A/Z099.01S/G100S/G128S/Y217Q
    34 G097A/Z099.01S/S101D/G128S/Y217Q
    35 G097A/G100S/S101D/G128S/Y217Q
    36 G097A/G128A/P129V/Y217Q
    37 V093Z/K094S/V095C/L096S/G097A/G128A/P129V/Y217Q
    38 Z096.01D/G097G/A098R/G128A/P129V/Y217Q
    39 G097A/Z099.01S/G128A/P129V/Y217Q
    40 G097A/G100S/G128A/P129V/Y217Q
    41 G097A/S101D/G128A/P129V/Y217Q
    42 Z096.01D/G097G/A098R/Z099.01S/G128A/P129V/Y217Q
    43 Z096.01D/G097G/A098R/G100S/G128A/P129V/Y217Q
    44 Z096.01D/G097G/A098R/S101D/G128A/P129V/Y217Q
    45 G097A/Z099.01S/G100S/G128A/P129V/Y217Q
    46 G097A/Z099.01S/S101D/G128A/P129V/Y217Q
    47 G097A/G100S/S101D/G128A/P129V/Y217Q
    48 G097A/G128A/P129D/Y217Q
    49 V093Z/K094S/V095C/L096S/G097A/G128A/P129D/Y217Q
    50 Z096.01D/G097G/A098R/G128A/P129D/Y217Q
    51 G097A/Z099.01S/G128A/P129D/Y217Q
    52 G097A/G100S/G128A/P129D/Y217Q
    53 G097A/S101D/G128A/P129D/Y217Q
    54 Z096.01D/G097G/A098R/Z099.01S/G128A/P129D/Y217Q
    55 Z096.01D/G097G/A098R/G100S/G128A/P129D/Y217Q
    56 Z096.01D/G097G/A098R/S101D/G128A/P129D/Y217Q
    57 G097A/Z099.01S/G100S/G128A/P129D/Y217Q
    58 G097A/Z099.01S/S101D/G128A/P129D/Y217Q
    59 G097A/G100S/S101D/G128A/P129D/Y217Q
    60 G097A/G128A/P129Z/Y217Q
    61 V093Z/K094S/V095C/L096S/G097A/G128A/P129Z/Y217Q
    62 Z096.01D/G097G/A098R/G128A/P129Z/Y217Q
    63 G097A/Z099.01S/G128A/P129Z/Y217Q
    64 G097A/G100S/G128A/P129Z/Y217Q
    65 G097A/S101D/G128A/P129Z/Y217Q
    66 Z096.01D/G097G/A098R/Z099.01S/G128A/P129Z/Y217Q
    67 Z096.01D/G097G/A098R/G100S/G128A/P129Z/Y217Q
    68 Z096.01D/G097G/A098R/S101D/G128A/P129Z/Y217Q
    69 G097A/Z099.01S/G100S/G128A/P129Z/Y217Q
    70 G097A/Z099.01S/S101D/G128A/P129Z/Y217Q
    71 G097A/G100S/S101D/G128A/P129Z/Y217Q
    72 G097A/G128A/P129G/S130Z/Y217Q
    73 V093Z/K094S/V095C/L096S/G097A/G128A/P129G/S130Z/
    Y217Q
    74 Z096.01D/G097G/A098R/G128A/P129G/S130Z/Y217Q
    75 G097A/Z099.01S/G128A/P129G/S130Z/Y217Q
    76 G097A/G100S/G128A/P129G/S130Z/Y217Q
    77 G097A/S101D/G128A/P129G/S130Z/Y217Q
    78 Z096.01D/G097G/A098R/Z099.01S/G128A/P129G/S130Z/
    Y217Q
    79 Z096.01D/G097G/A098R/G100S/G128A/P129G/S130Z/Y217Q
    80 Z096.01D/G097G/A098R/S101D/G128A/P129G/S130Z/Y217Q
    81 G097A/Z099.01S/G100S/G128A/P129G/S130Z/Y217Q
    82 G097A/Z099.01S/S101D/G128A/P129G/S130Z/Y217Q
    83 G097AG100S/S101D/G128A/P129G/S130Z/Y217Q
    84 G097A/G128A/S130V/G131D/S132I/Y217Q
    85 V093Z/K094S/V095C/L096S/G097A/G128A/S130V/G131D/
    S132I/Y217Q
    86 Z096.01D/G097G/A098R/G128A/S130V/G131D/S132I/Y217Q
    87 G097A/Z099.01S/G128A/S130V/G131D/S132I/Y217Q
    88 G097A/G100S/G128A/S130V/G131D/S132I/Y217Q
    89 G097A/S101D/G128A/S130V/G131D/S132I/Y217Q
    90 Z096.01D/G097G/A098R/Z099.01S/G128A/S130V/G131D/
    S132I/Y217Q
    91 Z096.01D/G097G/A098R/G100S/G128A/S130V/G131D/S132I/
    Y217Q
    92 Z096.01D/G097G/A098R/S101D/G128A/S130V/G131D/S132I/
    Y217Q
    93 G097A/Z099.01S/G100S/G128A/S130V/G131D/S132I/Y217Q
    94 G097A/Z099.01S/S101D/G128A/S130V/G131D/S132I/Y217Q
    95 G097A/G100S/S101D/G128A/S130V/G131D/S132I/Y217Q
    96 G097A/G128A/A134T/Y217Q
    97 V093Z/K094S/V095C/L096S/G097A/G128A/A134T/Y217Q
    98 Z096.01D/G097G/A098R/G128A/A134T/Y217Q
    99 G097A/Z099.01S/G128A/A134T/Y217Q
    100 G097A/G100S/G128A/A134T/Y217Q
    101 G097A/S101D/G128A/A134T/Y217Q
    102 Z096.01D/G097G/A098R/Z099.01S/G128A/A134T/Y217Q
    103 Z096.01D/G097G/A098R/G100S/G128A/A134T/Y217Q
    104 Z096.01D/G097G/A098R/S101D/G128A/A134T/Y217Q
    105 G097A/Z099.01S/G100S/G128A/A134T/Y217Q
    106 G097A/Z099.01S/S101D/G128A/A134T/Y217Q
    107 G097A/G100S/S101D/G128A/A134T/Y217Q
    108 G097A/I122S/N123G/G128S/Y217Q
    109 V093Z/K094S/V095C/L096S/G097A/I122S/N123G/G128S/
    Y217Q
    110 Z096.01D/G097G/A098R/I122S/N123G/G128S/Y217Q
    111 G097A/Z099.01S/I122S/N123G/G128S/Y217Q
    112 G097A/G100S/I122S/N123G/G128S/Y217Q
    113 G097A/S101D/I122S/N123G/G128S/Y217Q
    114 Z096.01D/G097G/A098R/Z099.01S/I122S/N123G/G128S/Y217Q
    115 Z096.01D/G097G/A098R/G100S/I122S/N123G/G128S/Y217Q
    116 Z096.01D/G097G/A098R/S101D/I122S/N123G/G128S/Y217Q
    117 G097A/Z099.01S/G100S/I122S/N123G/G128S/Y217Q
    118 G097A/Z099.01SS101D/I122S/N123G/G128S/Y217Q
    119 G097A/G100S/S101D/I122S/N123G/G128S/Y217Q
    120 G097A/1122S/N123G/G128A/P129V/Y217Q
    121 V093Z/K094S/V095C/L096S/G097A/I122S/N123G/G128A/
    P129V/Y217Q
    122 Z096.01D/G097G/A098R/I122S/N123G/G128A/P129V/Y217Q
    123 G097A/Z099.01S/I122S/N123G/G128A/P129V/Y217Q
    124 G097A/G100S/I122S/N123G/G128A/P129V/Y217Q
    125 G097A/S101D/I122S/N123G/G128A/P129V/Y217Q
    126 Z096.01D/G097G/A098R/Z099.01S/I122S/N123G/G128A/
    P129V/Y217Q
    127 Z096.01D/G097G/A098R/G100S/I122S/N123G/G128A/P129V/
    Y217Q
    128 Z096.01D/G097G/A098R/S101D/I122S/N123G/G128A/P129V/
    Y217Q
    129 G097A/Z099.01S/G100S/I122S/N123G/G128A/P129V/Y217Q
    130 G097A/Z099.01S/S101D/I122S/N123G/G128A/P129V/Y217Q
    131 G097A/G100S/S101D/I122S/N123G/G128A/P129V/Y217Q
    132 G097A/I122S/N123G/G128A/P129D/Y217Q
    133 V093Z/K094S/V095C/L096S/G097A/I122S/N123G/G128A/
    P129D/Y217Q
    134 Z096.01D/G097G/A098R/I122S/N123G/G128A/P129D/Y217Q
    135 G097A/Z099.01S/I122S/N123G/G128A/P129D/Y217Q
    136 G097A/G100S/I122S/N123G/G128A/P129D/Y217Q
    137 G097A/S101D/I122S/N123G/G128A/P129D/Y217Q
    138 Z096.01D/G097G/A098R/Z099.01S/I122S/N123G/G128A/
    P129D/Y217Q
    139 Z096.01D/G097G/A098R/G100S/I122S/N123G/G128A/P129D/
    Y217Q
    140 Z096.01D/G097G/A098R/S101D/I122S/N123G/G128A/P129D/
    Y217Q
    141 G097A/Z099.01S/G100S/I122S/N123G/G128A/P129D/Y217Q
    142 G097A/Z099.01S/S101D/I122S/N123G/G128A/P129D/Y217Q
    143 G097A/G100S/S101D/I122S/N123G/G128A/P129D/Y217Q
    144 G097A/I122S/N123G/G128A/P129Z/Y217Q
    145 V093Z/K094S/V095C/L096S/G097A/I122S/N123G/G128A/
    P129Z/Y217Q
    146 Z096.01D/G097G/A098R/I122S/N123G/G128A/P129Z/Y217Q
    147 G097A/Z099.01S/I122S/N123G/G128A/P129Z/Y217Q
    148 G097A/G100S/I122S/N123G/G128A/P129Z/Y217Q
    149 G097A/S101D/I122S/N123G/G128A/P129Z/Y217Q
    150 Z096.01D/G097G/A098R/Z099.01S/I122S/N123G/G128A/
    P129Z/Y217Q
    151 Z096.01D/G097G/A098R/G100S/I122S/N123G/G128A/P129Z/
    Y217Q
    152 Z096.01D/G097G/A098R/S101D/I122S/N123G/G128A/P129Z/
    Y217Q
    153 G097A/Z099.01S/G100S/I122S/N123G/G128A/P129Z/Y217Q
    154 G097A/Z099.01S/S101D/I122S/N123G/G128A/P129Z/Y217Q
    155 G097A/G100S/S101D/I122S/N123G/G128A/P129Z/Y217Q
    156 G097A/G128S/P129V/Y217Q
    157 V093Z/K094S/V095C/L096S/G097A/G128S/P129V/Y217Q
    158 Z096.01D/G097G/A098R/G128S/P129V/Y217Q
    159 G097A/Z099.01S/G128S/P129V/Y217Q
    160 G097A/G100S/G128S/P129V/Y217Q
    161 G097A/S101D/G128S/P129V/Y217Q
    162 Z096.01D/G097G/A098R/Z099.01S/G128S/P129V/Y217Q
    163 Z096.01D/G097G/A098R/G100S/G128S/P129V/Y217Q
    164 Z096.01D/G097G/A098R/S101D/G128S/P129V/Y217Q
    165 G097A/Z099.01S/G100S/G128S/P129V/Y217Q
    166 G097A/Z099.01S/S101D/G128S/P129V/Y217Q
    167 G097A/G100S/S101D/G128S/P129V/Y217Q
    168 G097A/G128S/P129D/Y217Q
    169 V093Z/K094S/V095C/L096S/G097A/G128S/P129D/Y217Q
    170 Z096.01D/G097G/A098R/G128S/P129D/Y217Q
    171 G097A/Z099.01S/G128S/P129D/Y217Q
    172 G097A/G100S/G128S/P129D/Y217Q
    173 G097A/S101D/G128S/P129D/Y217Q
    174 Z096.01D/G097G/A098R/Z099.01S/G128S/P129D/Y217Q
    175 Z096.01D/G097G/A098R/G100S/G128S/P129D/Y217Q
    176 Z096.01D/G097G/A098R/S101D/G128S/P129D/Y217Q
    177 G097A/Z099.01S/G100S/G128S/P129D/Y217Q
    178 G097A/Z099.01S/S101D/G128S/P129D/Y217Q
    179 G097A/G100S/S101D/G128S/P129D/Y217Q
    180 G097A/G128S/P129Z/Y217Q
    181 V093Z/K094S/V095C/L096S/G097A/G128S/P129Z/Y217Q
    182 Z096.01D/G097G/A098R/G128S/P129Z/Y217Q
    183 G097A/Z099.01S/G128S/P129Z/Y217Q
    184 G097A/G100S/G128S/P129Z/Y217Q
    185 G097A/S101D/G128S/P129Z/Y217Q
    186 Z096.01D/G097G/A098R/Z099.01S/G128S/P129Z/Y217Q
    187 Z096.01D/G097G/A098R/G100S/G128S/P129Z/Y217Q
    188 Z096.01D/G097G/A098R/S101D/G128S/P129Z/Y217Q
    189 G097A/Z099.01S/G100S/G128S/P129Z/Y217Q
    190 G097A/Z099.01S/S101D/G128S/P129Z/Y217Q
    191 G097AG100S/S101D/G128S/P129Z/Y217Q
    • EXAMPLE 5
  • Stain Removal Performance of Combinatorial Variants of BPN′ 3
  • The stain removal activity of combinatorial variants of BPN′ 3 generated as described in Example 4 was tested as described in Example 3. As described throughout, functionality of BPN′ variants, was quantified as a performance index (Pi), which is the ratio of performance of a variant to parent protein BPN′3. BPN′3 variants showing a Pi value greater or equal than 0.5 for BMI stain removal performance 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. Results are shown in Table 5-1.
  • TABLE 5-1
    Stain Removal Performance and Expression of Combinatorial Variants
    of BPN'3
    BMI
    pH 8/
    Variants TCA 16° C.
    V072G/G097A/G128A/Y217Q 0.93 1.08
    G097A/G128S/Y217Q 1.16 1.06
    G128A/Y217Q 1.07 1.05
    G097A/G128S/P129D/Y217Q 0.85 1.03
    G097A/G128A/A134T/Y217Q 0.56 1.03
    M124V/L126A/Y217Q 1.05 1.03
    G097A/G128A/P129D/Y217Q/A232P 0.46 1.01
    G097A/G128A/P129D/Y217Q 0.57 1.00
    G097A/G128S/P129V/A134T/Y217Q 0.29 0.95
    G097A/G128S/P129V/Y217Q 0.31 0.95
    V068I/G097A/G128S/P129D/Y217Q 0.41 0.85
    G097A/S101D/G128S/S204F/Y217Q/S236F/T254P 0.41 0.79
    G097A/S101D/G128S/Y217Q 0.43 0.76
    G097A/G128A/P129V/Y217Q 0.30 0.70
    G097A/S101D/G128A/Y217Q 0.50 0.64
    G097A/S101D/G128S/P129D/Y217Q 0.35 0.63
    G097A/S101D/G128A/A134T/Y217Q 0.40 0.58
    G097A/Z099.01S/A114S/G128S/Y217Q 0.21 0.53
    G097A/S101D/G128A/P194L/Y217Q 0.35 0.49
    G097A/Z099.01S/G128S/Y217Q 0.39 0.38
    G097A/S101D/G128A/P129D/Y217Q 0.36 0.36
    Q019R/G097A/Z099.01S/G128S/P129D/Y217Q 0.33 0.33
    G097A/G100S/G128S/Y217Q 0.46 0.25
    G097A/S101D/G128S/P129V/Y217Q 0.39 0.22
    G097A/Z099.01S/G128A/A134T/Y217Q 0.35 0.16
    G097A/G100S/1107V/G128S/P129D/Y217Q 0.37 0.15
    G097A/G100S/G128S/Y217Q/A230V 0.38 0.13
    G097A/G100S/G128A/Y217Q/K237N 0.39 0.12
    G097A/Z099.01S/G128S/P129D/Y217Q 0.37 0.12
    G097A/G100S/G128S/P129D/Y217Q 0.37 0.07
    Z096.01D/G097A/A098R/G128A/Y217Q 0.36
    Figure US20150056682A1-20150226-P00007
    G097A/Z099.01S/I122S/N123G/G128A/Y217Q 0.37
    Figure US20150056682A1-20150226-P00007
    G097A/Z099.01S/I122S/N123G/G128A/Y217Q/T220A/ 0.38
    Figure US20150056682A1-20150226-P00007
    A232T
    P005S/G097A/Z099.01S/S101D/G128A/Y217Q 0.42
    Figure US20150056682A1-20150226-P00007
    G097A/S101D/G128A/P129V/Y217Q 0.46
    Figure US20150056682A1-20150226-P00007
    V093Z/K094S/V095C/L096S/G097A/G128A/Y217Q 0.50
    Figure US20150056682A1-20150226-P00007
    G097A/G100S/S101D/G128A/Y217Q/A232P 0.51
    Figure US20150056682A1-20150226-P00007
    Z096.01D/G097A/A098R/S101D/G128A/Y217Q 0.53
    Figure US20150056682A1-20150226-P00007
    G097A/Z099.01S/G100S/G128A/Y217Q 0.53
    Figure US20150056682A1-20150226-P00007
    • EXAMPLE 6
  • Construction of Combinatorial Variants of BPN′ 3 at Positions 128, 129, and 130
  • Combinatorial libraries focused around positions 128/129/130 in BPN′G97A-G128A-Y217Q (BPN′3) were created using site specific saturation mutagenesis and/or site specific insertion and/or deletion. The variants generated contain substitution, insertion, and deletion mutations. Libraries L1, L2 and L3 are based on WT position S130S and L4 is based on S130A. Four saturation libraries focused around positions 128/129 and 130 were constructed as shown in Table 6-1.
  • TABLE 6-1
    Construction of Saturation Library at
    Positions 128, 129 and 130 in BPN′3
    Position Position Position Short Degenerate
    Library 128 Insertion 129 130 Name Primer Names
    L1 NNS NNS S XX 128/129 deg
    QC F/R
    L2 deletion NNS S -X 128/129 del
    QC F/R
    L3 NNS A NNS S XAX 128 A 129
    QC F/R
    L4 deletion NNS A -XA 128/129 del
    A QC F/R
  • Prior to library construction, two stop codons were introduced at positions 128 and 129 in the BPN3′ sequence to inactivate the protease. This was done to ensure a low background of the parent plasmid in the libraries. The parent plasmid (pHPLT-BPN′3) was methylated using two micrograms of plasmid DNA and methylase (New England Biolabs), according to the NEB protocol. Methylated DNA was then purified with DNA Clean and Concentrator kit from Zymo Research. Mutagenesis was performed using methods known in the art (See, Amin et al., Biotechniques 35: 1134-1140, [2003]) using a modified version of the QuikChange multi site-directed mutagenesis (QCMS) kit from Stratagene.
  • The mutagenesis reaction contained 60 ng template plasmid, 0.5 ul forward primer 128/129 Stop F (25 uM), 0.5 ul reverse primer 128/129 Stop R (25 uM), 1 ul dNTP's (QCMS kit), 2.5 ul 10× QCMS reaction buffer, 18.5 ul deionized water, and 1 ul of enzyme blend (QCMS kit), for a total volume of 25 ul. For the mutagenesis reaction, the thermocycler program used was 1 cycle at 95° for 1 min., followed by 25 cycles of 95° C. for 1 min, 55° C. for 1 min., and 68° C. for 11 minutes (MJ Research thermocycler). The template DNA was digested by DpnI (QCMS kit, Qiagen) and 1.5 uL of the reaction was amplified by rolling circle amplification (RCA) using the Templiphi kit (Amersham) using manufacturer's protocol.
  • TABLE 6-2 
    Primers
    SEQ ID
    NO: Primer Name Primer Sequence
    189 128/129 Stop F /5Phos/CATGAGCCTGGGATAATGAA
    GCGGCAGTGCGGCACTTAAAGCAG
    190 128/129 Stop R /5Phos/GTGCCGCACTGCCGCTTCAT
    TATCCCAGGCTCATGTTGATTACATC
  • One microliter of the amplified DNA was used to transform 100 μL of competent Bacillus subtilis cells (genotype: AaprE, AnprE, AspoIIE, amyE::xylRPxylAcomK-phleo). Aliquots of 20 uL or 80 uL of the transformation mixture were plated on Luria Agar plates supplemented with 10 μg/ml neomycin+1.6% skim milk (Teknova). After growth, 4 colonies not forming halos on the skim milk plates were picked to identify the plasmid with the correct sequence. The correct variant with the two stop codons at positions 128/129, called “pHPLT-BPN′ 128/129 Stop” was chosen as the parent plasmid for the construction of combinatorial libraries described in Table 6-1.
  • Plasmid pHPLT-BPN′ 128/129 Stop was methylated and used as template with the degenerate primer pairs shown in Table 6-3.
  • TABLE 6-3 
    NNS Mutagenesis Primer Sequences
    SEQ
    ID
    NO. Primer Name Sequence
    191 128/129 deg QC F /5Phos/CAT GAG CCT GGG ANN SNN SAG CGG CAG TGC GGC
    ACT TAA AGC AG
    192 128/129 deg QC R /5Phos/CCG CAC TGC CGC TSN NSN NTC CCA GGC TCA TGT
    TGA TTA CAT C
    193 128/129 del QC F /5Phos/CAT GAG CCT GGG ANN SAG CGG CAG TGC GGC ACT
    TAA AGC AG
    194 128/129 del QC R /5Phos/CCG CAC TGC CGC TSN NTC CCA GGC TCA TGT TGA
    TTA CAT C
    195 128 A 129 QC F /5Phos/CAT GAG CCT GGG ANN SGC GNN SAG CGG CAG TGC
    GGC ACT TAA AGC AG
    196 128 A 129 QC R /5Phos/CCG CAC TGC CGC TSN NCG CSN NTC CCA GGC TCA
    TGT TGA TTA CAT C
    197 128/129 del A QC F /5Phos/CAT GAG CCT GGG ANN SGC AGG CAG TGC GGC ACT
    TAA AGC AG
    198 128/129 del A QC R /5Phos/GTG CCG CAC TGC CTG CSN NTC CCA GGC TCA TGT
    TGA TTA CAT C
  • Overlapping forward and reverse primers (PAGE-purified) with the NNS codon(s) in the middle and 17-20 flanking bases were designed for each chosen library, and the sequences for each forward (F) and reverse (R) primer are shown in Table 6-3. Quik-change mutagenesis and rolling circle amplification for the 4 reactions was performed as described above. Transformations were plated on Luria Agar plates supplemented with 10 μg/ml neomycin +1.6% skim milk. All halo forming colonies were picked into two microtiter plates containing 125 ul LB medium supplemented with 10 ug/ml neomycin. The clones were sequenced by Quintara Biosciences. For protein expression, cultures were grown overnight in micro-filter plates (.22um, Millipore) containing 180 ul of a enriched semi-defined media based on MOPs buffer, with urea as major nitrogen source, glucose as the main carbon source, 1% soytone for robust cell growth, and supplemented with 2.5ug/ml neomycin. Cultures were grown for 64 hours at 37 C, 250rpm, and 70% humidity. Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
    • EXAMPLE 7
  • Stain Removal Performance of Combinatorial Variants of BPN′ 3 at positions 128, 129, and 130
  • The stain removal activity of combinatorial variants of BPN′ 3 at positions 128, 129, and 130 generated as described in Example 6 was tested as described in Example 3. As described throughout, functionality of BPN′ variants, was quantified as a performance index (Pi), which is the ratio of performance of a variant to parent protein BPN′3. BPN′3 variants showing a Pi value greater or equal than 0.5 for BMI stain removal performance and/or TCA precipitation showed improved cleaning benefits and/or expression. Results are shown in Table 7-1.
  • TABLE 7-1
    Stain Removal Performance and Expression of BPN' 3
    Variants at Positions 128, 129, and 130
    Variant TCA BMI
    G097A/G128S/P129T/Y217Q 0.77 1.04
    G097A/G128A/P129S/Y217Q 0.70 1.02
    G097A/G128S/P129C/Y217Q 0.61 1.00
    G097A/G128S/P129Q/Y217Q 0.96 1.00
    G097A/G128S/P129E/Y217Q 0.99 0.99
    G097A/P129S/Y217Q 0.87 0.98
    G097A/G128S/P129A/Y217Q 0.65 0.98
    G097A/G128S/P129K/Y217Q 0.90 0.96
    G097A/P129N/Y217Q 0.80 0.96
    G097A/P129E/Y217Q 1.01 0.95
    G097A/G128S/P129K/G131S/Y217Q 0.95 0.93
    G097A/G128S/P129R/Y217Q 0.83 0.92
    G097A/G128S/P129V/Y217Q 0.48 0.90
    G097A/G128S/P129Y/Y217Q 0.41 0.89
    G097A/G128A/P129R/Y217Q 0.69 0.88
    G097A/G128H/P129S/Y217Q 0.59 0.85
    G097A/G128N/P129S/Y217Q/V270A 0.51 0.84
    G097A/G128S/P129L/Y217Q 0.45 0.81
    G097A/P129R/Y217Q 0.70 0.80
    G097A/P129W/Y217Q 0.51 0.79
    G097A/P129Z/S130A/Y217Q 0.51 0.78
    G097A/G128H/P129R/Y217Q 0.58 0.76
    G097A/G128H/P129T/Y217Q 0.57 0.73
    G097A/P129G/Y217Q 0.55 0.70
    G097A/G128N/P129A/Y217Q 0.42 0.70
    G097A/G128H/Y217Q 0.58 0.68
    G097A/G128S/P129W/Y217Q 0.36 0.63
    G097A/P129Z/Y217Q 0.47 0.60
    G097A/G128N/Z128.01A/P129S/Y217Q 0.39 0.60
    G097A/G128N/Y217Q 0.47 0.59
    G097A/G128A/P129L/Y217Q 0.39 0.58
    G097A/G128T/P129N/Y217Q 0.39 0.56
    G097A/G128H/P129A/Y217Q 0.50 0.53
    G097A/G128M/P129R/Y217Q 0.53 0.53
    G097A/G128Q/P129K/Y217Q 0.49 0.53
    G097A/G128T/P129Q/Y217Q 0.42 0.47
    G097A/G128T/P129H/Y217Q 0.41 0.46
    G097A/G128T/P129R/Y217Q 0.40 0.44
    G097A/G128D/P129E/Y217Q 0.46 0.43
    G097A/G128T/P129T/Y217Q 0.37 0.42
    G097A/G128T/P129A/Y217Q 0.39 0.38
    G097A/G128Z/P129Z/Y217Q 0.45 0.36
    G097A/G128S/Z128.01A/P129S/Y217Q 0.42 0.31
    G097A/G128N/P129M/Y217Q 0.42 0.26
    G097A/G128N/P129V/Y217Q 0.46 0.26
    G097A/G128E/P129K/Y217Q 0.52 0.25
    G097A/G128N/Z128.01A/P129F/Y217Q 0.30 0.22
    G097A/G128C/P129R/Y217Q 0.77 0.21
    G097A/G128T/P129M/Y217Q 0.34 0.20
    G097A/G128R/Z128.01A/P129S/Y217Q 0.41 0.20
    G097A/G128S/Z128.01A/P129A/Y217Q 0.33 0.15
    G097A/G128D/Y217Q 0.41 0.15
    G097A/G128T/P129Y/Y217Q 0.38 0.14
    G097A/G128R/P129S/Y217Q 0.47 0.13
    G097A/G128R/Z128.01A/P129T/Y217Q 0.42 0.12
    G097A/G128R/Z128.01A/P129A/Y217Q 0.38 0.11
    G097A/G128T/P129W/Y217Q 0.37 0.11
    G097A/G128Y/Z128.01A/P129R/Y217Q 0.41 0.10
    G097A/G128C/P129D/Y217Q 0.84 0.10
    V084L/G097A/P129Z/Y217Q 0.46 0.10
    G097A/G128R/P129D/Y217Q 0.51 0.09
    G097A/G128A/Z128.01A/P129H/E195A/Y217Q 0.35 0.09
    G097A/G128S/Z128.01A/P129Y/Y217Q 0.40 0.09
    G097A/G128N/P129L/Y217Q 0.51 0.08
    G097A/G128C/P129H/Y217Q 0.71 0.07
    G097A/G128T/Z128.01A/P129T/Y217Q 0.38 0.05
    G097A/G128C/Z128.01A/P129A/Y217Q 0.39 0.05
    G097A/G128S/Z128.01A/P129I/Y217Q 0.41 0.05
    G097A/G128S/Z128.01A/P129E/Y217Q/Q271H 0.41 0.05
    G097A/G128S/Z128.01A/P129L/Y217Q 0.42 0.05
    G097A/G128Y/Z128.01A/P129S/Y217Q 0.43 0.05
    G097A/G128S/Z128.01A/P129M/R186H/Y217Q 0.44 0.05
    G097A/G128E/Z128.01A/P129D/Y217Q 0.44 0.05
    G097A/G128T/P129L/Y217Q 0.46 0.05
    G097A/G128F/P129D/Y217Q 0.47 0.05
    G097A/G128V/Z128.01A/P129R/Y217Q 0.47 0.05
    G097A/G128L/Z128.01A/P129S/Y217Q 0.49 0.05
    G097A/G128Y/P129T/Y217Q 0.51 0.05
    G097A/G128C/P129Y/Y217Q 0.53 0.05
    G097A/G128C/P129T/Y217Q 0.59 0.05
    Parent: G097A/G128A/Y217Q 1.00 1.00
    • EXAMPLE 8
  • Construction of Combinatorial Variants of BPN′
  • Combinatorial variants of BPN′ were created by extension (fusion) PCR using the following
  • BPN′ mutants as parent molecules—BPN′ G97A/G128A/Y217Q, BPN′ M124V/L126A/Y217Q, BPN′ G128A/Y217Q, and BPN′ N123G/Y217Q to create insertions or substitutions at positions 96, 98, 129 and/or 222. For each combinatorial mutant created, the parent DNA molecule, mutations introduced and PCR primers used are listed in the Table 8-1. To create each mutant, two fragments 1 and 2 were generated by the PCR primers (Table 8-2) in the 5′ and 3′ regions of the gene using the respective parent molecule listed in Table 8-1. These fragments were mixed and amplified by the flanking primers P4973 and P4950 (Table 8-2) to restore the full-length gene.
  • Each amplification reaction contained 30pmol of each PCR primer and 100 ng of the template DNA. Amplifications were carried out using Vent DNA polymerase (New England Biolabs). The PCR mix (20 ul) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15s, annealing at 55° C. for 15s and extension at 72° C. for 40s. Following amplification, the 5′ and 3′ fragments were gel-purified by the QIAGEN gel-band purification kit, mixed and amplified by the flanking primers P4973 and P4950. The full-length DNA fragment produced was gel-purified by the QIAGEN gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′ vector containing the respective parent molecules, which were also cut with the same restriction enzymes. Ligation mixtures were amplified using rolling circle amplification according to manufacturer's recommendation (Epicentre). One microliter of the ligation mixture was mixed with 5 ul of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 ul of the reaction buffer and 0.2 ul of the enzyme was added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform Bacillus subtilis cells (genotype: AaprE, ΔnprE, AspoIIE, amyE::xylRPxylAcomK-phleo) as described in Example 2. Transformations were plated on LB plates containing 1.6% skim milk and 10 ppm Neomycin and incubated overnight at 37° C. Only colonies with halos were picked and grown in microtiter plates for further analysis as described in Example 2 Protein concentration of culture supernatants was determined by TCA precipitation as described in Example 1.
  • TABLE 8-1
    Parent DNA Molecule, Mutations Introduced and PCR Primers for the
    Generation of Combinatorial Variants in BPN'
    Parent Molecule/ Mutations Primers Used for PCR Amplification
    Variant DNA Template Introduced Fragment 1 Fragment 2
    1 G97A/G128A/Y217Q Z098.001S P4947, P4952 P4951, P4948
    2 G97A/G128A/Y217Q Z096.001D/A098R P4947, P4956 P4955, P4948
    3 G97A/G128A/Y217Q P129V P4947, P4960 P4959, P4948
    4 M124V/L126A/Y217Q Z098.001S P4947, P4954 P4953, P4948
    5 M124V/L126A/Y217Q Z096.001D/A098R P4947, P4958 P4957, P4948
    6 M124V/L126A/Y217Q P129V P4947, P4962 P4961, P4948
    7 G128A/Y217Q Z098.001S P4947, P4954 P4953, P4948
    8 G128A/Y217Q Z096.001D/A098R P4947, P4958 P4957, P4948
    9 G128A/Y217Q P129V P4947, P4966 P4965, P4948
    10 N123G/Y217Q Z098.001S P4947, P4954 P4953, P4948
    11 N123G/Y217Q Z096.001D/A098R P4947, P4958 P4957, P4948
    12 N123G/Y217Q P129V P4947, P4964 P4963, P4948
    13 G97A/G128A/Y217Q M222Q P4947, P4968 P4967, P4948
  • TABLE 8-2 
    PCR primer Sequences Used for the Creation of
    Combinatorial Variants of BPN′.
    SEQ
    ID Primer
    NO: Name Sequence
    199 P4947 CTTTAGTCTAGCAAAAGGAGAG
    200 P4948 GTGTTTTTCTTGGAATTGTGCTG
    201 P4949 CGACTCGAGCCATCCAGATC
    202 P4950 CTTGTCTCCAAGCTTAAAATAAAA
    203 P4951 AAAGTTCTTGCAGCAAGTGACGGATCAGGCCAATACTCA
    204 P4952 GCCTGATCCGTCACTTGCTGCAAGAACTTTAACGGCGT
    AG
    205 P4953 TAAAGTTCTTGGCGCAAGTGACGGATCAGGCCAATACT
    CA
    206 P4954 CCTGATCCGTCACTTGCGCCAAGAACTTTAACGGCGTA
    207 P4955 GTTAAAGTTCTTGATGCACGTGACGGATCAGGCCAATA
    CTCA
    208 P4956 GATCCGTCACGTGCATCAAGAACTTTAACGGCGTAGAG
    CGA
    209 P4957 GTTAAAGTTCTTGATGGTCGTGACGGATCAGGCCAATA
    CTCA
    210 P4958 GATCCGTCACGACCATCAAGAACTTTAACGGCGTAGAG
    CGAA
    211 P4959 TGAGCCTGGGAGCAGTAAGCGGCAGTGCGGCACTTAAA
    212 P4960 GCACTGCCGCTTACTGCTCCCAGGCTCATGTTGATTAC
    213 P4961 TTAGCGCAGGAGGAGTAAGCGGCAGTGCGGCACTTAAA
    214 P4962 GCACTGCCGCTTACTCCTCCTGCGCTAACGTTGATTA
    215 P4963 GAGCCTGGGAGGAGTAAGCGGCAGTGCGGCACTTAAA
    216 P4964 TGCCGCACTGCCGCTTACTCCTCCCAGGCTCATGCCGA
    TT
    217 P4965 GAGCCTGGGAGCAGTAAGCGGCAGTGCGGCACTTAAA
    218 P4966 GCACTGCCGCTTACTGCTCCCAGGCTCATGTTGATT
    219 P4967 AACGGGACTTCCCAAGCCTCGCCGCATGTAGCTG
    220 P4968 ACATGCGGCGAGGCTTGGGAAGTCCCGTTTTGCGCAC
    221 P4973 AAAGGATCCTAATCGGCGCTTTTC
    • EXAMPLE 9
  • Stain Removal Performance of Combinatorial Variants of BPN′
  • The stain removal activity of combinatorial variants of BPN′ generated as described in Example 8 was tested as described in Example 3. As described throughout, functionality of BPN′ variants was quantified as a performance index (Pi), which is the ratio of performance of a variant to BPN′3 (BPN′ G97A-G128A-Y217Q). Variants showing a Pi value greater or equal than 0.5 for BMI stain removal performance 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. Results are shown in Table 9-1.
  • TABLE 9-1
    Stain Removal Performance and Expression
    of BPN' Variants
    BMI
    Variants TCA pH 8/16° C.
    G097A/G128A/Y217Q/M222Q 0.56 0.88
    M124V/L126A/P129V/Y217Q 0.69 0.79
    G128A/P129V/Y217Q 0.36 0.75
    G097A/G128A/P129V/Y217Q 0.27 0.75
    N123G/P129V/Y217Q 0.47 0.37
    V301/Z096.01D/G097A/A098R/G128A/Y217Q 0.54 0.19
    Z096.01D/A098R/G128A/Y217Q 0.47 0.19
    Z096.01D/A098R/M124V/L126A/Y217Q 0.49 0.17
    G097A/Z098.01S/G128A/Y217Q 0.71 0.17
    Z096.01D/A098R/N123G/Y217Q 0.54 0.16
    Z096.01D/G097A/A098R/G128A/Y217Q/S236F 0.78 0.12
    Z098.01S/M124V/L126A/Y217Q 0.45 0.11
    Z098.01S/G128A/Y217Q 0.56 0.07
    Z096.01D/G097A/A098R/G128A/Y217Q 0.55 0.07
    V0931/Z098.01S/M124V/L126A/Y217Q 0.64 0.06
    A073V/Z098.01S/N123G/Y217Q 0.52 0.05
    G065D/Z098.01S/G128A/Y217Q 0.66
    Figure US20150056682A1-20150226-P00007
    I011L/Z098.01S/G128A/Y217Q 0.53
    Figure US20150056682A1-20150226-P00007
    L082F/Z096.01D/A098R/N123G/Y217Q 0.41
    Figure US20150056682A1-20150226-P00007
    Z098.01S/N123G/A138V/Y217Q 0.44
    Figure US20150056682A1-20150226-P00007
    Z098.01S/N123G/Y217Q 0.52
    Figure US20150056682A1-20150226-P00007
  • All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. Those of skill in the art readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It is readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
  • The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by herein.
  • The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not excised material is specifically recited herein.

Claims (16)

1. A cleaning composition comprising a subtilisin variant comprising at least one modification, wherein said modification is selected from: G100C, S101D, S101H, Q103D, M124G, L126G, S132H, S161N, Q275Z, and/or at least one set of modifications selected from A001T/L096V/G097S/A098D/D099I, A001V/G097S/A098R/D099G, V004M/A098D/D099G, V004M/D099R/G100S/Z100.01L, V004M/G127C/G128S/P129R, V004M/M119C/D120S, V008I/A098G/D099R, V008I/A098N, V008I/M119C/D120G, Q010L/G097R/A098C/D099S, K012N/A098R/D099V, K012R/G097D/A098H/D099G, A013T/S101R/Q103S, A013V/G097N/A098H/D099G/G160S, A013V/G097S/Z097.01R/A098R, A013V/G100R/S101G, A013V/N118G/M119I, A013V/P129Z/S130H/S132G, P014L/N062D/S101R/Q103S, P014S/A098S/D099G, A015T/V068N/A069G, L016M/G128R/P129R/Z129.01G, H017Q/G127R/P129G, H017Y/A098R/D099I, H017Y/A098R/D099Z/S101G, H017Y/P129R, H017Y/S130Y/G131S/S132L, Q019H/G128R/Z128.01R/P129D, Q019H/Z128.01G/Z128.02G, G020D/G097R/A098G/D099C, G020D/P129Z/S130G/G131R/S132C, N025Y/S101L/Q103N, A029V/G097R/A098S, S033I/T066G/H067R/V068D/A216E, S038F/A098R/D099S, V044I/G097C/A098D/D099G, V044I/G097R/A098R, V044I/G100S/S101G, A045V/G097C/A098R/D099S, A048V/G097L/A098G/D099S, A048V/G100R/S101G, A048V/G128S/P129L/Z129.01G, M050I/A098G, S053F/S130I/S132G, S053T/D099H/S101Z, E054D/A098R/D099G, T055I/S101R/Q103R, T055S/T066I/Z066.01G/Z066.02G, Q059H/D099S/S101G, Q059H/M119S/D120R, Q059K/M119V/V121C, Q059L/P129G, N061D/G128R/P129V/Z129.01G, S063T/G097D/A098R/D099S, G065R/Z101.01R/Z101.02G, T066S/H067S, V068C/A069G, V068C/A069G/H238Q, V068G/A069G, V068G/A069G/V150I, V068G/A069S, V068I/A069G/A144V, V068I/G097D/A098G/D099Z/G100R, V068L/A069G, V068L/A069S, V068S/A069G, V068S/A069G/A116T, A069G/T071G, A069G/T071I, L075G/N076G/N077Z, L075R/N076G/N077Z, N076D/D099R/Z100.01G, I079F/A098G, V081A/S101R/Q103V, V081I/G097R/A098G/D099C, V084D/G097S/A098G/D099N, V084I/G127C/P129G, V084I/G127L, A085V/A098G, A085V/G097R/A098S/D099N, A085V/G128S/P129R/Z129.01G, A085V/N218I, A085V/S101G, A085V/S204P, A085V/Z099.01H/S101R, P086S/G131D/S132G, P086T/S101D/Q103S/V147I, A088S/S101R/Q103V, A088T/A098R/D099R, A088T/D099G/Z100.01G, A088T/G127N/P129R, A088T/G127S/P129R/Q206R/A273S, A088T/P129S/G146D, A088T/S101G, A088T/S130R/G131H/S132N, A088V/G100Z/S101C/Q103R, A088V/P129Z, A092G/S249R, A092G/V093C, A092G/V093I, A092G/V093L, A092L/V093G/K094Z, A092S/K094V, A092S/V093C, A092S/V093G, A092S/V093S, A092V/V093L, V093I/G128D/P129R, V093I/P129Z, V093R/Z093.01G/K094R, K094E/V095D, K094S/V148I, L096I/N118R/M119C/D120G/A151V, Z096.01D/A098R, Z096.01H/A098G, Z096.01N/A098H, Z096.01R/A098C, Z096.01R/A098G, Z096.01R/A098G/V192I, Z096.01R/A098R, Z096.01R/A098R/V192A, Z096.01R/A098S, Z096.01R/A098S/G157S, Z096.01R/Z096.02G, Z096.01R/Z096.02G/V147I, Z096.01S/A098G, Z096.01S/A098N, G097C/A098D/D099S, G097C/A098G/D099G, G097C/A098G/D099N, G097C/A098G/D099S, G097C/A098G/D099S/G100Z, G097C/A098G/D099V, G097C/A098N/D099S, G097C/A098R, G097C/A098R/D099G, G097C/A098R/D099H, G097C/A098R/D099H, G097C/A098R/D099N, G097C/A098R/D099R, G097C/A098R/D099R/N109D/A114S, G097C/A098S/D099H/A151V, G097C/A098V/D099S, G097C/Z097.01G/A098H/T253I, G097C/Z097.01S/A098G, G097D/A098C, G097D/A098G/D099G, G097D/A098G/D099R, G097D/A098H/D099R, G097D/A098H/D099V, G097D/A098R/D099G, G097D/A098R/D099R, G097D/A098R/D099S, G097D/A098R/D099Z, G097D/G100C/S101R, G097D/G100R/S101G, G097H/A098C/D099G, G097H/A098D/D099N, G097H/A098G, G097H/A098G/D099G, G097H/A098G/D099R, G097H/A098G/D099Z, G097H/A098G/D099Z/A151V, G097H/A098G/D099Z/G100R, G097H/A098G/D099Z/G100V, G097H/A098G/D099Z/S101N/G166S, G097H/A098R, G097H/A098R/D099G, G097H/A098R/D099L, G097H/A098R/D099N, G097H/A098S/D099G, G097H/A098S/D099I, G097H/A098S/D099L, G097H/A098S/D099S/K237E, G097L/A098C/D099S, G097L/A098G/D099S, G097L/A098G/D099S/G100Z, G097L/A098G/D099Z, G097L/A098H, G097L/A098R/D099N, G097L/A098V, G097N/A098G/D099C, G097N/A098G/D099R, G097N/A098G/D099R/V270I, G097N/A098G/D099S, G097N/A098G/D099V, G097N/A098G/D099V/A151V, G097N/A098G/D099V/S101F, G097N/A098H/D099N,G097N/A098R/D099R, G097N/A098R/D099S, G097N/A098R/D099V, G097N/A098S/D099S, G097R/A098C, G097R/A098C/D099S, G097R/A098F/A200V, G097R/A098G, G097R/A098G/D099C,G097R/A098G/D099G, G097R/A098G/D099G/A273T, G097R/A098G/D099S, G097R/A098G/D099S/A133D, G097R/A098G/D099V, G097R/A098H/D099C/S182N, G097R/A098H/D099G, G097R/A098N/D099G, G097R/A098N/D099Z, G097R/A098R, G097R/A098R/D099C, G097R/A098R/D099G, G097R/A098R/D099S, G097R/A098S/D099C, G097R/A098S/D099G, G097R/A098S/D099L, G097R/A098S/D099R, G097R/A098S/D099S, G097R/A098S/D099V, G097R/A098V/D099G, G097R/Z097.01H/A098C, G097R/Z097.01R/A098G, G097R/Z097.01R/A098G/V147I, G097R/Z097.01R/A098S, G097R/Z097.01R, G097R/Z097.01S/A098G, G097R/Z097.01S/A098R, G097S/A098C, G097S/A098C/D099G, G097S/A098C/D099H, G097S/A098C/D099L, G097S/A098C/D099R, G097S/A098C/D099S, G097S/A098C/D099Z, G097S/A098G/D099F, G097S/A098G/D099G, G097S/A098G/D099G, G097S/A098G/D099S, G097S/A098G/D099Z, G097S/A098G/D099Z/G100R, G097S/A098H/D099C, G097S/A098H/D099G, G097S/A098N/D099G, G097S/A098R, G097S/A098R/D099C, G097S/A098R/D099G, G097S/A098R/D099L, G097S/A098R/D099V, G097S/A098R/D099Y, G097S/A098S/D099G, G097S/A098S/D099G/A133V/A153V/S236Y, G097S/A098S/D099G/A153V, G097S/A098S/D099Z, G097S/Z097.01D/A098G/A144T/Q271H, G097S/Z097.01G/A098V, G097S/Z097.01S/A098G, G097S/Z097.01S/A098G/A273T, G097S/Z097.01Y, G097V/A098R, G097V/A098R/D099G, G097V/A098S/D099S, G097Y/A098H/D099N, G097Z/A098S/D099H, Z097.01D/A098R/D099G, Z097.01D/A098S/D099L, Z097.01D/A098V/D099G, Z097.01G, Z097.01G/A098G, Z097.01G/A098G/D099S, Z097.01G/A098R, Z097.01G/A098R/D099C, Z097.01G/A098R/D099G, Z097.01G/A098R/D099S, A098C/D099G, A098C/D099G/A116T, A098C/D099H, A098C/D099L, A098C/D099L/K213R, A098C/D099L, A098C/D099N, A098C/D099R, A098C/D099S, A098C/D099S/P194L, A098D/D099C, A098D/D099G, A098D/D099G/G100L, A098D/D099H/G100S, A098D/D099H/H238Y, A098D/D099R, A098D/D099R/G100D,A098D/D099R/G160S, A098F/D099S/G100L, A098G/D099C, A098G/D099G, A098G/D099G/A228T, A098G/D099G/G100H, A098G/D099G/N243D, A098G/D099G/S101P, A098G/D099G/T244S, A098G/D099G, A098G/D099H, A098G/D099L, A098G/D099R, A098G/D099R/A116T, A098G/D099R/G100D, A098G/D099R/V148I, A098G/D099S, A098G/D099S/L267M, A098G/D099V, A098G/D099Z, A098H/D099C, A098H/D099G, A098H/D099G/A216T, A098H/D099G/G100D, A098H/D099G/G100N/L267M, A098H/D099G/G100S, A098H/D099G/V143I, A098H/D099R, A098H/D099R/G100S, A098H/D099S/G100C, A098H/D099S/G100N, A098H/D099Y, A098I/D099G, A098I/D099S, A098L/D099G, A098L/D099S, A098N/D099G, A098N/D099L, A098N/D099S, A098R/A144T, A098R/A274V, A098R/D099C, A098R/D099G, A098R/D099G/G100C, A098R/D099G/G100D, A098R/D099G/G100L/S145T, A098R/D099G/G100N, A098R/D099G/G100R, A098R/D099G/G100S, A098R/D099G/V150I, A098R/D099H, A098R/D099I, A098R/D099L, A098R/D099N, A098R/D099N/G100L, A098R/D099R, A098R/D099R/G100D, A098R/D099R/S101Z, A098R/D099R/S130F, A098R/D099R, A098R/D099S, A098R/D099S/G100L/A153V, A098R/D099S, A098R/D099V, A098R/D099Z/S101G, A098R/G100C/A137T, A098R/G100S, A098S/D099C, A098S/D099G, A098S/D099G/G100C, A098S/D099G/G100D, A098S/D099G/G100R/L250I, A098S/D099G/G100S, A098S/D099G/Z099.01R, A098S/D099H, A098S/D099H/M124V, A098S/D099H/Z099.01R/G169A/V180A, A098S/D099R, A098S/D099R/G100L, A098S/D099R/G154S, A098S/D099R/G166S, A098S/D099R/Z100.01G, A098S/D099S, A098S/D099S/Z099.01R, A098S/D099V, A098T/P129Z/S130R/S132G, A098T/S101G, A098V/D099G, A098V/D099G/G100D, A098V/D099G/G100D/A153V, A098V/D099G/G100S, A098Y/D099R/G100L, A098Y/D099R/G100Z/S101Z/G102D, A098Z/S101R, Z098.01N, Z098.01R, Z098.01R/G100R, D099C/K136N, D099C/S101D, D099C/S101R, D099C/S101Z, D099C/Z099.01S, D099C/Z100.01G, D099F/S101Z, D099G/G100C, D099G/G100D/S101R, D099G/G100H, D099G/G100R/S101V, D099G/G100S/S101R, D099G/S101G, D099G/S101H, D099G/S101V, D099G/S101Z, D099G/S101Z/N269D, D099G/Z099.01N, D099G/Z099.01R/G100S, D099G/Z099.01S, D099G/Z100.01G, D099G/Z100.01G/S183N/Q275K, D099H/S101Z, D099H/S101Z/V150I, D099H/Z100.01G/A273V, D099H/Z100.01G/Z100.02G, D099I/Z099.01S, D099L/N118D, D099L/S101G, D099L/S101V, D099L/Z100.01G, D099N/A116T, D099N/G100R/S101V, D099N/S101G, D099N/S101H, D099N/S101Z, D099N/Z099.01S, D099N/Z100.01G, D099R/S101D, D099R/S101G, D099R/S101V, D099R/S101Z, D099R/S101Z/A133T, D099R/Z099.01N, D099R/Z099.01S, D099R/Z100.01G, D099R/Z100.01N, D099S/S101G, D099S/S101I, D099S/S101Z, D099S/Z099.01H, D099S/Z099.01L, D099S/Z099.01V, D099S/Z100.01G, D099S/Z100.01G/Z100.02G, D099V/S101D, D099V/S101V, D099V/S101Z, Z099.01H/S101L, Z099.01H/Z099.02R, Z099.01N/S101R, Z099.01S, Z099.01S/S101V, Z099.01S/Z099.02G/A228T, G100D/S101G, G100D/S101R, G100H/G102V, G100H/S101G, G100H/S101R, G100L/S101G, G100L/S101R, G100N/S101D, G100N/S101L, G100N/S101R, G100R/L267R/I268V, G100R/S101G, G100R/S101G/G160S/S182N, G100R/S101G/Q103Z/Y104Z/S161N, G100R/S101G/S163N, G100R/S101N, G100R/S101R, G100R/S101R/Q103Z/V270I, G100R/S101Z/G102C/Q103D, G100S/S101C, G100S/S101G, G100S/S101R, G100S/S101R, G100S/S101V, G100S/S101V/A231T, Z100.01G, Z100.01L, Z100.01N, Z100.01R, Z100.01R/S101G, S101C/Q103I, S101C/Q103S, S101D/Q103C, S101D/Q103D, S101D/Q103F, S101D/Q103H, S101D/Q103H, S101D/Q103R, S101D/Q103R/H238Y, S101D/Q103S, S101D/Q103V, S101F/Q103G, S101G/A151V, S101G/A179V, S101G/G102S, S101G/Q103G, S101G/Q103L, S101G/Q103N, S101G/Q103R, S101G/Q103R/A179T, S101G/Q103R/G157D, S101G/Q103S, S101G/Q103S/L233S, S101G/Q103S/L233S, S101G/Q245H, S101G/S249N, S101G/V139I, S101G/Z101.01R, S101G/Z101.01R/A200T, S101G/Z102.01G, S101H/G102S, S101H/Q103D, S101H/Q103G, S101H/Q103G/E251Q, S101H/Q103H, S101H/Q103I, S101H/Q103R, S101H/Q103R/A274Z/Q275Z, S101H/Q103S, S101H/Q103V, S101I/Q103R, S101L/G102S/Q103R, S101L/Q103D, S101L/Q103G, S101L/Q103R, S101L/Q103R/A138T, S101L/Q103R/V121I/K213E, S101L/Q103S, S101L/Q103V, S101L/Z101.01R, S101N/Q103R, S101N/Z102.01G, S101R/A151V, S101R/A273V, S101R/E156G, S101R/G102L/Q103G, S101R/G102S,S101R/G102S/Q103R, S101R/G102S/Q103V, S101R/G131D, S101R/Q103C, S101R/Q103D,S101R/Q103D/S248R, S101R/Q103G, S101R/Q103G/A116T, S101R/Q103G/V147I/A153V, S101R/Q103G, S101R/Q103L, S101R/Q103N, S101R/Q103R, S101R/Q103R/S161N, S101R/Q103R/V148I, S101R/Q103S, S101R/Q103S/L126F, S101R/Q103V, S101V/Q103G, S101V/Q103H, S101V/Q103N/Z275.01K, S101V/Q103R, S101V/Q103V, S101Y/G102S/Q103R, S101Y/Q103L, S101Y/Q103S, S101Y/Q103V, S101Z/G102I/Q103H/Y104G/L135F, S101Z/G102V/Q103R/Y104L, S101Z/Q103D, S101Z/Q103F, S101Z/Q103L, S101Z/Q103R, G102C/Q103L/Y104S, G102R/Q103C/Y104C/V192I, G102R/Q103G/Y104R, G102R/Q103R/Z103.01G, G102Z/Q103S, Z102.01G/Q103R, Z102.01G/Q103R/L267V, Z102.01G/Z102.02R/Z102.03G, Q103H/Y104N/Q275Z, Q103N/E156D, Q103R/I122L/N123S/M124V, Q103R/Y104F, Q103Y/S182N, S105G/P129S/S130H/G131H, W106C/A116D/N117S/N118C, N109S/M119C/D120G/V121L, A116G/N117Z/N118R/M119C, A116H/N117G/N118R, N117C/N118G/M119S, N117C/N118G/M119S/A216V, N117G/N118G/M119C, N117G/N118R/M119C, N117H/N118C/M119C, N117H/N118D/M119S, N117H/N118G/M 119C, N117H/N118G/M119L, N117H/N118G/M119S, N117H/N118G/M119V, N117H/N118H/M119V, N117H/N118R/M119C, N117H/N118R/M119S, N117H/N118S/M119C, N117H/N118S/M119S, N117H/N118S/M119V, N117I/N118G/M119V, N117L/N118G/M119V, N117R/N118G/M119C, N117R/N118G/M119I/V270I, N117R/N118G/M119S, N117R/N118H/M119V, N117R/N118R/M119C, N117R/N118R/M119I, N117R/N118R/M119S, N117R/N118R/M119V, N117R/N118S/M119I, N117R/N118S/M119L, N117S/N118G/M119C, N117S/N118G/M119S, N117S/N118H/M119V, N117S/N118R/M119V, N117V/N118G/M119C, N117V/N118H/M119C, N117Y/N118G/M119S, N117Y/N118G/M119V, Z117.01G/N118R/M119C, N118C/M119V/D120S, N118C/Z118.01G/M119C, N118D/M119C/D120L, N118D/M119S/D120R, N118G/M119C/D120G, N118G/M119C/D120L, N118G/M119I/D120R, N118G/M119N, N118G/M119S/D120G, N118G/M119S/D120R, N118G/M119V/D120G, N118G/M119V/D120S/S182N, N118H/M119C/D120R, N118H/M119I/D120G, N118H/M119N/V148I, N118H/M119S/D120G, N118H/M119V/D120G, N118R/M119C, N118R/M119C/D120G, N118R/M119C/D120R, N118R/M119S, N118R/M119S/D120G, N118R/M119S/D120R, N118R/M119S/D120S, N118R/M119V/D120C, N118R/M119V/D120G, N118R/M119V/D120G/A232T, N118S/M119C/D120R, M119C/D120G, M119C/D120G/V121I, M119C/D120G/V121L, M119C/D120H, M119C/D120H/A187V, M119C/D120R, M119H/V121I, M119I/D120C, M119I/D120G, M119I/D120R, M119L/D120G, M119S/D120R, M119S/D120S/V121I, M119S/V121I, M119V/D120C, M119V/D120G, M119V/D120R, M119V/D120S/V198L, D120G/I122C, D120G/I122C/L217S, D120G/I122G, D120G/I122V, D120H/I122L, D120H/V121C/I122V, D120N/V121L/I122V, D120R/A151V, D120R/I122V, D120R/V121I, D120R/V121I/I122C, D120R/V121I/I122C/A228T, D120S/I122C/A133S, V121C/I122C/N123C, V121C/I122G/N123V, V121C/I122S/N123V, V121C/I122V/N123S, V121I/I112C/N123C, V121I/I122C/V150I, V121I/I122G/N123C, V121I/I122S/N123C, V121I/I122S/N123I, V121I/I122V/N123S, V121L/I122S/N123V, V121L/N123C, I122C/N123C, I122C/N123S/M124L, I122D/N123V/M124L, I122G/N123C/M124L, I122G/N123G/Z123.01C, I122H/N123V/M124LI122S/N123S, I122V/N123G/M124C/A138V, I122V/N123S/M124I, I122V/S183G, N123C/M124S, N123C/M124V, N123S/M124L, N123S/M124S, M124C/L126S, M124C/L126S/T244I, M124C/L126V, M124I/L126H, L126F/P129Z, L126F/P129Z/S182N, L126I/P129Z, L126R/G127R, Z126.01S, G127C/G128S/P129R, G127C/P129G, G127C/P129G/A200T, G127C/P129R, G127C/P129R/A153T, G127D/P129G, G127D/P129S, G127H/P129G, G127H/P129R, G127L/P129R, G127N/P129C, G127N/P129G, G127N/P129G/A133T, G127N/P129G/A151V/K213N, G127N/P129R, G127N/P129S/A273V, G127N/P129V, G127R/A230V, G127R/P129D, G127R/P129D/L217S, G127R/P129G, G127R/P129R, G127R/P129S, G127S/P129D, G127S/P129F, G127S/P129G, G127S/P129G/G166D, G127S/P129G/I175T, G127S/P129R, G127S/P129S, G127S/P129V, G127V/P129G, G127V/P129R, Z127.01G/Z127.02H/P129D, Z127.01G/Z127.02H/P129G, Z127.01G/Z127.02H/P129R, Z127.01H/G128C/P129C/A230V, Z127.01H/P129G, Z127.01H/P129G/V148I, Z127.01H/P129R, Z127.01N/G128C/P129V, Z127.01N/G128S/P129S, Z127.01N/G128S/P129V, Z127.01N/P129R, Z127.01R/G128S/P129S, Z127.01R/P129G, Z127.01S/P129D, Z127.01S/P129G, Z127.01S/P129R, G128D/P129G, G128D/P129R/Z129.01R, G128D/P129S/G154S, G128H/P129C, G128H/P129H/Z129.01G, G128H/P129H/Z129.01R, G128H/P129R, G128H/P129R/Z129.01G, G128H/P129R/Z129.01R, G128H/P129S/Z129.01G, G128H/P129S/Z129.01R, G128H/P129S/Z129.01R/S248N, G128H/P129Y, G128H/Z128.01R/P129D, G128H/Z128.01R/P129S, G128H/Z128.01R/P129S/A144T, G128H/Z129.01R, 128N/P129R/Z129.01G, G128N/P129R/Z129.01R, G128N/P129S/Z129.01D, G128N/Z128.01R/P129S, G128R/P129G, G128R/P129H/Z129.01G, G128R/P129L/Z130.01S, G128R/P129R, G128R/P129R/Z129.01G, G128R/P129R/Z129.01G/A216V, G128R/P129S, G128R/P129S/Z129.01G, 128R/P129Z/S130R/S132G, G128R/Z128.01R/P129D, G128R/Z128.01R/P129F, G128S/P129C, G128S/P129C/Z129.01R, G128S/P129D, G128S/P129D/S248R, G128S/P129D/T244N, G128S/P129G, G128S/P129G/A187V, G128S/P129H/Z129.01R, G128S/P129R/L209F, G128S/P129R/Z129.01G, G128S/P129S/Z129.01D, G128S/P129S/Z129.01R, G128S/P129V, G128Y/P129R/Z129.01G, Z128.01G, Z128.01G/P129D, Z128.01G/P129R, Z128.01G/P129S/A134T, Z128.01G/Z128.02G, Z128.01R/P129D, Z128.01R/Z128.02G/P129S, Z128.01R/Z128.02H/P129G/G160S, Z128.01R/Z128.02R/P129D, Z128.01Y/Z128.02H/P129R, P129C/S130C/S132Z, P129D/Z129.01G/Z129.02D/G160D, P129D/Z129.01G/Z129.02G, P129G/G131Z, P129G/S130G/Z131.01G, P129G/S130H/S132Z, P129G/S130R/S132Z, P129G/V270I, P129G/Z129.01G, P129G/Z129.01R/Z129.02G, P129H/G131Z, P129H/G131Z/S132C, P129H/G131Z/S132H, P129H/S132Z, P129H/Z130.01D/Z130.02S, P129R/G131Z/S132H, P129R/S130D/S132Z, P129R/S130N/G131Z, P129R/S132T, P129R/S132Z, P129R/Z129.01G, P129R/Z129.01G/Z129.02G, P129R/Z129.01G/Z129.02G/A144T, P129R/Z129.01G/Z129.02G/G146D, P129R/Z129.01G/Z129.02R, P129R/Z129.01G, P129R/Z129.01R/Z129.02G, P129S/G131ZP129S/S130H/S132Z, P129S/Z129.01C, P129S/Z129.01R/Z129.02S, P129S/Z130.01N/Z130.02S, P129V/S132Z, P129Z, P129Z/G131R/S132C, P129Z/S130G, P129Z/S130G/G131H/S132H, P129Z/S130G/G131V, P129Z/S130H, P129Z/S130H/S132G, P129Z/S130H/S132N, P129Z/S130L, P129Z/S130R, P129Z/S130R/G131C, P129Z/S130R/G131C, P129Z/S130R/G131D/S132C, P129Z/S130R/G131H/A144E, P129Z/S130R/G131H/S132D, P129Z/S130R/G131R/S132D, P129Z/S130R/L267M, P129Z/S130R/S132C, P129Z/S130R/S132G, P129Z/S130V/S132G, P129Z/S130Z/G131N/S132G, S130D/G131C/S132D, S130D/G131R, S130D/G131R/S132D, S130D/G131Z, S130D/G131Z/S132H, S130F/G131N/S132V, S130F/G131S/S132V, S130F/S132V, S130G/G131D/S132C, S130G/G131H/S132D, S130G/G131S/S132I, S130G/S132G, S130H/G131C/S132G, S130H/G131H/S132R, S130H/G131R/S132C, S130H/G131S, S130H/G131S/S132C, S130H/S132Z, S130I/G131S/S132H, S130I/S132G, S130N/G131S/S132C, S130R/G131R/S132C, S130R/G131S/S132C/S161N, S130R/S132G, S130R/S132G/S183N, S130R/S132Z, S130V/G131D/S132I, S130V/G131H/S132C, S130V/S132N, S130V/S132Z, S130Y/G131H/S132C, S130Y/S132G, S130Z/G131H/S132H, G131H/S132L/W241R, G131N/S132C, G131S/S132G, G131Y/S132G, and A142V/G211V, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
2. A cleaning composition comprising a subtilisin variant comprising at least one modification, wherein said modification is selected from: V072G/G097A/G128A/Y217Q, G097A/G128S/Y217Q, G097A/G128S/P129D/Y217Q, G097A/G128A/A134T/Y217Q, M124V/L126A/Y217Q, G097A/G128A/P129D/Y217Q/A232P, G097A/G128A/P129D/Y217Q, G097A/G128S/P129V/A134T/Y217Q, G097A/G128S/P129V/Y217Q, V068I/G097A/G128S/P129D/Y217Q, G097A/S101D/G128S/S204F/Y217Q/S236F/T254P, G097A/S101D/G128S/Y217Q, G097A/G128A/P129V/Y217Q, G097A/S101D/G128A/Y217Q, G097A/S101D/G128S/P129D/Y217Q, G097A/S101D/G128A/A134T/Y217Q, G097A/Z099.01S/A114S/G128S/Y217Q, G097A/S101D/G128A/P194L/Y217Q, G097A/Z099.01S/G128S/Y217Q, G097A/S101D/G128A/P129D/Y217Q, Q019R/G097A/Z099.01S/G128S/P129D/Y217Q, G097A/G100S/G128S/Y217Q, G097A/S101D/G128S/P129V/Y217Q, G097A/Z099.01S/G128A/A134T/Y217Q, G097A/G100S/I107V/G128S/P129D/Y217Q, G097A/G100S/G128S/Y217Q/A230V, G097A/G100S/G128A/Y217Q/K237N, G097A/Z099.01S/G128S/P129D/Y217Q, G097A/G100S/G128S/P129D/Y217Q, Z096.01D/G097A/A098R/G128A/Y217Q, G097A/Z099.01S/I122S/N123G/G128A/Y217Q, G097A/Z099.01S/I122S/N123G/G128A/Y217Q/T220A/A232T, P005S/G097A/Z099.01S/S101D/G128A/Y217Q, G097A/S101D/G128A/P129V/Y217Q, V093Z/K094S/V095C/L096S/G097A/G128A/Y217Q, G097A/G100S/S101D/G128A/Y217Q/A232P, Z096.01D/G097A/A098R/S101D/G128A/Y217Q, and G097A/Z099.015/G100S/G128A/Y217Q, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
3. A cleaning composition comprising a subtilisin variant comprising at least two modifications, wherein said modifications are selected from: G097A/G128S/P129T/Y217Q, G097A/G128A/P129S/Y217Q, G097A/G128S/P129C/Y217Q, G097A/G128S/P129Q/Y217Q, G097A/G128S/P129E/Y217Q, G097A/P129S/Y217Q, G097A/G128S/P129A/Y217Q, G097A/G128S/P129K/Y217Q, G097A/P129N/Y217Q, G097A/P129E/Y217Q, G097A/G128S/P129K/G131S/Y217Q, G097A/G128S/P129R/Y217Q, G097A/G128S/P129V/Y217Q, G097A/G128S/P129Y/Y217Q, G097A/G128A/P129R/Y217Q, G097A/G128H/P129S/Y217Q, G097A/G128N/P129S/Y217Q/V270A, G097A/G128S/P129L/Y217Q, G097A/P129R/Y217Q, G097A/P129W/Y217Q, G097A/P129Z/S130A/Y217Q, G097A/G128H/P129R/Y217Q, G097A/G128H/P129T/Y217Q, G097A/P129G/Y217Q, G097A/G128N/P129A/Y217Q, G097A/G128H/Y217Q, G097A/G128S/P129W/Y217Q, G097A/P129Z/Y217Q, G097A/G128N/Z128.01A/P129S/Y217Q, G097A/G128N/Y217Q, G097A/G128A/P129L/Y217Q, G097A/G128T/P129N/Y217Q, G097A/G128H/P129A/Y217Q, G097A/G128M/P129R/Y217Q, G097A/G128Q/P129K/Y217Q, G097A/G128T/P129Q/Y217Q, G097A/G128T/P129H/Y217Q, G097A/G128T/P129R/Y217Q, G097A/G128D/P129E/Y217Q, G097A/G128T/P129T/Y217Q, G097A/G128T/P129A/Y217Q, G097A/G128Z/P129Z/Y217Q, G097A/G128S/Z128.01A/P129S/Y217Q, G097A/G128N/P129M/Y217Q, G097A/G128N/P129V/Y217Q, G097A/G128E/P129K/Y217Q, G097A/G128N/Z128.01A/P129F/Y217Q, G097A/G128C/P129R/Y217Q, G097A/G128T/P129M/Y217Q, G097A/G128R/Z128.01A/P129S/Y217Q, G097A/G128S/Z128.01A/P129A/Y217Q, G097A/G128D/Y217Q, G097A/G128T/P129Y/Y217Q, G097A/G128R/P129S/Y217Q, G097A/G128R/Z128.01A/P129T/Y217Q, G097A/G128R/Z128.01A/P129A/Y217Q, G097A/G128T/P129W/Y217Q, G097A/G128Y/Z128.01A/P129R/Y217Q, G097A/G128C/P129D/Y217Q, V084L/G097A/P129Z/Y217Q, G097A/G128R/P129D/Y217Q, G097A/G128A/Z128.01A/P129H/E195A/Y217Q, G097A/G128S/Z128.01A/P129Y/Y217Q, G097A/G128N/P129L/Y217Q, G097A/G128C/P129H/Y217Q, G097A/G128T/Z128.01A/P129T/Y217Q, G097A/G128C/Z128.01A/P129A/Y217Q, G097A/G128S/Z128.01A/P129I/Y217Q, G097A/G128S/Z128.01A/P129E/Y217Q/Q271H, G097A/G128S/Z128.01A/P129L/Y217Q, G097A/G128Y/Z128.01A/P129S/Y217Q, G097A/G128S/Z128.01A/P129M/R186H/Y217Q, G097A/G128E/Z128.01A/P129D/Y217Q, G097A/G128T/P129L/Y217Q, G097A/G128F/P129D/Y217Q, G097A/G128V/Z128.01A/P129R/Y217Q, G097A/G128L/Z128.01A/P129S/Y217Q, G097A/G128Y/P129T/Y217Q, G097A/G128C/P129Y/Y217Q, G097A/G128C/P129T/Y217Q, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
4. A cleaning composition comprising a subtilisin variant comprising at least two modifications, wherein said modifications are selected from: G097A/G128A/Y217Q/M222Q, M124V/L126A/P129V/Y217Q, G128A/P129V/Y217Q, G097A/G128A/P129V/Y217Q, N123G/P129V/Y217Q, V30I/Z096.01D/G097A/A098R/G128A/Y217Q, Z096.01D/A098R/G128A/Y217Q, Z096.01D/A098R/M124V/L126A/Y217Q, G097A/Z098.01S/G128A/Y217Q, Z096.01D/A098R/N123G/Y217Q, Z096.01D/G097A/A098R/G128A/Y217Q/S236F, Z098.01S/M124V/L126A/Y217Q, Z098.01S/G128A/Y217Q, Z096.01D/G097A/A098R/G128A/Y217Q, V093I/Z098.01S/M124V/L126A/Y217Q, A073V/Z098.01S/N123G/Y217Q, G065D/Z098.01S/G128A/Y217Q, I011L/Z098.01S/G128A/Y217Q, L082F/Z096.01D/A098R/N123G/Y217Q, Z098.01S/N123G/A138V/Y217Q, Z098.01S/N123G/Y217Q, wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
5. A cleaning composition comprising a subtilisin variant comprising the substitution Y217L and further comprising at least one modification set forth in any of claims 1-4, and wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
6. (canceled)
7. A cleaning composition comprising a subtilisin variant comprising the substitutions G97A/G128A/Y217Q and further comprising at least one modification set forth in any of claims 1-4, and wherein the positions correspond to the positions of BPN′ subtilisin of SEQ ID NO:2.
8. (canceled)
9. The cleaning composition of claim 1, wherein said cleaning composition is a laundry detergent.
10. The laundry detergent of claim 9, wherein said laundry detergent is a heavy duty liquid laundry detergent.
11. The cleaning composition of claim 1, wherein said cleaning composition is a dish detergent.
12. The cleaning composition of claim 1, further comprising one or more additional enzymes or enzyme derivatives selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof.
13. The cleaning composition of claim 1, further comprising at least one stabilizing agent.
14. The cleaning composition of claim 1, comprising at least 0.0001 weight percent of the subtilisin variant, and optionally, at least one suitable adjunct ingredient.
15. A method of cleaning, said method comprising the steps of:
a) contacting a surface and/or an article comprising a fabric with the cleaning composition of claim 1; and
b) optionally washing and/or rinsing said surface or article.
16-17. (canceled)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150064767A1 (en) * 2008-06-06 2015-03-05 Danisco Us Inc. Compositions and methods comprising variant microbial proteases

Families Citing this family (126)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4703113B2 (en) * 2002-01-16 2011-06-15 ジェネンコー・インターナショナル・インク Multiple substitution protease mutant
US20090060933A1 (en) * 2004-06-14 2009-03-05 Estell David A Proteases producing an altered immunogenic response and methods of making and using the same
EP2647692A3 (en) 2008-11-11 2014-01-22 The Procter and Gamble Company Compositions and methods comprising serine protease variants
KR20110095260A (en) 2008-11-11 2011-08-24 다니스코 유에스 인크. Bacillus subtilisin comprising one or more combinable mutations
SG177601A1 (en) * 2009-07-16 2012-02-28 Glaxo Group Ltd Improved anti-serum albumin binding single variable domains
JP6204018B2 (en) * 2009-09-25 2017-09-27 ノボザイムス アクティーゼルスカブ Use of protease variants
CA2782613C (en) 2009-12-09 2016-08-23 The Procter & Gamble Company Fabric and home care products
WO2011072099A2 (en) 2009-12-09 2011-06-16 Danisco Us Inc. Compositions and methods comprising protease variants
US20120067373A1 (en) * 2010-04-15 2012-03-22 Philip Frank Souter Automatic Dishwashing Detergent Composition
CN108410585A (en) * 2010-05-06 2018-08-17 宝洁公司 The consumer goods with ease variants
CN103764823B (en) * 2011-05-05 2018-05-11 丹尼斯科美国公司 Composition and method comprising serine protease variants
CN106065381B (en) * 2011-05-05 2019-07-26 宝洁公司 Composition and method comprising serine protease variants
WO2013076269A1 (en) * 2011-11-25 2013-05-30 Novozymes A/S Subtilase variants and polynucleotides encoding same
EP2607468A1 (en) 2011-12-20 2013-06-26 Henkel AG & Co. KGaA Detergent compositions comprising subtilase variants
WO2013092635A1 (en) 2011-12-20 2013-06-27 Novozymes A/S Subtilase variants and polynucleotides encoding same
CN104114698A (en) * 2012-02-17 2014-10-22 诺维信公司 Subtilisin variants and polynucleotides encoding same
EP2628785B1 (en) * 2012-02-17 2016-05-18 Henkel AG & Co. KGaA Detergent compositions comprising subtilase variants
WO2014099523A1 (en) 2012-12-21 2014-06-26 Danisco Us Inc. Alpha-amylase variants
WO2014099525A1 (en) 2012-12-21 2014-06-26 Danisco Us Inc. Paenibacillus curdlanolyticus amylase, and methods of use, thereof
ES2676895T5 (en) 2013-03-11 2022-04-27 Danisco Us Inc Combinatorial variants of alpha-amylase
EP3636662B1 (en) 2013-05-29 2022-07-13 Danisco US Inc. Novel metalloproteases
WO2014194032A1 (en) 2013-05-29 2014-12-04 Danisco Us Inc. Novel metalloproteases
EP3004342B1 (en) 2013-05-29 2023-01-11 Danisco US Inc. Novel metalloproteases
EP3004341B1 (en) 2013-05-29 2017-08-30 Danisco US Inc. Novel metalloproteases
US10647974B2 (en) 2013-06-27 2020-05-12 Novozymes A/S Subtilase variants and polynucleotides encoding same
WO2014207224A1 (en) 2013-06-27 2014-12-31 Novozymes A/S Subtilase variants and polynucleotides encoding same
BR112016005286A2 (en) * 2013-09-12 2017-09-12 Danisco Us Inc compositions and methods comprising lg12 clade protease variants
EP3058055A1 (en) 2013-10-15 2016-08-24 Danisco US Inc. Clay granule
DK3068879T3 (en) 2013-11-14 2020-03-16 Danisco Us Inc STABLE ENZYMER OBTAINED BY GLYCING REDUCTION
DK3553173T3 (en) 2013-12-13 2021-08-23 Danisco Us Inc SERINE PROTEASES OF BACILLUS GIBSONII-CLADE
DK3080262T3 (en) 2013-12-13 2019-05-06 Danisco Us Inc SERIN PROTEAS OF BACILLUS SPECIES
US10005850B2 (en) 2013-12-16 2018-06-26 E I Du Pont De Nemours And Company Use of poly alpha-1,3-glucan ethers as viscosity modifiers
CN106029700B (en) 2013-12-18 2019-04-12 纳幕尔杜邦公司 The poly- α -1 of cation, 3- glucan ether
WO2015112339A1 (en) 2014-01-22 2015-07-30 The Procter & Gamble Company Fabric treatment composition
WO2015112338A1 (en) 2014-01-22 2015-07-30 The Procter & Gamble Company Method of treating textile fabrics
EP3097174A1 (en) 2014-01-22 2016-11-30 The Procter & Gamble Company Method of treating textile fabrics
EP3097175B1 (en) 2014-01-22 2018-10-17 The Procter and Gamble Company Fabric treatment composition
US20150232785A1 (en) 2014-02-14 2015-08-20 E I Du Pont De Nemours And Company Polysaccharides for viscosity modification
EP3116914B8 (en) 2014-03-11 2021-04-21 E. I. du Pont de Nemours and Company Oxidized poly alpha-1,3-glucan as detergent builder
MX2016012044A (en) 2014-03-21 2017-06-29 Danisco Us Inc Serine proteases of bacillus species.
US9714403B2 (en) 2014-06-19 2017-07-25 E I Du Pont De Nemours And Company Compositions containing one or more poly alpha-1,3-glucan ether compounds
EP3158043B1 (en) 2014-06-19 2021-03-10 Nutrition & Biosciences USA 4, Inc. Compositions containing one or more poly alpha-1,3-glucan ether compounds
US10626388B2 (en) 2014-07-04 2020-04-21 Novozymes A/S Subtilase variants and polynucleotides encoding same
EP3207129B1 (en) 2014-10-17 2019-11-20 Danisco US Inc. Serine proteases of bacillus species
WO2016065238A1 (en) 2014-10-24 2016-04-28 Danisco Us Inc. Method for producing alcohol by use of a tripeptidyl peptidase
EP3224357A1 (en) 2014-10-27 2017-10-04 Danisco US Inc. Serine proteases of bacillus species
EP3550017B1 (en) 2014-10-27 2021-07-14 Danisco US Inc. Serine proteases
WO2016069552A1 (en) 2014-10-27 2016-05-06 Danisco Us Inc. Serine proteases
US20170335306A1 (en) 2014-10-27 2017-11-23 Danisco Us Inc. Serine proteases
EP3212783B1 (en) 2014-10-27 2024-06-26 Danisco US Inc. Serine proteases
WO2016081437A1 (en) 2014-11-17 2016-05-26 The Procter & Gamble Company Benefit agent delivery compositions
EP3227444B1 (en) * 2014-12-04 2020-02-12 Novozymes A/S Subtilase variants and polynucleotides encoding same
CN107002057A (en) * 2014-12-04 2017-08-01 诺维信公司 Liquid cleansing composition including ease variants
CN107002058A (en) * 2014-12-15 2017-08-01 诺维信公司 Subtilase variants
US10400230B2 (en) * 2014-12-19 2019-09-03 Novozymes A/S Protease variants and polynucleotides encoding same
EP3237631A1 (en) 2014-12-23 2017-11-01 E. I. du Pont de Nemours and Company Enzymatically produced cellulose
EP3268471B1 (en) * 2015-03-12 2019-08-28 Danisco US Inc. Compositions and methods comprising lg12-clade protease variants
WO2016176241A1 (en) 2015-04-29 2016-11-03 The Procter & Gamble Company Detergent composition
CN117683589A (en) 2015-04-29 2024-03-12 宝洁公司 Method for washing fabrics
EP3088503B1 (en) 2015-04-29 2018-05-23 The Procter and Gamble Company Method of treating a fabric
ES2683906T3 (en) 2015-04-29 2018-09-28 The Procter & Gamble Company Method of treating a tissue
DK3088505T3 (en) 2015-04-29 2020-08-03 Procter & Gamble PROCEDURE FOR TREATMENT OF A TEXTILE FABRIC
WO2016183509A1 (en) 2015-05-13 2016-11-17 Danisco Us Inc. AprL-CLADE PROTEASE VARIANTS AND USES THEREOF
WO2016201069A1 (en) 2015-06-09 2016-12-15 Danisco Us Inc Low-density enzyme-containing particles
FI3307427T3 (en) 2015-06-09 2023-11-09 Danisco Us Inc Osmotic burst encapsulates
WO2016201040A1 (en) 2015-06-09 2016-12-15 Danisco Us Inc. Water-triggered enzyme suspension
US11499146B2 (en) 2015-06-17 2022-11-15 Danisco Us Inc. Bacillus gibsonii-clade serine proteases
EP4141113A1 (en) 2015-11-05 2023-03-01 Danisco US Inc Paenibacillus sp. mannanases
WO2017079756A1 (en) 2015-11-05 2017-05-11 Danisco Us Inc Paenibacillus and bacillus spp. mannanases
WO2017083229A1 (en) 2015-11-13 2017-05-18 E. I. Du Pont De Nemours And Company Glucan fiber compositions for use in laundry care and fabric care
EP3374400B1 (en) 2015-11-13 2022-04-13 Nutrition & Biosciences USA 4, Inc. Glucan fiber compositions for use in laundry care and fabric care
EP3374488B1 (en) 2015-11-13 2020-10-14 DuPont Industrial Biosciences USA, LLC Glucan fiber compositions for use in laundry care and fabric care
WO2017089093A1 (en) 2015-11-25 2017-06-01 Unilever N.V. A liquid detergent composition
WO2017100720A1 (en) 2015-12-09 2017-06-15 Danisco Us Inc. Alpha-amylase combinatorial variants
EP3390625B1 (en) 2015-12-18 2023-09-06 Danisco US Inc. Polypeptides with endoglucanase activity and uses thereof
DE102016205670A1 (en) * 2016-04-06 2017-10-12 Henkel Ag & Co. Kgaa New protease with improved washing performance
JP2019518440A (en) * 2016-05-03 2019-07-04 ダニスコ・ユーエス・インク Protease variant and use thereof
CN109477112B (en) 2016-05-31 2024-09-06 丹尼斯科美国公司 Protease variants and uses thereof
JP7152319B2 (en) 2016-06-17 2022-10-12 ダニスコ・ユーエス・インク Protease variants and uses thereof
EP3535365A2 (en) 2016-11-07 2019-09-11 Danisco US Inc. Laundry detergent composition
US10550443B2 (en) 2016-12-02 2020-02-04 The Procter & Gamble Company Cleaning compositions including enzymes
EP3330349B1 (en) 2016-12-02 2024-06-19 The Procter & Gamble Company Cleaning compositions including enzymes
WO2018102478A1 (en) 2016-12-02 2018-06-07 The Procter & Gamble Company Cleaning compositions including enzymes
US11946081B2 (en) 2016-12-21 2024-04-02 Danisco Us Inc. Bacillus gibsonii-clade serine proteases
US20200392477A1 (en) 2016-12-21 2020-12-17 Danisco Us Inc. Protease variants and uses thereof
CN110621778A (en) 2017-03-15 2019-12-27 丹尼斯科美国公司 Trypsin-like serine protease and uses thereof
US20200040283A1 (en) 2017-03-31 2020-02-06 Danisco Us Inc Delayed release enzyme formulations for bleach-containing detergents
EP3645696A1 (en) 2017-06-30 2020-05-06 Danisco US Inc. Low-agglomeration, enzyme-containing particles
CN109819650A (en) * 2017-09-25 2019-05-28 春布里泽·格奥尔基·格奥尔基耶维奇 With antiviral and antibacterial activity thermostable composite and application thereof
CN111373039A (en) 2017-11-29 2020-07-03 丹尼斯科美国公司 Subtilisin variants having improved stability
BR112020012584A2 (en) 2017-12-21 2020-11-24 Danisco Us Inc. hot melt granules containing enzyme which comprise a thermotolerant desiccant
BR112020016068A2 (en) 2018-02-08 2020-12-08 Danisco Us Inc. THERMALLY RESISTANT MATRIX WAX PARTICLES FOR ENZYME ENCAPSULATION
WO2019245704A1 (en) 2018-06-19 2019-12-26 Danisco Us Inc Subtilisin variants
US20210363470A1 (en) 2018-06-19 2021-11-25 Danisco Us Inc Subtilisin variants
US20210189295A1 (en) 2018-08-30 2021-06-24 Danisco Us Inc Enzyme-containing granules
WO2020068486A1 (en) 2018-09-27 2020-04-02 Danisco Us Inc Compositions for medical instrument cleaning
CN111019925B (en) * 2018-10-10 2022-08-23 上饶市康可得生物科技有限公司 Method for screening protease variants and protease variants obtained
WO2020073553A1 (en) * 2018-10-10 2020-04-16 上饶市康可得生物科技有限公司 Method for screening protease variant and obtained protease variant
EP3887515A1 (en) 2018-11-28 2021-10-06 Danisco US Inc. Subtilisin variants having improved stability
CN109481336B (en) * 2018-12-19 2021-10-26 广州立白企业集团有限公司 Oral care compositions and uses thereof
EP3976776A1 (en) 2019-05-24 2022-04-06 Danisco US Inc. Subtilisin variants and methods of use
WO2020247582A1 (en) 2019-06-06 2020-12-10 Danisco Us Inc Methods and compositions for cleaning
IT201900023433A1 (en) 2019-12-10 2021-06-10 Lonati Spa WITHDRAWER DEVICE TO TAKE A TUBULAR KNITTED PRODUCT FROM A CIRCULAR MACHINE FOR KNITWEAR, Hosiery OR SIMILAR
CN116323935A (en) 2020-08-27 2023-06-23 丹尼斯科美国公司 Enzymes and enzyme compositions for cleaning
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections
US20240117275A1 (en) 2021-01-29 2024-04-11 Danisco Us Inc. Compositions for cleaning and methods related thereto
CN112877304B (en) * 2021-03-15 2022-07-26 合肥师范学院 Construction and application of bacterial laccase mutant LacAT and expression strain thereof
WO2023278297A1 (en) 2021-06-30 2023-01-05 Danisco Us Inc Variant lipases and uses thereof
EP4396320A2 (en) 2021-09-03 2024-07-10 Danisco US Inc. Laundry compositions for cleaning
EP4402258A2 (en) 2021-09-13 2024-07-24 Danisco US Inc. Bioactive-containing granules
CN118679251A (en) 2021-12-16 2024-09-20 丹尼斯科美国公司 Subtilisin variants and methods of use
EP4448750A2 (en) 2021-12-16 2024-10-23 Danisco US Inc. Subtilisin variants and uses thereof
WO2023114939A2 (en) 2021-12-16 2023-06-22 Danisco Us Inc. Subtilisin variants and methods of use
WO2023168234A1 (en) 2022-03-01 2023-09-07 Danisco Us Inc. Enzymes and enzyme compositions for cleaning
CN114733656B (en) * 2022-04-25 2024-01-23 江苏泓润生物质能科技有限公司 Synchronous split-phase deslagging and impurity removing system for pretreated kitchen waste
WO2023250301A1 (en) 2022-06-21 2023-12-28 Danisco Us Inc. Methods and compositions for cleaning comprising a polypeptide having thermolysin activity
WO2024050339A1 (en) 2022-09-02 2024-03-07 Danisco Us Inc. Mannanase variants and methods of use
WO2024050343A1 (en) 2022-09-02 2024-03-07 Danisco Us Inc. Subtilisin variants and methods related thereto
WO2024050346A1 (en) 2022-09-02 2024-03-07 Danisco Us Inc. Detergent compositions and methods related thereto
WO2024102698A1 (en) 2022-11-09 2024-05-16 Danisco Us Inc. Subtilisin variants and methods of use
WO2024163584A1 (en) 2023-02-01 2024-08-08 Danisco Us Inc. Subtilisin variants and methods of use
EP4410941A1 (en) * 2023-02-01 2024-08-07 The Procter & Gamble Company Detergent compositions containing enzymes
WO2024163600A1 (en) * 2023-02-01 2024-08-08 Danisco Us Inc. Subtilisin variants and methods of use
WO2024186819A1 (en) 2023-03-06 2024-09-12 Danisco Us Inc. Subtilisin variants and methods of use
WO2024191711A1 (en) 2023-03-16 2024-09-19 Nutrition & Biosciences USA 4, Inc. Brevibacillus fermentate extracts for cleaning and malodor control and use thereof
CN117417922B (en) * 2023-12-13 2024-03-15 北京科为博生物科技有限公司 High-temperature-resistant alkaline protease and gene and application thereof

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6436690B1 (en) * 1993-09-15 2002-08-20 The Procter & Gamble Company BPN′ variants having decreased adsorption and increased hydrolysis wherein one or more loop regions are substituted
US20040147008A1 (en) * 2002-11-06 2004-07-29 Novozymes A/S Subtilase variants
US20050054843A1 (en) * 2001-12-31 2005-03-10 Estell David A Proteases producing an altered immunological response and methods of making and using the same
US20050148059A1 (en) * 2001-12-31 2005-07-07 Estell David A. Protease producing an altered immunogenic response and methods of making and using the same
US20080009431A1 (en) * 2006-06-05 2008-01-10 Jean-Pol Boutique Enzyme stabilization
US20110251073A1 (en) * 2008-06-06 2011-10-13 Cascao-Pereira Luis G Compositions And Methods Comprising Variant Microbial Proteases
US8728790B2 (en) * 2009-12-09 2014-05-20 Danisco Us Inc. Compositions and methods comprising protease variants
US20140193888A1 (en) * 2009-12-09 2014-07-10 The Procter & Gamble Company Fabric and home care products

Family Cites Families (113)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1296839A (en) 1969-05-29 1972-11-22
GB1372034A (en) 1970-12-31 1974-10-30 Unilever Ltd Detergent compositions
DK187280A (en) 1980-04-30 1981-10-31 Novo Industri As RUIT REDUCING AGENT FOR A COMPLETE LAUNDRY
GR76237B (en) 1981-08-08 1984-08-04 Procter & Gamble
US4760025A (en) 1984-05-29 1988-07-26 Genencor, Inc. Modified enzymes and methods for making same
US5411873A (en) * 1984-05-29 1995-05-02 Genencor, Inc. Process for producing heterologous polypeptides
US5801038A (en) * 1984-05-29 1998-09-01 Genencor International Inc. Modified subtilisins having amino acid alterations
US5972682A (en) 1984-05-29 1999-10-26 Genencor International, Inc. Enzymatically active modified subtilisins
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
DK154572C (en) 1985-08-07 1989-04-24 Novo Industri As ENZYMATIC DETERGENT ADDITIVE, DETERGENT AND METHOD FOR WASHING TEXTILES
EP0218272B1 (en) 1985-08-09 1992-03-18 Gist-Brocades N.V. Novel lipolytic enzymes and their use in detergent compositions
DK122686D0 (en) 1986-03-17 1986-03-17 Novo Industri As PREPARATION OF PROTEINS
ATE116365T1 (en) * 1986-04-30 1995-01-15 Genencor Int MUTANT OF A NON-HUMAN CARBONYL HYDROLASE, FOR THESE DNA SEQUENCES AND VECTORS CODING AND HOSTS TRANSFORMED BY THESE VECTORS.
US5322770A (en) 1989-12-22 1994-06-21 Hoffman-Laroche Inc. Reverse transcription with thermostable DNA polymerases - high temperature reverse transcription
US4810414A (en) 1986-08-29 1989-03-07 Novo Industri A/S Enzymatic detergent additive
US4914031A (en) * 1987-04-10 1990-04-03 Amgen, Inc. Subtilisin analogs
DE3851875T2 (en) 1987-05-29 1995-04-13 Genencor Int CUTINASE CONTAINING DETERGENT COMPOSITIONS.
ATE125865T1 (en) 1987-08-28 1995-08-15 Novo Nordisk As RECOMBINANT HUMICOLA LIPASE AND METHOD FOR PRODUCING RECOMBINANT HUMICOLA LIPASES.
JPS6474992A (en) 1987-09-16 1989-03-20 Fuji Oil Co Ltd Dna sequence, plasmid and production of lipase
ATE129523T1 (en) 1988-01-07 1995-11-15 Novo Nordisk As SPECIFIC PROTEASES.
JP3079276B2 (en) 1988-02-28 2000-08-21 天野製薬株式会社 Recombinant DNA, Pseudomonas sp. Containing the same, and method for producing lipase using the same
US4977252A (en) 1988-03-11 1990-12-11 National Starch And Chemical Investment Holding Corporation Modified starch emulsifier characterized by shelf stability
WO1990009446A1 (en) 1989-02-17 1990-08-23 Plant Genetic Systems N.V. Cutinase
US5665587A (en) * 1989-06-26 1997-09-09 Novo Nordisk A/S Modified subtilisins and detergent compositions containing same
JPH04500385A (en) * 1989-06-26 1992-01-23 ユニリーバー・ナームローゼ・ベンノートシヤープ Enzyme detergent composition
EP0528828B2 (en) 1990-04-14 1997-12-03 Genencor International GmbH Alkaline bacillus lipases, coding dna sequences therefor and bacilli which produce these lipases
US5354559A (en) 1990-05-29 1994-10-11 Grain Processing Corporation Encapsulation with starch hydrolyzate acid esters
US5766898A (en) * 1990-12-05 1998-06-16 Novo Nordisk A/S Proteins with changed epitopes and methods for the production thereof
DE69133035T2 (en) 1991-01-16 2003-02-13 The Procter & Gamble Company, Cincinnati Compact detergent compositions with highly active cellulases
US5340735A (en) 1991-05-29 1994-08-23 Cognis, Inc. Bacillus lentus alkaline protease variants with increased stability
DE4231726A1 (en) * 1992-09-23 1994-03-24 Cognis Bio Umwelt Mutated subtilisin-like serine proteases
ATE217346T1 (en) * 1992-09-24 2002-05-15 Genencor Int NEW CLEANING AGENT COMPOSITIONS CONTAINING ALKALINE PROTEASES
ATE237681T1 (en) 1992-12-01 2003-05-15 Novozymes As ACCELERATION OF ENZYME REACTIONS
US5646101A (en) 1993-01-18 1997-07-08 The Procter & Gamble Company Machine dishwashing detergents containing an oxygen bleach and an anti-tarnishing mixture of a paraffin oil and sequestrant
DK77393D0 (en) 1993-06-29 1993-06-29 Novo Nordisk As ENZYMER ACTIVATION
US5698504A (en) 1993-07-01 1997-12-16 The Procter & Gamble Company Machine dishwashing composition containing oxygen bleach and paraffin oil and benzotriazole compound silver tarnishing inhibitors
US5486303A (en) 1993-08-27 1996-01-23 The Procter & Gamble Company Process for making high density detergent agglomerates using an anhydrous powder additive
AU7870394A (en) 1993-09-15 1995-04-03 Procter & Gamble Company, The Subtilisin bpn' variants with decreased adsorption and increased hydrolysis
US5677272A (en) * 1993-10-14 1997-10-14 The Procter & Gamble Company Bleaching compositions comprising protease enzymes
MA23346A1 (en) * 1993-10-14 1995-04-01 Genencor Int VARIANTS OF THE SUB-USE
AU8079794A (en) * 1993-10-14 1995-05-04 Procter & Gamble Company, The Protease-containing cleaning compositions
DE4342680A1 (en) 1993-12-15 1995-06-22 Pfeiffer Erich Gmbh & Co Kg Discharge device for media
US5861271A (en) 1993-12-17 1999-01-19 Fowler; Timothy Cellulase enzymes and systems for their expressions
US5691295A (en) 1995-01-17 1997-11-25 Cognis Gesellschaft Fuer Biotechnologie Mbh Detergent compositions
ES2364776T3 (en) 1994-02-24 2011-09-14 HENKEL AG &amp; CO. KGAA IMPROVED AND DETERGENT ENZYMES THAT CONTAIN THEM.
DE69535736T2 (en) 1994-02-24 2009-04-30 Henkel Ag & Co. Kgaa IMPROVED ENZYMES AND DETERGENTS CONTAINED THEREOF
US5686014A (en) 1994-04-07 1997-11-11 The Procter & Gamble Company Bleach compositions comprising manganese-containing bleach catalysts
ES2180645T3 (en) 1994-06-17 2003-02-16 Genencor Int CLEANING METHOD BASED ON COMPOSITIONS THAT CONTAIN A CAPABLE ENZYME TO DEGRADE THE CELL WALLS OF THE PLANTS AND ITS USE IN CLEANING METHODS.
GB2294268A (en) 1994-07-07 1996-04-24 Procter & Gamble Bleaching composition for dishwasher use
GB9416841D0 (en) * 1994-08-19 1994-10-12 Finnfeeds Int Ltd An enzyme feed additive and animal feed including it
US5879584A (en) 1994-09-10 1999-03-09 The Procter & Gamble Company Process for manufacturing aqueous compositions comprising peracids
US5516448A (en) 1994-09-20 1996-05-14 The Procter & Gamble Company Process for making a high density detergent composition which includes selected recycle streams for improved agglomerate
US5691297A (en) 1994-09-20 1997-11-25 The Procter & Gamble Company Process for making a high density detergent composition by controlling agglomeration within a dispersion index
US5489392A (en) 1994-09-20 1996-02-06 The Procter & Gamble Company Process for making a high density detergent composition in a single mixer/densifier with selected recycle streams for improved agglomerate properties
ATE190090T1 (en) 1994-12-09 2000-03-15 Procter & Gamble COMPOSITIONS CONTAINING DIACYL PEROXIDE PARTICLES FOR AUTOMATIC DISHWASHING
US5534179A (en) 1995-02-03 1996-07-09 Procter & Gamble Detergent compositions comprising multiperacid-forming bleach activators
US5574005A (en) 1995-03-07 1996-11-12 The Procter & Gamble Company Process for producing detergent agglomerates from high active surfactant pastes having non-linear viscoelastic properties
US5569645A (en) 1995-04-24 1996-10-29 The Procter & Gamble Company Low dosage detergent composition containing optimum proportions of agglomerates and spray dried granules for improved flow properties
US5597936A (en) 1995-06-16 1997-01-28 The Procter & Gamble Company Method for manufacturing cobalt catalysts
CN1192774A (en) 1995-06-16 1998-09-09 普罗格特-甘布尔公司 Automatic dishwashing compositions comprising cobalt catalysts
US5565422A (en) 1995-06-23 1996-10-15 The Procter & Gamble Company Process for preparing a free-flowing particulate detergent composition having improved solubility
DE19530816A1 (en) 1995-08-23 1997-02-27 Cognis Bio Umwelt Use of mutant subtilisin protease in cosmetic products
US5576282A (en) 1995-09-11 1996-11-19 The Procter & Gamble Company Color-safe bleach boosters, compositions and laundry methods employing same
ES2174105T5 (en) 1995-09-18 2007-03-01 THE PROCTER &amp; GAMBLE COMPANY LIBERATION SYSTEMS.
US5879564A (en) 1995-11-14 1999-03-09 Cytec Technology Corp. High performance polymer flocculating agents
MA24136A1 (en) 1996-04-16 1997-12-31 Procter & Gamble MANUFACTURE OF SURFACE AGENTS.
EP2278001B1 (en) * 1996-11-04 2013-10-23 Novozymes A/S Protease variants and compositions
EP0932667B1 (en) * 1996-11-04 2008-10-01 Novozymes A/S Subtilase variants and compositions
JP4489190B2 (en) 1997-03-07 2010-06-23 ザ、プロクター、エンド、ギャンブル、カンパニー Bleach composition containing metal bleach catalyst and bleach activator and / or organic percarboxylic acid
WO1998039335A1 (en) 1997-03-07 1998-09-11 The Procter & Gamble Company Improved methods of making cross-bridged macropolycycles
WO1999011770A1 (en) * 1997-08-29 1999-03-11 Novo Nordisk A/S Protease variants and compositions
RU2252958C2 (en) * 1997-10-23 2005-05-27 Джененкор Интернэшнл, Инк. Subtilisin variant (variants), dna encoding the same, expression vector, cleaning composition, animal feed, composition for cloth treatment
MA25044A1 (en) * 1997-10-23 2000-10-01 Procter & Gamble WASHING COMPOSITIONS CONTAINING MULTISUBSTITUTED PROTEASE VARIANTS.
US5935826A (en) 1997-10-31 1999-08-10 National Starch And Chemical Investment Holding Corporation Glucoamylase converted starch derivatives and their use as emulsifying and encapsulating agents
EP1032655B1 (en) 1997-11-21 2005-06-29 Novozymes A/S Protease variants and compositions
US6773907B2 (en) * 1997-11-21 2004-08-10 Peter Kamp Hansen Subtilase enzymes
JP2002500019A (en) 1997-12-24 2002-01-08 ジェネンコア インターナショナル インコーポレーテッド Improved analytical method for preferred enzymes and / or preferred detergent compositions
US6936249B1 (en) * 1998-04-15 2005-08-30 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
CN101024826B (en) 1998-06-10 2014-09-03 诺沃奇梅兹有限公司 Novel mannanases
US6461849B1 (en) * 1998-10-13 2002-10-08 Novozymes, A/S Modified polypeptide
US6376450B1 (en) 1998-10-23 2002-04-23 Chanchal Kumar Ghosh Cleaning compositions containing multiply-substituted protease variants
EP1135392A2 (en) 1998-11-30 2001-09-26 The Procter & Gamble Company Process for preparing cross-bridged tetraaza macrocycles
JP4647787B2 (en) * 1998-12-18 2011-03-09 ノボザイムス アクティーゼルスカブ Subtilase enzyme subgroups I-S1 and I-S2 with additional amino acid residues in the active site loop region
WO2000037599A1 (en) * 1998-12-18 2000-06-29 Novozymes A/S Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in the active site loop region
DE60040283D1 (en) * 1999-05-20 2008-10-30 Novozymes As SUBTILASE ENZYMES OF I-S1 AND I-S2 SUB-GROUPS WITH AT LEAST ONE ADDITIONAL AMINO ACID BETWEEN POSITION 97 AND 98
CN1399677A (en) * 1999-07-22 2003-02-26 宝洁公司 Subtilisin protease variants having amino acid substitutions in defined epitope regions
MXPA02008192A (en) * 2000-02-23 2002-11-29 Procter & Gamble Liquid laundry detergent compositions having enhanced clay removal benefits.
JP2004508011A (en) * 2000-04-03 2004-03-18 マキシジェン，　インコーポレイテッド Subtilisin mutant
US7226770B2 (en) * 2000-04-28 2007-06-05 Novozymes A/S Lipolytic enzyme variant
US20020182734A1 (en) 2000-08-11 2002-12-05 Diaz-Torres Maria R. Bacillus transformation, transformants and mutant libraries
US6541235B1 (en) * 2000-09-29 2003-04-01 University Of Maryland Biotechnology Institute Calcium free subtilisin mutants
US6440991B1 (en) 2000-10-02 2002-08-27 Wyeth Ethers of 7-desmethlrapamycin
US6582914B1 (en) 2000-10-26 2003-06-24 Genencor International, Inc. Method for generating a library of oligonucleotides comprising a controlled distribution of mutations
DK1373296T3 (en) * 2001-03-23 2012-01-09 Procter & Gamble Proteins producing an altered immunogenic response, and methods for their preparation and use
MXPA04004479A (en) * 2001-11-13 2004-08-11 Procter & Gamble Topical compositions containing enzymes stabilized with inhibitors.
JP4703113B2 (en) * 2002-01-16 2011-06-15 ジェネンコー・インターナショナル・インク Multiple substitution protease mutant
US20050239043A1 (en) * 2002-02-26 2005-10-27 Harding Fiona A Subtilisin carlsberg proteins with reduced immunogenicity
TWI319007B (en) * 2002-11-06 2010-01-01 Novozymes As Subtilase variants
US20040214737A1 (en) * 2003-04-25 2004-10-28 John Billman Foamy composition for pretreatment of stains on fabrics
EP1627885B8 (en) * 2003-04-30 2015-02-18 Innovative Medicine and Any Peptide Pharmaceuticals Corp Polypeptide capable of assisting digestive organ absorption
BRPI0416797A (en) 2003-11-19 2007-04-17 Genencor Int serine proteases, nucleic acids encoding serine enzymes and vectors and host cells incorporating them
CN103333870A (en) 2003-12-03 2013-10-02 丹尼斯科美国公司 Perhydrolase enzyme
BRPI0509671A (en) 2004-04-08 2007-10-30 Genencor Internation Inc mutant alpha-amylases
DE102004027091A1 (en) * 2004-06-02 2005-12-29 Henkel Kgaa Performance enhanced alkaline protease variants and detergents and cleaners containing these performance enhanced alkaline protease variants
EP1904628B1 (en) * 2005-07-08 2011-10-19 Novozymes A/S Subtilase variants
DK2390321T3 (en) 2005-10-12 2015-02-23 Procter & Gamble The use and manufacture of a storage stable neutral metalloprotease
CN101460618A (en) * 2006-04-20 2009-06-17 诺维信公司 Savinase variants having an improved wash performance on egg stains
RU2509152C2 (en) * 2006-07-18 2014-03-10 ДАНИСКО ЮЭс, ИНК., ДЖЕНЕНКОР ДИВИЖН Dishware washing method
EP2647692A3 (en) * 2008-11-11 2014-01-22 The Procter and Gamble Company Compositions and methods comprising serine protease variants
EP2551335A1 (en) * 2011-07-25 2013-01-30 The Procter & Gamble Company Enzyme stabilized liquid detergent composition
US20130123162A1 (en) * 2011-11-10 2013-05-16 The Procter & Gamble Company Consumer products

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6436690B1 (en) * 1993-09-15 2002-08-20 The Procter & Gamble Company BPN′ variants having decreased adsorption and increased hydrolysis wherein one or more loop regions are substituted
US20050054843A1 (en) * 2001-12-31 2005-03-10 Estell David A Proteases producing an altered immunological response and methods of making and using the same
US20050148059A1 (en) * 2001-12-31 2005-07-07 Estell David A. Protease producing an altered immunogenic response and methods of making and using the same
US20040147008A1 (en) * 2002-11-06 2004-07-29 Novozymes A/S Subtilase variants
US20080009431A1 (en) * 2006-06-05 2008-01-10 Jean-Pol Boutique Enzyme stabilization
US20150159147A1 (en) * 2008-06-06 2015-06-11 Danisco Us Inc. Compositions And Methods Comprising Variant Microbial Proteases
US20110256609A1 (en) * 2008-06-06 2011-10-20 Basler Joshua R Compositions And Methods Comprising Variant Microbial Proteases
US20110256610A1 (en) * 2008-06-06 2011-10-20 Viktor Yuryevich Alekseyev Compositions And Methods Comprising Variant Microbial Proteases
US20150064767A1 (en) * 2008-06-06 2015-03-05 Danisco Us Inc. Compositions and methods comprising variant microbial proteases
US20110251073A1 (en) * 2008-06-06 2011-10-13 Cascao-Pereira Luis G Compositions And Methods Comprising Variant Microbial Proteases
US20150247140A1 (en) * 2008-06-06 2015-09-03 Danisco Us Inc. Compositions and Methods Comprising Variant Microbial Proteases
US8728790B2 (en) * 2009-12-09 2014-05-20 Danisco Us Inc. Compositions and methods comprising protease variants
US20140193888A1 (en) * 2009-12-09 2014-07-10 The Procter & Gamble Company Fabric and home care products
US8785171B2 (en) * 2009-12-09 2014-07-22 The Procter & Gamble Company Fabric and home care products comprising cold water proteases
US9157052B2 (en) * 2009-12-09 2015-10-13 Danisco Us Inc. Methods for cleaning using a variant protease derived from subtilisin
US20160083710A1 (en) * 2009-12-09 2016-03-24 Danisco Us Inc. Compositions and methods comprising protease variants

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150064767A1 (en) * 2008-06-06 2015-03-05 Danisco Us Inc. Compositions and methods comprising variant microbial proteases
US20150159147A1 (en) * 2008-06-06 2015-06-11 Danisco Us Inc. Compositions And Methods Comprising Variant Microbial Proteases
US20150247140A1 (en) * 2008-06-06 2015-09-03 Danisco Us Inc. Compositions and Methods Comprising Variant Microbial Proteases
US10563189B2 (en) * 2008-06-06 2020-02-18 The Procter & Gamble Company Compositions and methods comprising variant microbial proteases

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