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US20090209026A1 - Alpha-amylase variants with altered properties - Google Patents

Alpha-amylase variants with altered properties Download PDF

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Publication number
US20090209026A1
US20090209026A1 US12/263,958 US26395808A US2009209026A1 US 20090209026 A1 US20090209026 A1 US 20090209026A1 US 26395808 A US26395808 A US 26395808A US 2009209026 A1 US2009209026 A1 US 2009209026A1
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amylase
alpha
variant
activity
seq
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Mee-Young Yoon
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Danisco US Inc
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Danisco US Inc
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Publication of US20090209026A1 publication Critical patent/US20090209026A1/en
Priority to US13/151,905 priority patent/US20110269210A1/en
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    • 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
    • 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
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • This disclosure relates to novel alpha-amylases.
  • it relates to methods of using certain variant alpha-amylase activities, and blends thereof for stain removal and as a component of detergent compositions for washing.
  • Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes that catalyze hydrolysis of starch and related linear or branched 1,4-glucosidic oligo- and polysaccharides.
  • Amylases can be used for a variety of purposes.
  • amylases are used commercially in the initial stages of starch processing (e.g., liquefaction); in wet milling processes; and in alcohol production from carbohydrate sources. They are also used as cleaning agents or adjuncts in detergent matrices; in the textile industry for starch desizing; in baking applications; in the beverage industry; in oilfields in drilling processes; in recycling processes, e.g. for de-inking paper, and in animal feed.
  • alpha-amylase variants with improved properties for specific uses, such as starch liquefaction and textile desizing.
  • amylases that provide, e.g., manufacturing and/or performance advantages over the industry standard enzymes (e.g., from Bacillus licheniformis ), for various uses, including commercial desizing as well as cleaning/washing and stain or starch removal processes.
  • detergents and cleaning aids or formulations comprising improved amylases and additional components, such as surfactant, chelators, and the like.
  • the present disclosure relates, inter alia, to novel ⁇ -amylolytic enzymes variants of parent ⁇ -amylase such as an AmyS-like ⁇ -amylase, in particular variants exhibiting altered properties that are advantageous in connection with the cleaning or washing processes, or the removal of starch, for example in desizing woven material.
  • the variant is altered, as compared to a parent AmyS-like alpha-amylase or a reference amylase, in one or more of net charge, substrate specificity, substrate cleavage, substrate binding, thermal stability, activity at one or more pH's, stability at one or more pH's, stability in oxidizing conditions, Ca 2+ requirements, specific activity, catalytic rate, catalytic efficiency, activity in the presence of a chelator, thermal or pH stability in the presence of a chelator, utility for desizing, or utility for a cleaning process, or amount of expression in a protein expression system, and other properties of interest.
  • one or more alterations may result in a variant that has reduced Ca 2+ dependency and/or an altered pH/activity profile and/or altered thermostability, as compared to a parent ⁇ -amylase, such as an AmyS-like amylase.
  • a variant of a parent Geobacillus stearothermophilus alpha-amylase wherein the variant has an amino acid sequence which has at least about 95% homology to a parent Geobacillus stearothermophilus alpha-amylase and comprises a substitution of amino acid 242, wherein the amino acid positions in the peptide sequence are numbered relative to a reference amylase (e.g., SEQ ID NO: 1 or 2), and wherein the variant has alpha-amylase activity.
  • a reference amylase e.g., SEQ ID NO: 1 or 2
  • compositions comprising: a) at least one variant alpha-amylase comprising an amino acid sequence at least about 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant having detectable alpha-amylase activity, and b) at least one of an additional enzyme, a detergent, a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner.
  • the reference amylase is SEQ ID NO: 1 or 2
  • the composition is a component of a product for use in laundry, dish, or hard-surface cleaning, desizing, or fabric or stain treatment.
  • the composition comprises an additional enzyme is a protease, a lipase, an amylase, a cellulase, a peroxidase, an oxidase, a pectinase, a lyase, a cutinase, a laccase, or a combination thereof.
  • an additional enzyme is a protease, a lipase, an amylase, a cellulase, a peroxidase, an oxidase, a pectinase, a lyase, a cutinase, a laccase, or a combination thereof.
  • the surfactant is nonionic, anionic, cationic, or zwitterionic.
  • the variant alpha-amylase is preferably a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant.
  • the variant has altered stability to oxidation and the variant alpha-amylase further includes deletion or substitution of one or more methionine residues including residues located at amino positions 8, 9, 96, 200, 206, 284, 307, 311, 316, and 438 of a parent AmyS-like alpha-amylase, where the reference alpha-amylase in SEQ ID NO: 2.
  • the variant alpha-amylase further comprises a sequence modification at one or more amino acid positions corresponding to amino acid positions 97, 179, 180, 193, 319, 349, 358, 416, 428, or 443 of the reference alpha-amylase.
  • the variant comprises one or more of substitution at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443.
  • variant alpha-amylases comprising a substitution of an N193 or a V416 or both, e.g., a substitution of N193F or V416G, or both.
  • the variants feature deletion of one or more amino acids, e.g., at positions F178, R179, G180,1181, G182 and K183.
  • the variant alpha-amylase has altered metal ion dependence or altered stability or activity in an absence of added calcium or a presence of a chelator in certain embodiments.
  • the variant alpha-amylase preferably has at least 95%, 98%, or even 99% or more homology to SEQ ID NO: 2, and comprises a substitution of amino acid 242 relative to numbering in a reference alpha-amylase comprising SEQ ID NO: 1, and wherein the variant alpha-amylase has alpha-amylase activity.
  • the parent AmyS-like alpha-amylase is SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference alpha-amylase is SEQ ID NO: 1 or 2 in one embodiment.
  • the variant alpha-amylase has improved performance in a wash process at a pH ⁇ about 8, relative to the parent AmyS-like alpha-amylase.
  • the variant alpha-amylase can feature, in various embodiments, a set of substitutions of a) Q97E, Q319E, Q358E, Q443E; b) Q97E, Q319R, Q358E, Q443R; c) Q97E, Q319R, Q358E; d) Q97E, Q319R, Q443E; e) Q97E, Q319R, Q443R; f) Q97E, Q358R; g) Q97E, Q443E; h) Q319R, Q358E, Q443E; or i) Q319R, Q358R, Q443E.
  • compositions that are detergent or cleaning formulations comprising at least one variant amylase comprising an amino acid sequence at least about 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, wherein the variant has detectable alpha-amylase activity; wherein the reference amylase is SEQ ID NO: 1 or 2.
  • the variant is an S242 variant comprising at least a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T substitution.
  • this disclosure provided methods of desizing a woven material subsequent to a weaving process comprising contacting the woven material with a variant alpha-amylase comprising an amino acid sequence at least about 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase.
  • the variant preferably has detectable alpha-amylase activity.
  • the contacting is performed under conditions and for a time that are effective for at least partially removing sizing from the woven material.
  • the variant alpha-amylase is altered, as compared to a parent AmyS-like alpha-amylase or a reference alpha-amylase, in one or more of: (a) net charge, (b) substrate specificity, (c) substrate cleavage, (d) substrate binding, (e) thermal stability, (f) activity at one or more pH's, (g) stability at one or more pH's, (h) stability in oxidizing conditions, (i) Ca 2+ requirements, (j) specific activity, (k) catalytic rate, (l) catalytic efficiency, (m) activity in a presence of a chelator, (n) thermal or pH stability in the presence of a chelator, (o) effectiveness for desizing, or (p) amount of expression in a protein expression system.
  • the parent AmyS-like alpha-amylase is SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference alpha-amylase is SEQ ID NO: 1 or 2 in various embodiments.
  • the variant alpha-amylase is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant.
  • the variant alpha-amylase further comprises one or more of substitution at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443, wherein the reference alpha-amylase is SEQ ID NO: 1 or 2.
  • the methods comprise contacting one or more items to be washed or cleaned with a composition comprising a variant alpha-amylase comprising an amino acid sequence at least about 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase.
  • the contacting is performed under conditions and for a time effective for at least partially washing or cleaning the one or more items.
  • the variant has detectable alpha-amylase activity.
  • at least one item is soiled with at least one starch-containing material, the removal of which is aided by the variant amylase.
  • the composition further comprises one or more of an additional enzyme, a detergent, a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner.
  • an additional enzyme e.g., a detergent, a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner.
  • the parent AmyS-like alpha-amylase is SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference alpha-amylase is SEQ ID NO: 1 or 2.
  • the variant alpha-amylase is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant.
  • the variant alpha-amylase has improved performance in a wash process at a pH ⁇ about 8, relative to the parent AmyS-like alpha-amylase.
  • the variant alpha-amylase comprises one or more of substitution at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443.
  • the variant alpha-amylase comprises a set of substitutions of a) Q97E, Q319E, Q358E, Q443E; b) Q97E, Q319R, Q358E, Q443R; c) Q97E, Q319R, Q358E; d) Q97E, Q319R, Q443E; e) Q 97E, Q319R, Q443R; f) Q97E, Q358R; g) Q97E, Q443E; h) Q319R, Q358E, Q443E; or i) Q319R, Q358R, Q443E.
  • the method can also comprise use of variant alpha-amylases comprising deletion of one or more amino acids at positions F178, R179, G180, I181, G182, or K183.
  • the variant alpha-amylase has altered metal ion dependence or altered stability, or activity in an absence of added calcium or the presence of a chelator.
  • kits comprising a) one or more variant alpha-amylases comprising an amino acid sequence at least about 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant having detectable alpha-amylase activity, and b) at least one of an additional enzyme, a detergent, a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner.
  • the kit further comprises instructions for use, e.g., for using the kit components in a process for desizing a woven material, or for washing or cleaning one or more items soiled with a starch-containing substance.
  • FIG. 1 shows alignment of amino acid sequences among several candidate parent alpha-amylases (AmyS-like amylases) for use herein. Positions corresponding to any amino acid position (e.g., 1 through 520) of the amylase from Geobacillus stearothermophilus (SEQ ID NO: 1) can be readily determined.
  • SEQ ID NO: 1 alpha-amylase from G. stearothermophilus “BSG”; SEQ ID NO: 2, truncated amylase from G. stearothermophilus (AmyS, SPEZYME XTRA); SEQ ID NO: 3 , G. stearothermophilus (S242A variant amylase); SEQ ID NO: 4, G.
  • S242Q variant amylase SEQ ID NO: 5
  • G. stearothermophilus S242E variant amylase
  • SEQ ID NO: 6 Yamane 707 amylase
  • SEQ ID NO: 7 mature LAT amylase
  • SEQ ID NO: 9 B. amyloliquefaciens amylase, BAN
  • FIG. 2 shows the pHPLT-AmyS plasmid.
  • FIG. 3 shows percent residual activity of S242 variants after heat stress at 95° C. for 30 minutes. Variant positions P, S, W, and Y were missing and replaced by wild-type AmyS (Spezyme® Xtra (labeled “Z”)). A positive control, G. stearothermophilus with ⁇ 179-180 with the C-terminus truncated by 29 amino acids (i.e., SEQ ID NO: 2) is also shown. Lines indicate 2 ⁇ and 3 ⁇ above the standard deviation of the percent residual activity of the wild-type enzyme. S242A and S242Q clearly show higher residual activities than the wild-type.
  • FIG. 4 Panels A, B, C, D, E, F, G, H, and I show pair-wise alignments and consensus sequences for several sequences from FIG. 1 , and feature, respectively, Consensus Sequences 2, 3, 4, 5, 6, 7, 8, 9, and 10, or SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and 30, respectively.
  • FIG. 5 shows the thermal melting curves and the melting points for the wild-type and amylase variants without added calcium.
  • FIG. 6 shows the thermal melting curves and the melting points in the presence of 2 mM added calcium for both the wild-type and the amylase variants.
  • FIG. 7 shows the activity profile at 4, 10, and 20 minutes for Spezyme Xtra and two variants, relative to Liquozyme SC.
  • FIG. 8 shows the activity profile of four variants relative to the S242Q variant for three time points.
  • FIG. 9 is a graph depicting the performance of S242Q (filled circles) and its variants (open circles), as a function of charge, in the rice starch microswatch assay under North American laundry conditions using S242Q combinatorial charge library, rice starch microswatch cleaning in Tide 2 ⁇ , at 20° C. Reference is made to Example 10.
  • FIG. 10 is a graph depicting the performance of a truncated Bacillus sp. TS-23 amylase (closed circles) with the following mutations: Q98R, M201L, S243Q R309A, Q320R, Q359E, and K444E and its charge variants (open circles) (see co-pending U.S. Patent Application No. PCT/US2008/007103, filed 6 Jun. 2008) in the rice starch microswatch assay as a function of charge under Western European laundry conditions with TS23t combinatorial charge library, rice starch microswatch cleaning in Persil at 40° C. Reference is made to Example 10.
  • FIG. 11 is a graph depicting the performance of S242Q (closed circles) and its variants (open circles) in the BODIPY-starch assay as a function of charge.
  • S242Q combinatorial charge library (CCL) specific activity on BODIPY-starch, standard assay conditions Reference is made to Example 10.
  • FIG. 12 Panel A is a graph depicting the relative BODIPY-starch hydrolysis as a function of relative shake tube expression (i.e., relative BODIPY-starch hydrolysis vs. relative shake tube expression); Panel B is a graph depicting the relative microswatch-starch hydrolysis as a function of relative shake tube expression (i.e., relative microswatch-starch hydrolysis vs. relative shake tube expression).
  • Example 13 is a graph depicting the relative BODIPY-starch hydrolysis as a function of relative shake tube expression (i.e., relative BODIPY-starch hydrolysis vs. relative shake tube expression).
  • FIG. 13 Panel A is a graph depicting the relative shake tube expression as a function of charge; Panel B is a graph depicting the relative BODIPY-starch hydrolysis as a function of charge. Reference is made to Example 13.
  • FIG. 14 Panel A is a graph depicting the relative shake tube expression as a function of charge; Panel B is a graph depicting the relative microswatch cleaning activity as a function of charge. Reference is made to Example 13.
  • FIG. 15 shows the effects of added Ca 2+ on desizing performance of variant S242Q compared to that of Ethyl and Xtra in the LAUNDER-O-METER under conditions of 85° C., for 30 min. at 0.01 ppm active protein.
  • the desizing was performed in the presence of 0 or 5 ppm CaCl 2 . See Example 14.
  • FIG. 16 shows the effects of added Ca 2+ on desizing performance of variant S242Q compared to that of Ethyl and Xtra in the LAUNDER-O-METER under conditions of 97° C., for 30 min. at 0.01 ppm active protein. The desizing was performed in the presence of 0 or 5 ppm CaCl 2 . See Example 14.
  • the present disclosure relies on routine techniques and methods used in the field of genetic engineering and molecular biology.
  • the following resources include descriptions of general methodology useful in accordance with what is disclosed herein: Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (2nd Ed., 1989); Kreigler, GENE TRANSFER AND EXPRESSION; A LABORATORY MANUAL (1990) and Ausubel et al., Eds. CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY (1994).
  • isolated means that the isolated substance, e.g. a compound or a sequence, is modified by the hand of man relative to that compound or sequence as found in nature.
  • an isolated sequence is at least partially free, or even substantially free, from at least one other component with which the sequence is naturally associated as found in nature.
  • “Purified” when used to describe a material or substance means that the material or substance is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure.
  • starch refers to any carbohydrate composition comprising complex polysaccharides, comprising amylose and/or amylopectin with the formula (C 6 H 10 O 5 ) x , wherein “X” can be any number.
  • starch refers to any such carbohydrate that is naturally present in plants, including but not limited to grains, grasses, tubers, and roots, and more specifically from wheat, barley, corn, rye, rice, sorghum, cassaya, millet, potato, sweet potato, and tapioca.
  • Starch can also refer to synthetic starches or modified starches, such as chemically-modified starch for use as a detectable substrate for enzyme assays, or starches chemically- or enzymatically-modified to improve one or more properties for use.
  • phytic acid As used herein, “phytic acid” (or inositol hexakisphosphate (IP6)), is the principle storage form of phosphorus in many plant tissues, such as bran, seeds, and the like. Phytic acid is also referred to as “phytate” herein, especially when in salt form. Various other inositol phosphates such as inositol penta-(IP5), tetra-(IP4), and triphosphate (IP3) are also referred to herein as phytates. Phytates are generally indigestible by man and most monogastric animals.
  • Enzymes that degrade phytates are referred to herein as “phytases” or “fytases” are generally myo-inositol-hexaphosphate phosphohydrolases.
  • Phytase activity is defined as fytase units (FTU or U), where one FTU is defined as the quantity of enzyme that liberates 1 micromol of inorganic-P per minute from 0.0015 mol/l sodium phytate at pH 5.5, and 37° C. This definition provides a useful measure of quantity of phytase activity and represents a simple bench mark measurement.
  • Phytate-degrading enzymes of yeasts e.g., Schwanniomyces occidentalis, Pichia anomala, Arxula adeninivorans ), gram-negative bacteria (e.g., Escherichia coli, Pseudomonas spp., Klebsiella spp.), and gram-positive (e.g., Bacillus spp.) have been identified and characterized.
  • Phytases from many plants, and from filamentous fungi such as Penicillium spp., Aspergillus spp., Trichoderma spp. Mucor piriformis , and Cladosporium spp., are also known.
  • 3-phytases (EC 3.1.3.8) and 6-phytases (EC 3.1.3.26), depending on the site of initiation of hydrolysis, have been characterized. Also, phytase have been characterized, based on their pH “optima,” as either acid (pH optima around 5) or alkaline (pH optima around 9). A variety of commericial phytases are available, including ROVABIO (Genencor International).
  • Amylase refers to an enzyme that is capable of catalyzing the cleavage of a starch substrate, leading to a degradation or partial degradation of the starch. Amylases are generally hydrolases that cleave glycosidic linkages in starch. As used herein amylase includes any glucoamylase, alpha-amylase, ⁇ -amylase, for example, the wild-type alpha-amylases of Bacillus spp., especially B. licheniformis .
  • alpha-amylases (EC 3.2.1.1; ⁇ -D-(1 ⁇ 4)-glucan glucanohydrolase) are endo-acting enzymes defined as cleaving ⁇ -D-(1 ⁇ 4) ⁇ -glycosidic linkages within the starch molecule in a random fashion.
  • the exo-acting amylolytic enzymes such as ⁇ -amylases (EC 3.2.1.2; ⁇ -D-(1 ⁇ 4)-glucan maltohydrolase) and some product-specific amylases like maltogenic alpha-amylase (EC 3.2.1.133) cleave the substrate starch molecule from the non-reducing end.
  • ⁇ -Amylases ⁇ -glucosidases (EC 3.2.1.20; ⁇ -D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; ⁇ -D-(1 ⁇ 4)-glucan glucohydrolase), and product-specific amylases can produce malto-oligosaccharides of specific length from starch.
  • Wild-type alpha-amylase from Bacillus stearothermophilus or “AmyS” amylase is sometimes referred to herein as XTRA or SPEZYME XTRA, which are commercial AmyS products from Genencor International.
  • AmyS-like alpha-amylases are useful as parent amylases herein.
  • AmyS-like alpha-amylases constitute a class of alpha-amylases herein, based on the substantial homology found between them.
  • “AmyS-like alpha-amylase” is intended to indicate the class of alpha-amylases, in particular Bacillus alpha-amylases, especially Geobacillus stearothermophilus alpha-amylases, which, at the amino acid level, exhibit a substantial identity to the alpha-amylase having the amino acid sequence shown in SEQ ID NO: 2, herein. Spezyme Xtra is commercially available from Danisco US Inc, Genencor Division.
  • Geobacillus stearothermophilus has been referred to as Bacillus stearothermophilus in the literature and the two may be used interchangeably herein. All the alpha-amylases having the amino acid sequences provided herein as SEQ ID NOS: 1, 6, 7, 8, 9, 10, 11, 12, 15 and 16, respectively, are considered to be AmyS-like alpha-amylases and thus are suitable as parent alpha-amylases.
  • AmyS-like alpha-amylases also include alpha-amylases i) having amino acid sequences with at least about 60% homology (identity), such as at least about 70%, at least about 75%, or at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity, with at least one of the amino acid sequences shown in SEQ ID NOS: 1, 6, 7, 8, 9, 10, 11, 12, 15 and 16, and/or ii) that are encoded by a DNA sequence that hybridizes with a DNA sequence encoding any of the above-specified alpha-amylases, or those apparent from SEQ ID NOS: 9 (BAN), 5 (BSG), 3 (SP722), 1 (SP690), 7 (LAT), 11 (AA560) of WO 06/002643 or of the present specification, which encode any of the amino acid sequences shown in SEQ ID NOS: 1, 6, 7, 8, 9, 10, 11,
  • Still further homologous alpha-amylases useful as AmyS-like alpha-amylases and thus, as parent enzymes for producing variants herein, include the alpha-amylase produced by the B. lichenformis strain described in EP 0252666; (ATCC 27811), and the alpha-amylases identified in WO 91/00353 and WO 94/18314; commercial AmyS-like alpha-amylases are comprised in the products sold under the following tradenames: Spezyme® AA and ULTRAPHLOW (available from Danisco US Inc, Genencor Division), and KeistaseTM (available from Daiwa) and LIQUEZYME SC (available from Novozymes, Denmark).
  • Section 1.5 herein below provides further information regarding AmyS-like alpha-amylases.
  • Table A therein provides a list of several useful AmyS-like alpha-amylases, as well as a convenient method of comparing amino acid sequence identities therebetween. The skilled artisan will appreciate the similar tables can be constructed for other alpha-amylases to determine their suitability for use herein as apparent enzyme.
  • SAPU spectrophotometric acid protease unit
  • GAU Glucoamylase unit
  • variant may be used interchangeably with the term “mutant.”
  • variants can refer to either polypeptides or nucleic acids. Variants include substitutions, insertions, deletions, truncations, transversions, and/or inversions, at one or more locations relative to a reference sequence.
  • variant nucleic acids include sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences presented herein. For example, a variant nucleic acid sequence herein can be at least partially complementary to a sequence capable of hybridizing under stringent conditions (e.g., 50° C.
  • the term variant encompasses sequences that are complementary to sequences that are capable of hybridizing under highly stringent conditions (e.g., 65° C. and 0.1 ⁇ SSC) to the nucleotide sequences presented herein.
  • thermostable when used to describe an enzyme means the enzyme is more thermostable than a reference enzyme.
  • an alpha-amylase variant is more thermostable than a wild-type B. licheniformis alpha-amylase if the variant has a relatively higher enzymatic activity after a specific interval of time under the same experimental conditions, e.g., the same temperature, substrate concentration, etc.
  • a more thermostable enzyme has a higher heat capacity determined by differential scanning calorimetry, compared to a reference enzyme.
  • Melting temperature (T m ) of a polypeptide is a temperature at which the conformation of the polypeptide undergoes a measurable temperature-dependent change.
  • Protein conformation and T m can be analyzed, for example, by circular dichroism, one of the most general and basic tools to study protein folding.
  • Circular dichroism spectroscopy measures the absorption of circularly polarized light. In proteins, structures such as alpha helices and beta sheets are generally chiral, and thus absorb circularly polarized light. The light absorption provides a measure of the degree of foldedness of the protein. Changes in this absorption as a function of temperature or concentration of a denaturant can be used to study equilibrium unfolding of the protein.
  • This type of spectroscopy can also be combined with devices, such as stopped flow mixers, to measure kinetics of protein folding/unfolding.
  • Calcium dependent means that, a particular enzyme requires calcium to substantially exhibit catalytic activity.
  • calcium dependent encompasses a property of any enzyme that has a strict requirement for a divalent metal ion to exhibit catalytic activity, and also includes enzymes whose catalytic activity is substantially (e.g. more than 20%) increased in the presence of calcium or another divalent cation.
  • pH stable with respect to an enzyme can refer to the enzyme activity or the protein conformation of the enzyme.
  • pH stable means the enzyme remains catalytically-active at a specified pH or across a specified pH range.
  • an enzyme may be deemed “stable” at a pH wherein the protein is not irreversibly denatured. In such a case, the enzyme would become catalytically active when returned to a pH capable of supporting catalytic activity. pH stability may also be used in a relative or comparative manner, for example, with a reference enzyme.
  • an alpha-amylase variant can be more pH stable than a wild-type B.
  • licheniformis alpha-amylase when the variant has a relatively higher activity than the wild-type, e.g., when held at a given pH or assayed under the same conditions, including pH. pH's of most interest are typically either the conditions of actual use, or pH's that are at or near the boundaries or extremes of the enzyme's natural ability to remain catalytically active.
  • pH range means a range of pH values e.g., from more acid to more basic, or vice versa. With respect to an enzyme activity, a pH range indicates the upper and lower pH values at which the enzyme exhibits a specified level of activity—e.g. a minimum activity, a specified percentage of maximal activity, or a specified level of substrate conversion or product formation.
  • Recombinant when used in reference to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, is the result of, or has been modified by, the introduction of a heterologous sequence or the alteration of a native sequence, or that the cell is derived from a cell so modified or altered.
  • recombinant cells may express genes that are not found within the native (non-recombinant) form of the cell or may express native genes that are otherwise differently expressed (e.g. under-expressed, or over-expressed), abnormally expressed, or not expressed at all.
  • nucleotide sequence or “nucleic acid sequence” refers to any sequence of two or more nucleotides, ribonucleotides, or the like, or derivatives thereof. Nucleotide sequences include oligonucleotide and polynucleotide sequences, as well as variants, homologues, fragments and derivatives thereof. A nucleotide sequence may be single-, double-, or multi-stranded. The nucleotide sequence may be from any source or origin, e.g., genomic, synthetic, or recombinant, and includes genomic DNA, cDNA, synthetic DNA, and RNA, and the like as well as hybrids thereof. Nucleotide sequences may comprise one or more codons and may encode one or more polypeptides. Nucleotide sequences may preferentially assume one or more energetically preferred three-dimensional structures.
  • a “vector” refers to a nucleotide sequence frequently useful for experimental use in vitro, or for introduction of nucleic acids into one or more cell types.
  • Vectors include cloning vectors, in vivo or in vitro expression vectors, shuttle vectors, plasmids, phagemids, cosmids, phage particles, cassettes and the like.
  • an “expression vector” as used herein means a DNA construct comprising a DNA sequence which is operably-linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host.
  • control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
  • a polynucleotide or a polypeptide having a certain percent (e.g., at least about 80%, 85%, 90%, 95%, or 99%) of sequence identity with another sequence means that, when aligned, that percentage of bases or amino acid residues are the same in comparing the two sequences.
  • This alignment and the percent homology or identity can be determined using any suitable software program known in the art, for example those described in CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY (F. M. Ausubel et al. (eds) 1987, Supplement 30, section 7.7.18).
  • Such programs may include the GCG Pileup program, FASTA (Pearson et al. (1988) Proc. Natl, Acad. Sci.
  • sequences encompassed by the disclosure are also defined by the ability to hybridize under stringent hybridization conditions with the exemplified amyS sequence (e.g., SEQ ID NO:5 of WO 06/002643).
  • a nucleic acid is hybridizable to another nucleic acid sequence when a single stranded form of the nucleic acid can anneal to the other nucleic acid under appropriate conditions of temperature and solution ionic strength.
  • Hybridization and washing conditions are well known in the art (see, e.g., Sambrook (1989) supra, particularly chapters 9 and 11).
  • stringent conditions correspond to a T m of 65° C. and 0.1 ⁇ SSC, 0.1% SDS.
  • a “gene” refers to a DNA segment that is involved in producing a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).
  • Heterologous with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.
  • the protein is a commercially important industrial protein. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes.
  • Endogenous with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.
  • transformed As used herein, “transformed”, “stably transformed”, and “transgenic” used in reference to a cell means the cell comprises at least one non-native (e.g., heterologous) nucleic acid sequence.
  • a stably-transformed cell comprises at least one such nucleic acid sequence integrated into its genome, or in an episomal plasmid that is maintained through multiple generations.
  • expression refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene.
  • the process includes both transcription and translation.
  • a “signal sequence” means a sequence of amino acids covalently-bound to the N-terminal portion of a protein, which facilitates the transport of the protein, e.g., secretion of the mature form of the protein outside the cell.
  • the definition of a signal sequence is functional.
  • the mature form of the extracellular protein lacks the signal sequence which is cleaved off, e.g., during the secretion process.
  • derived encompasses the terms “originated from”, “obtained from” or “obtainable from”, and “isolated from”.
  • protein and “polypeptide” are used interchangeably herein.
  • the conventional one-letter or three-letter code for amino acid residues is used herein.
  • a “promoter” is a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene.
  • the promoter may be an inducible promoter or a constitutive promoter.
  • cbh1 from Trichoderma reesei an inducible promoter, can be used herein.
  • “Operably-linked” refers to juxtaposition wherein elements are in an arrangement allowing them to be functionally related, even where not in close physical proximity.
  • a promoter is operably-linked to a coding sequence if it is capable of controlling the coding sequence and does control the transcription of the sequence under conditions permissive thereof, or conducive thereto.
  • Selective marker refers to a gene capable of expression in a host, and which allows selecting those hosts expressing the marker gene.
  • selectable markers include but are not limited to gene that provide altered resistance to an antimicrobial agent (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer metabolic selectivity, for example, a nutritional advantage on the host cell, such as growth on a particular substrate as a sole source of carbohydrate.
  • “Introduced” in the context of inserting a nucleic acid sequence into a cell means “transfection”, or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • the genome of the cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
  • transiently expressed e.g., transfected mRNA
  • “Host,” “host strain,” or “host cell” means a suitable cell in which to place an expression vector or DNA construct comprising a polynucleotide, e.g., encoding a variant alpha-amylase.
  • Host strains are preferably bacterial cells.
  • “host cell” means cells and/or protoplasts created from the cells of a microbial strain, e.g., a Bacillus spp.
  • culturing refers to growing a population of microbial cells under suitable conditions in a medium capable of supporting such growth. In one embodiment, culturing refers to fermentative bioconversion of a starch substrate containing granular starch to an end-product (typically in a vessel or reactor).
  • enzyme conversion in general refers to the modification of a substrate by enzyme action.
  • enzyme action in general refers to the modification of a starch substrate by the action of an enzyme.
  • sacharification refers to enzymatic conversion of starch to glucose.
  • degree of polymerization refers to the number (n) of anhydroglucopyranose units in a given saccharide.
  • DPI the monosaccharides, such as glucose and fructose.
  • DP2 the disaccharides, such as maltose and sucrose.
  • a DP>3 denotes polymers with a degree of polymerization of greater than 3. The skilled artisan will understand that compounds with greater DE are more polymeric.
  • End-product or “desired end-product” refer to any intended product of an enzymatic reaction, e.g. a starch-derived molecule that is enzymatically converted from the starch substrate.
  • residual starch refers to any remaining starch (soluble or insoluble) left in a composition after fermentation of a starch-containing substrate.
  • specific activity means an enzyme unit defined as the number of moles of substrate converted to product by an enzyme preparation per unit time under specific conditions. Specific activity is expressed as units (U)/unit weight of protein, generally, U/mg protein.
  • Yield refers to the amount of end-product or desired end-products produced using the methods of the present disclosure. In some embodiments, the yield is greater than that produced using methods known in the art. In some embodiments, the term refers to the volume of the end product and in other embodiment the term refers to the concentration of the end product.
  • biologically-active refers to a compound or sequence that has a measurable effect on a biological system, e.g., a cell, an organ, or an organism.
  • ATCC refers to American Type Culture Collection located at Manassas, Va. 20108 (ATCC).
  • NRRL refers to the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research (and previously known as USDA Northern Regional Research Laboratory), Peoria, Ill.
  • “food” means any ingredient, component or composition that provides a nutritive value for an animal, including a human.
  • the term for the gene is generally italicized, (e.g., the gene that encodes amyL ( B. licheniformis AA) may be denoted as amyL).
  • the term for the protein is generally not italicized and the first letter is generally capitalized, (e.g., the protein encoded by the amyL gene may be denoted as AmyL or amyL).
  • nucleic acids are written left to right in 5′ to 3′ orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • a deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or ⁇ (A30-N33).
  • any amino acid residue may be substituted for the amino acid residue present in the position.
  • the alanine may be deleted or substituted for any other amino acid, i.e., any one of:
  • A30X means any one of the following substitutions: A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30V; or in short:
  • alpha-amylases produced by Bacillus spp. are highly homologous (identical) on the amino acid level and may be useful as parent enzymes herein.
  • the percent identity (based on amino acid sequence) of a number of known Bacillus alpha-amylases, relative to each other can be found in the below Table A:
  • the B. licheniformis alpha-amylase (SEQ ID NO: 7) has been found to be about 81% homologous with the B. amyloliquefaciens alpha-amylase (SEQ ID NO: 9), and about 65% homologous with the G. stearothermophilus alpha-amylase (BSG) (SEQ ID NO: 1).
  • Additional homologous alpha-amylases include SP690 and SP722 disclosed in WO 95/26397, and the #707 alpha-amylase derived from Bacillus spp.
  • AmyS-like alpha-amylases may be used as a parent alpha-amylase.
  • the parent alpha-amylase is derived from G. stearothermophilus , e.g., one of those referred to above, such as the G. stearothermophilus alpha-amylase having the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the parent alpha-amylase (i.e., backbone alpha-amylase) may also be a hybrid alpha-amylase, i.e., an alpha-amylase that comprises a combination of partial amino acid sequences derived from at least two alpha-amylases.
  • the parent hybrid alpha-amylase may be one, which on the basis of amino acid homology (identity) and/or DNA hybridization (as defined above), can be determined to belong to the AmyS-like alpha-amylase family described above.
  • the hybrid alpha-amylase is typically composed of at least one part of a AmyS-like alpha-amylase and part(s) of one or more other alpha-amylases selected from AmyS-like alpha-amylases or non-AmyS-like alpha-amylases of microbial (bacterial or fungal) and/or mammalian origin.
  • the parent hybrid alpha-amylase may comprise a combination of partial amino acid sequences deriving from at least two AmyS-like alpha-amylases, or from at least one AmyS-like and at least one non-AmyS-like bacterial alpha-amylase, or from at least one AmyS-like and at least one fuigal alpha-amylase.
  • the AmyS-like alpha-amylase from which a partial amino acid sequence derives may be any of the specific AmyS-like alpha-amylase referred to herein.
  • the parent alpha-amylase may comprise a C-terminal part of an alpha-amylase derived from a strain of B. licheniformis , and an N-terminal part of an alpha-amylase derived from a strain of G. stearothermophilus or from a strain of G. stearothermophilus (BSG).
  • BSG G. stearothermophilus
  • Homology may be determined as the degree of identity between two sequences indicating a relationship therebetween, e.g. a derivation of the first sequence from the second or vice versa.
  • the homology may be determined by visual inspection or manual calculations, but more conveniently by means of computer programs known in the art, such as GAP, a program provided in the GCG program package (described above).
  • GAP a program provided in the GCG program package (described above).
  • Gap GCG v8 may be used, for example with the default scoring matrix for identity and the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, respectively for nucleic acidic sequence comparison, and GAP creation penalty of 3.0 and GAP extension penalty of 0.1, respectively, for protein sequence comparison.
  • GAP uses the method of Needleman and Wunsch, (1970), J. Mol. Biol. 48: 443-453, to make alignments and to calculate the identity.
  • a structural alignment between Spezyme Xtra (SEQ ID NO: 2) and, e.g., another alpha-amylase may be used to identify equivalent/corresponding positions in other AmyS-like alpha-amylases.
  • One method of obtaining said structural alignment is to use the Pile Up program from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1.
  • Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., FEBS Lett. 224: 149-155, 1987) and reverse threading (Huber, T; Torda, A E, Protein Sci. 7(1) 142-149, 1998).
  • the oligonucleotide probe used in the characterization of the AmyS-like alpha-amylase above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the alpha-amylase in question.
  • Suitable conditions for assessing hybridization involve pre-soaking in 5 ⁇ SSC and pre-hybridizing for 1 hour at 40° C. in a solution of 20% formamide, 5 ⁇ Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 mM ATP for 18 hours at 40° C., followed by three times washing of the filter in 2 ⁇ SSC, 0.2% SDS at 40° C. for 30 minutes (low stringency), preferred at 50° C. (medium stringency), more preferably at 65° C. (high stringency), even more preferably at 75° C. (very high stringency). More details about the hybridization method can be found in Sambrook et al., M OLECULAR C LONING : A L ABORATORY M ANUAL , 2 nd Ed., Cold Spring Harbor, 1989.
  • derived from is intended not only to indicate an alpha-amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence.
  • the term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the alpha-amylase in question.
  • the parent alpha-amylase may be a variant of a naturally occurring alpha-amylase, i.e., a variant, which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase.
  • a variant described herein may, in one embodiment, comprise one or more modifications in addition to those outlined above.
  • one or more proline residues (Pro) present in the part of the alpha-amylase variant that is modified is/are replaced with a non-proline residue which may be any of the possible, naturally-occurring non-proline residues, and which preferably is an alanine, glycine, serine, threonine, valine or leucine.
  • one or more cysteine residues present in the parent alpha-amylase may be replaced with a non-cysteine residue such as serine, alanine, threonine, glycine, valine or leucine.
  • relevant amino acid residues which may be deleted or substituted with a view to improving the oxidation stability include the single cysteine residue (C363) and the methionine residues located in positions M8, M9, M96, M200, M206, M284, M307, M311, M316 and M438 in SEQ ID NO: 2.
  • Particularly interesting pair-wise deletions of this type are R179*+G180*; and I181*+G182* (SEQ ID NOS: 16 or 15, respectively) (or equivalents of these pair-wise deletions in another alpha-amylase meeting the requirements of a parent alpha-amylase in the context of the present disclosure).
  • residues of interest include N193F and V416G in the amino acid sequence shown in SEQ ID NO: 2.
  • AmyS-like alpha-amylases with altered properties.
  • Parent alpha-amylases specifically contemplated herein are AmyS-like alpha-amylases and parent hybrid AmyS-like alpha-amylases.
  • the Geobacillus stearothermophilus alpha-amylase (SEQ ID NO: 2) is used as the starting point, i.e., the parent amylase, but in other embodiments, the SP722, BLA, BAN, AA560, SP690, KSM AP1378, #707 and other Bacillus alpha-amylases may be used. Amino acid positions corresponding to positions in SEQ ID NO: 2 are readily determined in accordance herewith.
  • SEQ ID NO: 1 is presently a preferred sequence for such purpose, because it is the longest B. stearothermophilus sequence presently available herein.
  • this disclosure relates to variant with altered properties, e.g., as described above.
  • this disclosure provides a variant of a parent G. stearothermophilus alpha-amylase, comprising an alteration at one or more positions (using e.g., SEQ ID NO: 1 for the amino acid numbering) selected from the group of:
  • the alteration(s) are independently (i) an insertion of an amino acid downstream of the amino acid that occupies the position; (ii) a deletion of the amino acid that occupies the position; or (iii) a substitution of the amino acid that occupies the position with a different amino acid,
  • the variant has alpha-amylase activity
  • each position corresponds to a position of the amino add sequence of the parent amylase, e.g., a G. stearothermophilus alpha-amylase, e.g., having the amino acid sequence shown in SEQ ID NO: 2, e.g., a truncated alpha-amylase that is available commercially as SPEZYME XTRA from Genencor.
  • a G. stearothermophilus alpha-amylase e.g., having the amino acid sequence shown in SEQ ID NO: 2, e.g., a truncated alpha-amylase that is available commercially as SPEZYME XTRA from Genencor.
  • S242A Specifically contemplated herein are S242A, S242Q, S242N and S242E.
  • residues R179, G180, 1181, G182, K183 were chosen to explore the effect of mutations in the calcium-sodium binding region, and P245 was chosen because a proline in the middle of an alpha-helix is unusual.
  • mutations including amino acid substitutions and deletion
  • improved stability i.e., higher or lower
  • high temperatures i.e., about 70-120° C.
  • extreme pH i.e. low or high pH, i.e., pH 4-6 or pH 8-11, respectively
  • free calcium concentrations below 60 ppm include any of the mutations listed in the “Altered Properties” section.
  • the stability may be determined as described in the “Methods” section below.
  • Altered Ca 2+ stability means the stability of the enzyme under Ca 2+ depletion has been improved, i.e., higher or lower stability, relative to the parent enzyme.
  • mutations including amino acid substitutions and deletions
  • improved Ca 2+ stability i.e., higher or lower stability, at especially high pH (i.e., pH 8-10.5) include any of the mutations listed in the “Altered Properties” section.
  • important mutations with respect to obtaining variants exhibiting altered specific activity, in particular increased or decreased specific activity, especially at temperatures from about 10-60° C., preferably about 20-50° C., especially about 0-40° C., include any of the mutations listed in the in “Altered Properties” section.
  • the specific activity may be determined as described in the “Methods” section below.
  • the described variants may have altered oxidation stability, in particular higher oxidation stability, in comparison to the parent alpha-amylase. Increased oxidation stability is advantageous in, e.g., detergent compositions and decreased oxidation stability may be advantageous in compositions intended for starch liquefaction. Oxidation stability may be determined as described in the “Methods” section below.
  • Important positions and mutations with respect to obtaining variants with altered pH profile, in particular improved activity at especially high pH (i.e., pH 8-10.5) or low pH (i.e., pH 4-6) include mutations of amino residues located close to the active site residues.
  • Preferred specific mutations/substitutions include those listed above in the section “Altered Properties” for the positions in question. Suitable assays are described in the “Methods” section below.
  • Important positions and mutations with respect to obtaining variants with improved wash performance at especially high pH include the specific mutations/substitutions listed above in the section “Altered Properties” for the positions in question.
  • the wash performance may be tested as described below in the “Methods” section.
  • the DNA sequence encoding a parent ⁇ -amylase may be isolated from any cell or microorganism producing the ⁇ -amylase in question, using various methods well known in the art.
  • a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the ⁇ -amylase to be studied. If the amino acid sequence of the ⁇ -amylase is known, homologous, labeled oligonucleotide probes may be synthesized and used to identify ⁇ -amylase-encoding clones from a genomic library prepared from the organism in question.
  • a labeled oligonucleotide probe containing sequences homologous to a known ⁇ -amylase gene can be used as a probe to identify ⁇ -amylase-encoding clones, e.g., using hybridization and washing conditions of lower stringency.
  • Another method for identifying ⁇ -amylase-encoding clones is based on inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming ⁇ -amylase-negative bacteria with the resulting genomic DNA library, and plating the transformed bacteria onto agar containing a substrate for ⁇ -amylase, thereby allowing clones expressing the ⁇ -amylase to be readily identified.
  • an expression vector such as a plasmid
  • transforming ⁇ -amylase-negative bacteria with the resulting genomic DNA library
  • the DNA sequence encoding the enzyme may be prepared synthetically by established, standard methods, e.g. the phosphoamidite method described by S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22: 1859-1869 (1981) or the method described by Matthes et al., EMBO J. 3:801-895 (1984).
  • phosphoamidite method oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated, and cloned in appropriate vectors.
  • the DNA sequence may be of mixed origin comprising e.g., genomic and synthetic sequences, synthetic and cDNA sequences, or genomic and cDNA sequences, prepared by ligating fragments of synthetic, genomic, or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al. EMBO J. 3:801-895 (1988).
  • mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis.
  • a single-stranded gap of DNA, bridging the ⁇ -amylase-encoding sequence is created in a vector carrying the ⁇ -amylase gene.
  • the synthetic nucleotide, bearing the desired mutation is annealed to a homologous portion of the single-stranded DNA.
  • gene shuffling e.g., as described in WO 95/22625 (from Affymax Technologies N.V.) or in WO 96/00343 (from Novo Nordisk A/S), or other corresponding techniques resulting in hybrid enzymes comprising the mutation(s), e.g., substitution(s) and/or deletion(s), in question.
  • a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
  • the recombinant expression vector carrying the DNA sequence encoding an alpha-amylase variant for use herein may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may be integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence should be operably-connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA sequence encoding an alpha-amylase variant for use herein, especially in a bacterial host are the promoter of the lac operon of E.
  • the Streptomyces coelicolor agarase gene dagA promoters the promoters of the Bacillus licheniformis alpha-amylase gene (amyL), the promoters of the Geobacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.
  • useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A.
  • niger neutral alpha-amylase A. niger acid stable alpha-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • Expression vectors for use herein may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably-connected to the DNA sequence encoding the alpha-amylase variant. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBI 10, pE194, pAMB1 and pIJ702.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis , or one that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis , or one that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243.
  • Bacillus alpha-amylases mentioned herein comprise a pre-region permitting secretion of the expressed protease into the culture medium. If desirable, this pre-region may be replaced by a different pre-region or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective pre-regions.
  • Cells for use herein can be used as host cells in the recombinant production of an alpha-amylase variant.
  • the cell may be transformed with a DNA construct encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • Cells for use herein may be cells of a higher organism such as a mammal or an insect, but are preferably microbial cells, e.g., a bacterial or a fungal (including yeast) cell.
  • suitable bacteria are Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis , or Streptomyces lividans or Streptomyces murinus , or gram-negative bacteria such as E. coli .
  • the transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.
  • a yeast may favorably be selected from a species of Saccharomyces or Schizosaccharomyces , e.g. Saccharomyces cerevisiae .
  • a filamentous fungus may advantageously be selected from a species of Aspergillus , e.g., Aspergillus oryzae or Aspergillus niger .
  • Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
  • the disclosure relates to a method of producing an alpha-amylase variant, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase variant. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the ATCC).
  • the alpha-amylase variant secreted from the host cells may be recovered from the culture medium by known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • the below assays may be used to screening of AmyS-like alpha-amylase variants having altered stability at high or low pH and/or under Ca 2+ depleted conditions compared to the parent enzyme and AmyS-like alpha-amylase.
  • Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with 10 ⁇ g/mL kanamycin at 37° C. for at least 21 hours.
  • the cellulose acetate layer is located on the TY agar plate.
  • Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with glycine-NaOH buffer, pH 8.6-10.6 and incubated at room temperature (can be altered from about 10-60° C.) for 15 min.
  • the cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in glycine-NaOH buffer, pH 8.6-10.6.
  • the assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.
  • Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with a relevant antibiotic, e.g., kanamycin or chloramphenicol, at 37° C. for at least 21 hours.
  • a relevant antibiotic e.g., kanamycin or chloramphenicol
  • Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with carbonate/bicarbonate buffer about pH 8.5-10 and with different EDTA concentrations (about 0.001 mM to about 100 mM).
  • the filters are incubated at room temperature for 1 hour.
  • the cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use.
  • residual activity is detected on plates containing 1% agarose, 0.2% starch in carbonate/bicarbonate buffer about pH 8.5-10.
  • the assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for about 2 hours at room temperature. After removal of the filters, the assay plates are stained with about 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen
  • Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dasseli Germany) on TY agar plates with 10 ⁇ g/mL chloramphenicol at 37° C. for at least 21 hours.
  • the cellulose acetate layer is located on the TY agar plate.
  • Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter, and the nitrocellulose filter with bound variants is transferred to a container with citrate buffer, pH 4.5 and incubated at 80° C. for 20 minutes (when screening for variants in the wild-type backbone) or 85° C. for 60 minutes (when screening for variants of the parent alpha-amylase).
  • the cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on assay plates containing 1% agarose, 0.2% starch in citrate buffer, pH 6.0.
  • the assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at 50° C. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are re-screened twice under the same conditions as the first screen.
  • Positive transformants after rescreening are picked from the storage plate and tested in a secondary plate assay. Positive transformants are grown for 22 hours at 37° C. in 5 mL LB+chloramphenicol.
  • the Bacillus culture of each positive transformant and as a control a clone expressing the corresponding backbone are incubated in citrate buffer, pH 4.5 at 90° C. and samples are taken at 0, 10, 20, 30, 40, 60 and 80 minutes.
  • a 3- ⁇ L sample is spotted on an assay plate.
  • the assay plate is stained with 10% Lugol solution.
  • Improved variants are seen as variants with higher residual activity (detected as halos on the assay plate) than the backbone.
  • the improved variants are determined by nucleotide sequencing.
  • the stability of the variants may be assayed as follows: Bacillus cultures expressing the variants to be analyzed are grown for 21 hours at 37° C. in 10 mL LB+chloramphenicol. 800 ⁇ L culture is mixed with 200 microliters citrate buffer, pH 4.5. A number of 70 ⁇ L aliquots corresponding to the number of sample time points are made in PCR tubes and incubated at 70° C. or 90° C. for various time points (typically 5, 10, 15, 20, and 30 minutes) in a PCR machine. The 0 min sample is not incubated at high temperature.
  • Activity in the sample is measured by transferring 20 ⁇ L to 200 ⁇ L of the alpha-amylase PNP-G 7 substrate MPR3 ((Boehringer Mannheim Cat. No. 1660730) as described below under “Assays for Alpha-Amylase Activity”. Results are plotted as percentage activity (relative to the 0 time point) versus time, or stated as percentage residual activity after incubation for a certain period of time.
  • a B. subtilis strain harboring the relevant expression plasmid may be fermented and purified as follows: The strain is streaked on a LB-agar plate with 10 ⁇ g/mL kanamycin from ⁇ 80° C. stock, and grown overnight at 37° C. The colonies are transferred to 100 mL PS-1 media supplemented with 10 ⁇ g/mL chloramphenicol in a 500 mL shaking flask.
  • composition of PS-1 medium Pearl sugar 100 g/L Soy Bean Meal 40 g/L Na 2 HPO 4 , 12 H 2 O 10 g/L Pluronic TM PE 6100 0.1 g/L CaCO 3 5 g/L
  • the culture is shaken at 37° C. at 270 rpm for 5 days.
  • the specific activity can be determined using the PHADEBAS® assay (Magle Life Sciences) as activity/mg enzyme.
  • the manufactures instructions are followed (see also below under “Assay for Alpha-Amylase Activity”).
  • the pI can be determined by isoelectric focusing (e.g., using Pharmacia, Ampholine, pH 3.5-9.3).
  • amylase stability may be measured using the method as follows:
  • the enzyme is incubated under the relevant conditions. Samples are taken at various time points, e.g., after 0, 5, 10, 15 and 30 minutes and diluted 25 times (same dilution for all taken samples) in assay buffer (50 mM Britton buffer pH 7.3) and the activity is measured using the PHADEBAS assay (Magle Life Sciences) under standard conditions pH 7.3, 37° C.
  • the activity measured before incubation (0 minutes) is used as reference (100%).
  • the decline in percent is calculated as a function of the incubation time.
  • the table shows the residual activity after, e.g., 30 minutes of incubation.
  • PHADEBAS® Amylase Test supplied by Magle Life Sciences
  • PHADEBAS® Amylase Test supplied by Magle Life Sciences
  • the measured 620 nm absorbance after 10 or 15 minutes of incubation is in the range of 0.2 to 2.0 absorbance units at 620 ⁇ m. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and a blue color will be produced. The color intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.
  • Alpha-amylase activity is determined by a method employing the PNP-G 7 substrate.
  • PNP-G 7 which is a abbreviation for p-nitrophenyl-alpha, D-maltoheptaoside, is a blocked oligosaccharide which can be cleaved by an endo-amylase.
  • Kits containing PNP-G 7 substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim (Cat. No. 1054635).
  • reagent solution 10 mL of substrate/buffer solution is added to 50 mL enzyme/buffer solution as recommended by the manufacturer.
  • the assay is performed by transferring a 20 ⁇ L sample to a 96 well microtitre plate and incubating at 25° C. 200 ⁇ L reagent solution pre-equilibrated to 25° C. is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 30 seconds over 4 minutes at OD 405 nm in an ELISA reader.
  • the slope of the time dependent absorption-curve is directly proportional to the activity of the alpha-amylase in question under the given set of conditions.
  • the variant is incubated with different concentrations of LAS (linear alkyl benzene sulphonate; Nansa 1169/P) for 10 minutes at 40° C.
  • LAS linear alkyl benzene sulphonate
  • the residual activity is determined using the PHADEBAS® assay method or the alternative method employing the PNP-G 7 substrate.
  • LAS is diluted in 0.1 M phosphate buffer pH 7.5.
  • concentrations 500 ppm, 250 ppm, 100 ppm, 50 ppm, 25 ppm, and 10 ppm on no LAS.
  • the variant is diluted in the different LAS buffers to concentration of 0.01-5 mg/l in a total volume of 10 mL and incubated for 10 minutes in a temperature controlled water bath. The incubation is stopped by transferring a small aliquot into cold assay buffer. It is important that during activity measurement the LAS concentration is below 1 ppm, in order not to affect the activity measurement.
  • the residual activity is determined in duplicate using the above mentioned PHADEBAS® assay or alternative method. The activity is measured after subtraction of the blank. The activity with no LAS is 100%.
  • the alpha-amylase variants presented herein possess valuable properties allowing for a variety of industrial applications in cleaning processes and stain removal.
  • One or more of the variant enzymes or compositions described herein may also be used in detergents, in particular laundry detergent compositions and dishwashing detergent compositions, hard surface cleaning compositions.
  • the variants can also be used in compositions for desizing of textiles, fabrics or garments, for production of pulp and paper, beer making, ethanol production, and starch conversion processes as described above.
  • variants herein may also be useful for desizing of textiles, fabrics, and garments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP 119,920 hereby incorporated by reference), beer making or brewing, and in pulp and paper production or related processes.
  • the variant alkaline alpha-amylase may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where re-pulping occurs at pH above about 7 and where amylases facilitate the disintegration of the waste material through degradation of the reinforcing starch.
  • the alpha-amylase variants are especially useful in a process for producing a papermaking pulp from starch-coated printed-paper. The process may be performed as described in WO 95/14807, comprising the following steps:
  • step a) treating with a starch-degrading enzyme before, during or after step a), and
  • the alpha-amylases may also be very useful in modifying starch where enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alkaline alpha-amylase variants it is possible to modify the starch in the presence of the filler thus allowing for a simpler integrated process.
  • alpha-amylase variant may also be very useful in textile, fabric or garment desizing.
  • alpha-amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size, which has served as a protective coating on weft yarns during weaving. Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch breakdown is preferred because it does not involve any harmful effect on the fiber material.
  • the desizing processing is sometimes combined with the scouring and bleaching steps.
  • non-enzymatic auxiliaries such as alkali or oxidation agents are typically used to break down the starch, because traditional alpha-amylases are not very compatible with high pH levels and bleaching agents.
  • the non-enzymatic breakdown of the starch size does lead to some fiber damage because of the rather aggressive chemicals used. Accordingly, it would be desirable to use the alpha-amylase variants as they have an improved performance in alkaline solutions.
  • the alpha-amylases may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile.
  • Desizing and bleaching processes are well known in the art. For instance, such processes are described in WO 95/21247, U.S. Pat. No. 4,643,736, and EP 119,920, which are hereby incorporated by reference.
  • the variant alpha-amylases described herein may be added to and thus become a component of a detergent composition for various cleaning or washing processes, including laundry and dishwashing.
  • the detergent composition provided for herein may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pretreatment of stained fabrics and a rinse added fabric softener composition or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
  • a detergent additive comprising a variant enzyme described herein.
  • the detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a peroxidase, another amylolytic enzyme, e.g., another alpha-amylase, glucoamylase, maltogenic amylase, CGTase and/or a cellulase mannanase (such as MANNASTARTM from Danisco US Inc., Genencor Division)), pectinase, pectin lyase, cutinase, and/or laccase.
  • a protease e.g., a lipase, a peroxidase
  • another amylolytic enzyme e.g., another alpha-amylase, glucoamylase, maltogenic amylase, CGTase and/or a cellulase mannanase (such
  • the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
  • proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus , e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • trypsin-like pro-teases are trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • Other examples of useful proteases may be found in WO98/23732, WO99/20770, WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946.
  • Preferred commercially available protease enzymes include ALCALASE®, SAVINASE®, PRIMASE®, DURALASE®, ESPERASE®, and KANNASE® (from Novozymes A/S), MAXATASE®, MAXACAL, MAXAPEM®, PROPERASE®, PURAFECT®, PURAFECT OXP®, FN2®, FN3®, FN4® (Genencor International Inc.).
  • Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces ), e.g., from H. lanuginosa ( T. lanuginosus ) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase , e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.
  • lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
  • Preferred commercially available lipase enzymes include LIPOLASETM and LIPOLASE ULTRATM (Novozymes A/S).
  • Amylases One or more additional amylases may also be included. Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus , e.g., a special strain of B. licheniformis , described in more detail in GB 1,296,839. Examples of useful alpha-amylases are the variants described in WO 94/18314, WO 96/39528, WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424.
  • alpha-amylases are DURAMYLTM, LlQUEZYMETM TERMAMYTM, NATALASETM, FUNGAMYLTM and BANTM (Novozymes A/S), RAPIDASETM and PURASTARTM (from Genencor).
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Trichoderma, Humicola, Fusarium, Thielavia, Acremonium , e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.
  • Trichoderma reesei cellulases are disclosed in U.S. Pat. No. 4,689,297, U.S. Pat. No. 5,814,501, U.S. Pat. No. 5,324,649, WO 92/06221 and WO 92/06165.
  • Bacillus cellulases are disclosed in U.S. Pat. No. 6,562,612.
  • cellulases include CELLUZYME®, and CAREZYME® (Novozymes A/S), CLAZINASE®, and PURADAX HAI) (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).
  • Peroxidases/Oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus , e.g., from C. cinereus , and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
  • peroxidases include GUARDZYME® (Novozymes A/S).
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive e.g., a separate additive or a combined additive, can be formulated, e.g., granulate, a liquid, a slurry, etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of about 1000 to about 20000; ethoxylated nonyl-phenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid.
  • a liquid detergent may be aqueous, typically containing up to about 70% water and 0 to about 30% organic solvent, or non-aqueous.
  • the detergent composition comprises one or more surfactants, which may be non-ionic, semi-polar, anionic, cationic, and/or zwitterionic.
  • the surfactants are typically present at a level of from about 0.1% to about 60% by weight.
  • the detergent When included therein, the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • the detergent When included therein, the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
  • a non-ionic surfactant such as alcohol ethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
  • the detergent may contain 0 to about 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent may comprise one or more polymers.
  • examples are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid co-polymers.
  • the detergent may contain a bleaching system, which may comprise a H 2 O 2 source such as perborate or percarbonate that may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxyben-zenesul-fonate.
  • a bleaching system may comprise peroxy acids of, e.g., the amide, imide, or sulfone type.
  • the enzyme(s) of the detergent composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative, such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative, such as 4-formylphenyl boronic acid
  • the detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil re-deposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil re-deposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • any enzyme in particular, one or more of the variant enzymes described herein, may be added, e.g., at about 0.01 mg to about 100 mg of enzyme protein per liter of wash liquor. In one embodiment, about 0.055 mg of enzyme protein per liter of wash liquor are used. In other embodiments, about 0.1 mg to about 1.0 mg of enzyme protein per liter of wash liquor are used.
  • variant enzymes described herein may additionally be incorporated in the detergent formulations disclosed in WO 97/07202, which is hereby incorporated as reference.
  • the enzymes may also be used in dish wash detergent compositions, including the following:
  • POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 0.4-2.5% Sodium metasilicate 0-20% Sodium disilicate 3-20% Sodium triphosphate 20-40% Sodium carbonate 0-20% Sodium perborate 2-9% Tetraacetyl ethylene diamine (TAED) 1-4% Sodium sulphate 5-33% Enzymes 0.0001-0.1% 2) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 1-2% (e.g.
  • NTA Nitrilotrisodium acetate
  • TAED Tetraacetyl ethylene diamine
  • Liquid carrier selected from higher 25.0-45.0% glycols, polyglycols, polyoxides, glycolethers Stabilizer (e.g. a partial ester of phosphoric acid and a 0.5-7.0% C 16 -C 18 alkanol)
  • Foam suppressor e.g. silicone
  • Enzymes 0.0001-0.1% 8 NON-AQUEOUS LIQUID DISHWASHING COMPOSITION Liquid nonionic surfactant (e.g.
  • Stabilizing system e.g. mixtures of finely divided 0.5-7.0% silicone and low molecular weight dialkyl polyglycol ethers
  • Low molecule weight polyacrylate polymer 5.0-15.0%
  • Clay gel thickener e.g.
  • compositions Comprising the Variant Alpha-Amylases
  • compositions comprising:
  • a) at least one variant alpha-amylase comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant having detectable alpha-amylase activity, and
  • the variant is altered, as compared to a parent AmyS-like alpha-amylase or a reference amylase, in one or more of a variety of properties that can alter it use or performance for certain applications, e.g., commercial processes described herein.
  • the altered properties can include any property, for example, such as net charge, substrate specificity, substrate cleavage, substrate binding, thermal stability, activity at one or more pH's, stability at one or more pH's, stability in oxidizing conditions, Ca 2+ requirements, specific activity, catalytic rate, catalytic efficiency, activity in the presence of a chelator, thermal or pH stability in the presence of a chelator, utility for desizing, or utility for a cleaning process, or amount of expression in a protein expression system.
  • these altered properties preferably have utility to the end-user, or to the producer of the amylase, or both.
  • amylases of known or readily-determined sequence can be used as the reference amylase.
  • the reference amylase is SEQ ID NOS: 1 or 2.
  • the parent amylase and the reference amylase can be the same amylase in some embodiments.
  • the composition is, in certain embodiments, a component of a product for use in laundry, dish, or hard-surface cleaning, desizing, or fabric or stain treatment.
  • the composition may be part of a dishwashing detergent for application as a liquid, semi-solid, solid, etc, or it can be a granular or liquid laundry detergent formulation.
  • the composition comprises additional components as required for the intended application. Examples of many such formulations are provided herein, and still others will be familiar to those of skill in the art.
  • the composition comprises an additional enzyme that is a protease, a lipase, an amylase, a cellulase, a peroxidase, an oxidase, a pectinase, a lyase, a cutinase, or a laccase, or other useful enzyme.
  • an additional enzyme that is a protease, a lipase, an amylase, a cellulase, a peroxidase, an oxidase, a pectinase, a lyase, a cutinase, or a laccase, or other useful enzyme.
  • the skilled artisan will be familiar with these and other enzymes that may be useful in connection with the variant amylases provided herein.
  • the amounts of enzyme that are useful can be determined empirically for a given application, however, guidelines are provided herein, e.g., in the examples.
  • the composition comprises one or more surfactants.
  • the surfactant is generally nonionic, anionic, cationic, or zwitterionic, or a combination thereof.
  • the amylase variant is preferably a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant.
  • stability to oxidation and stability to chelators or altered metal ion concentrations are useful.
  • the variant amylase has altered stability to oxidation and the variant further includes deletion or substitution of one or more methionine residues including residues located at amino positions 8, 9, 96, 200, 206, 284, 307, 311, 316, and 438 of a parent amylase, where the reference amylase in SEQ ID NOS: 1 or 2.
  • Variant amylases can further comprise an amino acid sequence modification at one or more amino acid positions corresponding to amino acid positions 97, 179, 180, 193, 319, 349, 358, 416, 428, or 443 of the reference amylase (which is preferably SEQ ID NOS: 1 or 2).
  • the variant comprises or further comprises one or more of substitution at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443.
  • the variant in one embodiment comprises a substitution of an N193 or a V416 or both, for example, a substitution that is N193F, or V416G, or both.
  • Other embodiments include further modification such as deletion of one or more amino acids at positions F178, R179, G180, I181, G182 and K183. As described elsewhere herein, such deletions may be even more useful when provided in pair-wise fashion or more.
  • the variant has altered metal ion dependence, or altered stability or activity in the absence of added calcium, or in the presence of a chelator, or a combination thereof. Such variants may also have excellent utility in cleaning and washing processes.
  • the variant alpha-amylase has at least 95% homology to SEQ ID NO: 2 and comprises a substitution of amino acid 242 relative to numbering in a reference amylase comprising the amino acid sequence SEQ ID NO: 1.
  • the variant preferably has detectable alpha-amylase activity, particularly under the conditions of use.
  • the parent alpha-amylase is SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference amylase is SEQ ID NO: 1 or 2.
  • the variant amylase has improved performance in a wash process at very low and very high pH's.
  • the wash performance is improved at pH ⁇ about 8, relative to the parent amylase. More preferred are those variants with improved wash performance above about pH 8.5 to about pH 11.
  • the variant in one embodiment comprises a set of substitutions of a) Q97E, Q319E, Q358E, Q443E; b) Q97E, Q319R, Q358E, Q443R; c) Q97E, Q319R, Q358E; d) Q97E, Q319R, Q443E; e) Q 97E, Q319R, Q443R; f) Q97E, Q358R; g) Q97E, Q443E; h) Q319R, Q358E, Q443E; or i) Q319R, Q358R, Q443E relative to the reference amylase, e.g. a SEQ ID NO: 1 or 2 amylase sequence.
  • the reference amylase e.g. a SEQ ID NO: 1 or 2 amylase sequence.
  • detergent or cleaning formulations comprising at least one variant amylase comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase.
  • the amylase variants have a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, and have detectable alpha-amylase activity.
  • the reference amylase is SEQ ID NOS: 1 or 2.
  • the detergent or cleaning formulation preferably comprises an amylase variant that an S242 variant comprising at least a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T substitution.
  • the variant can comprise any one or combination of the variant features and alterations disclosed herein.
  • kits One embodiment of the kit comprises
  • variant alpha-amylases comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase, said variant having detectable alpha-amylase activity, and
  • kits further comprise instructions for using the kit in a process for desizing a woven material or washing or cleaning one or more items soiled with a starch-containing substance.
  • kits for making the described alpha-amylases are also provided.
  • the kits provide representative sequences e.g. amino acid sequences and/or nucleic acid derived therefrom, for use as parent alpha-amylases and reference amylases.
  • this disclosure provides methods of using the variant alpha-amylases in desizing of fabrics or other woven material, and in washing or cleaning processes.
  • this disclosure provides methods of desizing a woven material subsequent to a weaving process.
  • the methods generally comprise contacting the woven material with a variant alpha-amylase under conditions and for a time effective for at least partially removing sizing from the woven material.
  • the variant comprises an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and has a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase.
  • the variant has detectable alpha-amylase activity.
  • the variant is preferably altered in one more of its physical or enzymatic properties, as compared to a parent AmyS-like alpha-amylase or a reference amylase.
  • the amylase is altered in one or more characteristics of: net charge, substrate specificity, substrate cleavage, substrate binding, thermal stability, activity at one or more pH's, stability at one or more pH's, stability in oxidizing conditions, Ca 2+ requirements, specific activity, catalytic rate, catalytic efficiency, activity in the presence of a chelator, thermal or pH stability in the presence of a chelator, effectiveness for desizing, or amount of expression in a protein expression system.
  • the reference amylase is SEQ ID NOS: 1 or 2.
  • the parent alpha-amylase is SEQ ID NOS: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference amylase is SEQ ID NOS: 1 or 2.
  • the variant is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant.
  • the variant can further comprise one or more substitutions at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443, wherein the reference amylase is SEQ ID NO: 1 or 2.
  • the washing or cleaning processes can subject the enzymes, including amylases to extreme conditions and challenge the limits of the enzyme activity.
  • the methods provided comprise contacting one or more items to be washed or cleaned with a composition comprising a variant alpha-amylase comprising an amino acid sequence at least 95% identical to that of a parent AmyS-like alpha-amylase, and having a substitution at an amino acid position corresponding to position 242 of a reference alpha-amylase.
  • the variant preferably has detectable alpha-amylase activity, and the contacting step is under conditions and for a time effective for at least partially washing or cleaning the one or more items.
  • at least one of the one or more items is soiled with at least one starch-containing material, the removal of which is aided by the variant amylase.
  • the composition further comprises at least one component of a detergent composition or a cleaning formulation.
  • the composition comprises one or more of an additional enzyme, a detergent, a surfactant, a chelator, an oxidizing agent, an acidulant, an alkalizing agent, a source of peroxide, a source of hardness, a salt, a detergent complexing agent, a polymer, a stabilizing agent, or a fabric conditioner.
  • the parent alpha-amylase can be any of SEQ ID NOS: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16, and the reference amylase is SEQ ID NOS: 1 or 2.
  • the parent alpha-amylase is conveniently SEQ ID NOS: 1, 2, 15, or 16, while in others, the parent alpha-amylase is SEQ ID NOS: 6, 7, 8, 9, 10, 11, or 12.
  • the variant is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant.
  • the reference amylase is SEQ ID NO: 1 or 2
  • the variant is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant in certain embodiments.
  • the variant further comprises a sequence modification at one or more amino acid positions corresponding to amino acid positions 97, 179, 180, 193, 319, 349, 358, 416, 428, or 443 of the reference amylase.
  • the variant comprises one or more of substitution at positions as follows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443, or an arginine at 443 in various embodiments.
  • Substitution of an N193 or a V416 or both such as a substitution of N193F or V416G, or both are also useful in certain variants.
  • the variant comprises deletion of one or more amino acids at any of specific positions F178, R179, G180, I181, G182 and K183.
  • the variant preferably has altered metal ion dependence or altered stability, or activity in the absence of added calcium or the presence of a chelator.
  • the foregoing deletions of amino acids can also be used—alone or in combination with any of the foregoing alterations.
  • the variant generally has improved performance in a wash process relative to the parent amylase, for example under conditions such as pH>about 8.
  • the method includes the use of a variant that comprises a set of substitutions of a) Q97E, Q319E, Q358E, Q443E; b) Q97E, Q319R, Q358E, Q443R; c) Q 97E, Q319R, Q358E; d) Q97E, Q319R, Q443E; e) Q97E, Q319R, Q443R; f) Q97E, Q358R; g) Q97E, Q443E; h) Q319R, Q358E, Q443E; or i) Q319R, Q358R, Q443E.
  • a variant that comprises a set of substitutions of a) Q97E, Q319E, Q358E, Q443E; b) Q97E, Q319R, Q358E, Q443R; c) Q 97E, Q319R, Q358E; d)
  • the variants at position S242 of the mature sequence of AmyS were constructed using site directed mutagenesis.
  • the template for mutagenesis was methylated pHPLT-AmyS (see FIG. 2 ) using dam-Methylase from New England Biolabs (Massachusetts).
  • Degenerate primers (S242F(forward) and S242R(reverse), given below) were synthesized and diluted to 10 ⁇ M at Operon (Huntsville, Ala.) with complementary forward and reverse sequences both containing a 5′ phosphate group for ligation in the reaction.
  • the sequence of the parent alpha-amylase (SEQ ID NO: 2) is attached hereto.
  • S242 F SEQ ID NO: 17 5′[Phos]GTCAAGCATATTAAGTTCNNSTTTTTTCCTGATTGGTTG 3′
  • S242 R SEQ ID NO: 18 5′[Phos]CAACCAATCAGGAAAAAASNNGAACTTAATATGCTTGAC 3′
  • the reaction consisted of 18 ⁇ L of sterile distilled H 2 O, 2.5 ⁇ L of 10 ⁇ buffer from the kit, 1 ⁇ L dNTPs from the kit, 1.25 ⁇ L of the forward primers (of 10 ⁇ M stock), 1.25 ⁇ L of the reverse primers (of 10 ⁇ M stock), 1 ⁇ L of pHPLT-AmyS plasmid DNA as template ( ⁇ 70 ng), and 1 ⁇ L of the enzyme blend from the kit for a total of 26.5 ⁇ L.
  • the cycling conditions were 95° C. for 1 min once, then 95° C. for 1 min, 55° C. for 1 min, 65° C. for 10 min for 25 cycles.
  • DpnI 10 U/ ⁇ L was added to the Multi-site Quik-ChangeTM reaction mixture and incubated at 37° C. for 18 hours and then another 0.5 ⁇ L was added for an additional 3 hours.
  • DpnI digested reaction was used as template for rolling circle amplification with the TEMPLIPHI amplification kit (Amersham Biosciences, Piscataway, N.J.), and the reaction was performed according to the Amersham protocol.
  • One microliter of rolling circle DNA was transformed into 100 ⁇ L of Bacillus subtilis competent cells (2 protease deleted B. subtilis strain ( ⁇ aprE, ⁇ nprE, amyE::xylRPxylAcomK-phleo)) and shaken at 37° C. for 1 hour.
  • the entire transformation was next plated on LA+10 ppm Neo+1% insoluble starch plates (25 ⁇ L one plate, 75 ⁇ L on another plate) and incubated overnight at 37° C.
  • Ninety-six transformants were picked into 150 ⁇ L of LB+10 ppm Neo in a micro-titer plate and grown overnight at 37° C.
  • the overnight plate was stamped onto a large LA+10 ppm Neo+1% insoluble starch plate with a 96 pin replicating tool and submitted to Quintara Biosciences (Berkeley, Calif.) for colony PCR and sequencing.
  • the variants were picked into a 96 well micro-titer plates containing 125 ⁇ L of LB+10 ppm Neo, arraying the variants into a quad format with controls.
  • the arrayed micro-titer plate was grown for 6 hours at 37° C. and 250 rpm.
  • the micro-titer culture plate was used to inoculate a new micro-titer plate (micro-titer plate and plate lids from Enzyscreen, Leiden, The Netherlands) containing 150 ⁇ L of MBD medium for protein expression (G.
  • Colonies were streaked from the microtiter plates from Example 1 onto starch plates with 10 ppm Neomycin. The plates were incubated overnight at 37° C., and single colonies were picked and used to inoculate shake flasks (250 mL with 25 mL media) containing media (see below) and 20 ppm Neomycin. The cultures were grown at 37° C., 275 rpm, for about 8 hrs (until an OD (600 nm) of 2.0 was reached). The culture broths were mixed with 50% glycerol at 2:1 ratio, put into individually-labeled culture vials and frozen at ⁇ 80° C. Subsequent production of the selected alpha-amylases were made from these glycerol stocks.
  • Enzymes were purified from the fermentation broth using hydrophobic interaction chromatography as follows: the broth was concentrated 10-fold then diluted back to its original volume with 50 mM MES, 2 mM CaCl 2 , pH 6.8 with 1 M ammonium sulfate, then sterile-filtered using a glass fiber filter Samples were then loaded onto PHENYL SEPHAROSE FF high density column (20 ⁇ 95 mm; Amersham, GE Healthcare Bio-Sciences, Sweden) pre-equilibrated with the same buffer. Non-amylase proteins were removed with 10 column volumes of the same buffer without ammonium sulfate followed by 5 column volumes of water. Enzymes of interest were eluted with 50 mM MES, 2 mM CaCl 2 , pH 6.8 containing 40% propylene glycol.
  • Protein concentrations were determined either with a standard quantitative SDS page gel densitometry method or using an activity assay using a standard amylase assay kit from Megazyme (Wicklow, Ireland).
  • a standard curve generated using purified amylase (Bacillus 707 amylase; SEQ ID NO: 6) was used for comparing assay data.
  • This example shows that the variants described herein may have an altered property relative to the parent alpha-amylase.
  • a high-throughput thermal stability screen of G. stearothermophilus alpha-amylase (AmyS) variants was carried out.
  • Amylase expression was approximately 100 ppm in the culture supernatants of the expression plates. After 60-65 hours of growth at 37° C. in a humidified shaker (250 rpm and 70% relative humidity), the culture supernatants were clarified to remove cellular material using filter plates. The clarified supernatants were diluted 10-fold into buffer containing 50 mM NaOAc/2.6 mM CaCl 2 /0.002% Tween-20, at pH 5.8, to a final concentration of approximately 10 ppm. One aliquot of each supernatant was further diluted to 0.02 ppm, for determination of activity of the enzyme variants as described below using a fluorescently-labeled corn starch substrate.
  • Amylase activity was determined using the amylase ENZCHECK ULTRA AMYLASE assay kit essentially as described by the manufacturer (Invitrogen, San Diego Calif.). Final concentration of the amylase in the assay was approximately 0.02 ppm. Assay buffer was 50 mM NaOAc/2.6 mM CaCl 2 /0.002% Tween-20, pH 5.8. The substrate was BODIPY fluorescence dye conjugated 100 ⁇ g/mL DQTM starch from corn (Invitrogen—Eugene, Oreg.). Increased fluorescence, indicating amylase activity, was measured using a SpectraMAX M2 (Molecular Devices, Sunnyvale, Calif.). The reaction was monitored at room temperature for 5 minutes with the instrument recording in kinetic mode. Excitation wavelength was 485 nm; emission was monitored at 520 nm with a cutoff filter at 515 nm.
  • the wild-type AmyS (Xtra) showed 33-43% residual activity after being subject to thermal stress for 30 minutes at 95° C.
  • AmyS variants, S242A and S242Q retained 55-65% and 70-80% residual activities, respectively, following the same thermal stress conditions. See FIG. 3 and Table 3-1. These residual activity measurements indicate the two variants are more thermostable than the wild-type alpha-amylase.
  • Spezyme Xtra, S242A, S242E, and S242Q were purified from shake flask fermentation broth (see Example 2) using hydrophobic interaction chromatography.
  • the protein was eluted from the column in purified form using 50 mM MES, pH 6.8, containing 40% propylene glycol and 2 mM CaCl 2 .
  • the same sample was then re-scanned to check the reversibility of the process.
  • the thermal unfolding process was irreversible.
  • the buffer used was 10 mM sodium acetate, pH 5.5.
  • a 200° C./hr scan rate was used to minimize any artifacts that may have resulted from aggregation.
  • the thermal midpoint (T m ) of the DSC curves was used as an indicator of the thermal stability of the tested protein.
  • Table 4-1 shows the thermal melting points for the amylase proteins tested. The thermal melting curves and the melting points for the wild-type and variant amylases are shown in FIG. 5 .
  • the thermal unfolding for the amylase variants S242A, S242E, and S242Q in the absence and presence of 2 mM calcium chloride show considerable increase in the melting points for the variants when compared to that for the wild-type.
  • the wild-type amylase has a thermal melting point of 100.8° C. while the T m 's for S242A, S242E, and S242Q are 106.5° C., 107.8° C., and 110.1° C., respectively.
  • substitution of S242 with A results in an increase in the T m of 5.7° C.
  • substitution of S242 with E results in an increase in the T m of 7.0° C.
  • substitution of S242 with Q results in an increase in the T m of 9.3° C.
  • the wild-type amylase displayed a thermal melting point of 106.8° C. while the T m 's for S242A, S242E, and S242Q were 111.8° C., 112.2° C., and 113.8° C., respectively.
  • T m 's for S242A, S242E, and S242Q were 111.8° C., 112.2° C., and 113.8° C., respectively.
  • all four proteins had increased T m values.
  • the increase in T m for wild-type and the S242A variants in the presence of calcium was 6° C. and 5.3° C., respectively.
  • the increase in T m for the S242E variant was 4.4° C.
  • T m for the S242Q variant was 3.7° C.
  • This example shows that the tested variants have altered activity profiles relative not only to the parent alpha-amylase but also to an industry standard enzyme. Protein determinations were made on purified or plate samples. The variants and standard alpha-amylases were each assayed on the basis of equal protein concentration.
  • Either plate or purified variants were diluted to approximately 20 ppm using pH 5.6 malic acid buffer.
  • the substrate consisted of 15% cornstarch in the same 50 mM Malic acid buffer, pH 5.6.
  • Four hundred microliters of the starch suspension was equilibrated to 70° C. for 2.5 minutes.
  • Seven (7) 11L of the diluted enzyme was quickly added to the equilibrated starch at a final protein concentration of about 0.36 ppm.
  • the reaction mix was then put into a pre-heated 85° C. shaking heating block and mixed at 300 rpm.
  • the reactions were quenched with 50 ⁇ L of 125 mM NaOH at predetermined time intervals.
  • the reaction tubes were spun and the supernatant was diluted 10 fold into 10 mM NaOH, for analysis of DP profile by HPAEC-PAD.
  • Reactions were set up for 4, 10 and 20 minutes.
  • the 4 min reaction provides an indication of the enzyme initial conversion f product to substrate;
  • the 10 minute reaction provides an indication of the enzyme's thermal activity, and
  • the 20 minute reaction provides an indication of the enzyme's thermal stability.
  • BCA bisulfite-containing phosphatidylcholine
  • BCA bisulfite assay
  • Pierce assay was used to determine the protein concentration in samples on microtiter plate (MTP) scale.
  • the chemical and reagent solutions used were: BCA protein assay reagent, and Pierce dilution buffer (50 mM MES, pH 6.5, 2 mM CaCl 2 , 0.005% TWEEN®-80).
  • the equipment included a SpectraMAX (type 340; Molecular Devices) MTP reader.
  • the MTPs were obtained from Costar (type 9017).
  • Two-hundred (200) ⁇ L BCA Reagent was pipetted into each well, followed by 20 ⁇ L diluted protein. After thorough mixing, the MTPs were incubated for 30 minutes at 37° C. Air bubbles were removed before the optical density (OD) of the solution in the wells was read at 562 nm. To determine the protein concentration, the background reading was subtracted from the sample readings. The OD 562 was plotted for protein standards (purified enzyme) to produce a standard curve. The protein concentration of the samples were interpolated from the standard curve.
  • the Bradford dye reagent (Quick Start) assay was used to determine the protein concentration in samples on MTP scale.
  • the chemical and reagent solutions used were: Quick Start Bradford Dye Reagent (BIO-RAD Catalog No. 500-0205), Dilution buffer (10 mM NaCl, 0.1 mM CaCl 2 , 0.005% TWEEN(R-80).
  • the equipment used was a Biomek FX Robot (Beckman) and a SpectraMAX (type 340) MTP reader.
  • the MTPs were from Costar (type 9017).
  • the detergents used in this assay did not contain enzymes or the enzymes present in commercial detergents had been destroyed through heat deactivation as described elsewhere in this document.
  • the equipment used included an Eppendorf Thermomixer and a SpectraMAX (type 340) MTP reader.
  • the MTPs were obtained from Costar (type 9017).
  • AATCC HDL Detergent Preparation
  • the detergent solution was vigorously stirred for at least 15 minutes.
  • 5 mM HEPES (free acid) was added and the pH adjusted to 8.0.
  • Rice Starch Microswatch Assay for testing Amylase Performance Test detergents were prepared as described elsewhere in this document. The equipment used included a New Brunswick Innova 4230 shaker/incubator and a SpectraMAX (type 340) MTP reader. The MTPs were obtained from Corning (type 3641). Aged rice starch with orange pigment swatches (CS-28) were obtained from Center for Test Materials (Vlaardingen, Netherlands). Before cutting 0.25-inch circular microswatches, the fabric was washed with water. Two microswatches were placed in each well of a 96-well microtiter plate. The test detergent was equilibrated at 20° C. (North America) or 40° C. (Western Europe).
  • Alpha-amylase concentration and specific activity was determined, in some cases, by titration with an inhibitory polyclonal antibody.
  • Polyclonal antibodies raised to Bacillus stearothermophilus alpha-amylase (AmyS) were found to be strongly inhibitory of AmyS and the alpha-amylase from Bacillus sp. TS-23 (e.g., the binding is tight enough to produce a linear titration of activity loss). Therefore, this antibody can be used to measure enzyme concentration, which, in turn, is used to calculate specific activity.
  • a polyclonal antibody directed against purified AmyS was raised in a rabbit and purified by standard methods.
  • An empirical “apparent concentration” value of an antibody stock solution was determined by measuring the inhibition of a sample of AmyS of known specific activity. The antibody sample was used to determine the concentration and specific activity of AmyS and TS23t variants. These values were used to create normalized 96-well enzyme stock plates, in which all of the variants were diluted to a common concentration.
  • Electrophoretic mobility of variant protein samples was measured using the PHASTGEL system (GE Healthcare) on pre-cast native polyacrylamide gels (PHASTGEL Homogeneous) at either 7.5% or 12.5% concentration.
  • Buffer strips PASTGEL Native
  • Typical run conditions consisted of 400 V for 12.75 minutes with an anode-to-cathode distance of 3.7 cm.
  • electrophoretic mobility of variant protein samples was measured on 1 mm-thick 0.5-1.5% agarose gels at various pH values (i.e. 5.8, 8.0 and 10.0) through a choice of a suitable buffer system.
  • the electrophoresis was carried out under non-denaturing conditions.
  • the Cathode-Anode length was 13.9 cm.
  • a sample of 1-2 ⁇ g protein was mixed with 5% glycerol+0.05% bromophenol blue and loaded on each lane. Gels were run typically for 1 hour at 100 V.
  • Heat inactivation of commercial detergent formulas serves to destroy the enzymatic activity of any protein components while retaining the properties of non-enzymatic components.
  • this method was suitable for preparing commercially-purchased detergents for use in testing the enzyme variants.
  • 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 incubation time for heat inactivation of North American (NA) and Japanese (JPN) heavy duty granular laundry (HDG) detergent was 8 hours and that for Western European (WE) HDG detergent was 5 hours.
  • NA North American
  • WE Western European
  • HDG Western European
  • the incubation time for heat inactivation of NA and WE auto dishwashing (ADW) detergents was 8 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 suc-AAPF-pNA assay.
  • a standard protocol for assessing protein and carbohydrate soil cleaning was used whereby the soil level on a fabric swatch was measured before and after cleaning under standard conditions.
  • the fabric swatches consisted of woven cotton fabric soiled with either maize starch, rice starch or a blood, milk, and carbon black mixture. Swatches were purchased from Testfabrics, Inc. (West Pittiston, Pa.). Maize Starch (EMPA 161) and Blood, Milk, Carbon Black (EMPA 116) technical soils were produced by EMPA Test materials AG (St. Gallen, Switzerland). Rice Starch (CFT CS-28) soils were produced by the Center for Testmaterials BV (Vlaardingen, Netherlands).
  • % SR1 percent stain removal index
  • Enzyme was added directly into the wash solution and reactions were then initiated by addition of either 40 g/L or 200 g/L of soiled and ballast fabric.
  • the washing reactions were agitated at 100 rpm for 10, 15, or 40 minutes at 20° C., 25° C., 30° C., 40° C., or 50° C.
  • swatches were rinsed for 3 minutes in tap water, spun in a front-loading washing machine at 1000 rpm to remove excess water, and dried in a dryer at low heat on a permanent press cycle for approximately 45 minutes. Comparison of the extent of soil removal was assessed by reflectometry and expressed as the % soil removal index (% SR1). The control condition did not contain enzyme and the positive control consisted of various doses of benchmark commercial enzymes.
  • the BODIPY-starch assay was performed using the EnzChek® Ultra Amylase Assay Kit (E33651, Invitrogen).
  • a 1 mg/mL stock solution of the DQ starch substrate was prepared by dissolving the contents of the vial containing the lyophilized substrate in 100 ⁇ L of 50 mM sodium acetate buffer at pH 4.0. The vial was vortexed for about 20 seconds and left at room temperature, in the dark, with occasional mixing until dissolved.
  • 900 ⁇ L of assay buffer 50 mM sodium acetate with 2.6 mM CaCl 2 at pH 5.8 was added, and the vial was mixed by vortex for about 20 seconds.
  • the substrate solution was stored at room temperature, in the dark, until ready to use or at 4° C.
  • a 100 ⁇ g/mL of working solution of the DQ substrate was prepared from the 1 mg/mL substrate solution in the assay buffer.
  • 190 ⁇ L of 100 ⁇ g/mL substrate solution was added to each well in a flat-bottom 96-well microtiter plate.
  • 10 ⁇ L of each enzyme sample was added to a well, mixed for 30 seconds using a thermomixer at 800 rpm.
  • a blank sample containing buffer and substrate only (no-enzyme blank) was included in the assay.
  • the rate of change of fluorescence intensity was measured (excitation: 485 nm, emission: 520 nm) in a fluorescence microtiter plate reader at 25° C. for 5 minutes.
  • Substrates used included bagasse (sugarcane bagasse from Brazil, dilute-acid pre-treated by National Renewable Energy Laboratory, washed and buffered at pH 5), AFEX (ammonia fiber expansion corn stover), and PCS (dilute sulfuric acid pre-treated corn stover, washed and adjusted to pH 5). All substrates were brought to the desired percentage solids prior to use.
  • Amylase Binding Amylase charge ladder variants were purified and diluted to 200 ppm for testing. A 1% cellulose bagasse solution was prepared in borate buffer (40 mM, pH8.5, 0.016% Tween80). 150 ⁇ L of the bagasse solution was added into each well in a microtiter filtration plate. 150 ⁇ L of borate buffer was added into a set of separate wells, which served as controls. 10 ⁇ L of amylase charge ladder variants was added into the filtration plate, each condition was in duplicates. The plate was incubated at room temperature for 2 hours. The filtrate was collected and amylase activity in the supernatant was measured by BODIPY-starch assay.
  • borate buffer 40 mM, pH8.5, 0.016% Tween80
  • the coding region for the LAT signal peptide is shown below:
  • the amino acid sequence of the LAT signal peptide is shown below:
  • amino acid sequence of the mature truncated S242Q amylase with the substituted amino acid shown in italics was used as the basis for making the variant libraries described herein:
  • the PCR products were purified using QIAQUIK columns from Qiagen, and resuspended in 50 ⁇ L of deionized water.
  • 50 ⁇ L of the purified DNA was digested with HpaI (Roche) and PstI (Roche), and the resultant DNA resuspended in 30 ⁇ L of deionized water.
  • 10-20 ng/ ⁇ L of the DNA was cloned into plasmid pHPLT using PstI and HpaI cloning sites.
  • the ligation mixtures were directly transformed into competent B. subtilis cells (genotype: ⁇ vpr, ⁇ wprA, ⁇ mpr-ybfJ, ⁇ nprB). The B. subtilis cells (genotype: ⁇ vpr, ⁇ wprA, ⁇ mpr-ybfJ, ⁇ nprB). The B. subtilis cells (genotype: ⁇ vpr, ⁇ wprA
  • subtilis cells have a competency gene (comk) which is placed under a xylose inducible promoter, so xylose was used to induce competency for DNA binding and uptake (see Hahn et al., Mol. Microbiol., 21: 763-775, 1996).
  • This Example describes the methods used to express various recombinant enzymes of the transformed B. subtilis of the preceding Examples.
  • Alpha-Amylase Expression 2 mL scale.
  • B. subtilis clones containing S242Q (or a variant thereof) expression vectors were replicated with a steel 96-well replicator from glycerol stocks into 96-well culture plates (BD, 353075) containing 150 ⁇ L of LB media+10 ⁇ g/mL neomycin, grown overnight at 37° C., 220 rpm in a humidified enclosure. A 100 ⁇ L aliquot from the overnight culture was used to inoculate 2000 ⁇ L defined media+10 ⁇ g/mL neomycin in 5 mL plastic culture tubes.
  • the cultivation media was an enriched semi-defined media based on MOPS buffer, with urea as major nitrogen source, glucose as the main carbon source, and supplemented with 1% SOYTONE and 5 mM calcium for robust cell growth.
  • Culture tubes were incubated at 37° C., 250 rpm, for 72 hours. Following this incubation, the culture broths were centrifuged for 10 minutes at 3000 ⁇ g. The supernatant solution was decanted into 15 mL polypropylene conical tubes; 80 ⁇ L of each sample were aliquoted into 96 well plates for protein quantitation.
  • This Example describes the production of enzyme charge ladders and combinatorial charge libraries.
  • Enzyme Charge Ladders Multiple protein variants spanning a range of physical properties of interest are selected from existing libraries or are generated by site-directed mutagenesis techniques as known in the art (see e.g., U.S. patent application Ser. Nos., 10/576,331, 11/581,102, and 11/583,334, assigned to Genencor International. This defined set of probe proteins is then assayed in a test of interest.
  • amylase charge ladder variants are shown in the following tables and assayed as described herein. In these tables, the charge change is relative to the parent enzyme.
  • AmyS-S242Q Charge Ladder AmyS-S242Q Variant ⁇ Charge Q97E-Q319E-Q358E-Q443E ⁇ 4 Q97E-Q319E-Q358E ⁇ 3 Q97E-Q319E ⁇ 2 Q97E ⁇ 1 Q97R-Q319E 0 Parent AmyS-S242Q 0 Q97R +1 Q97R-Q319R +2 Q97R-Q319R-Q358R +3 Q97R-Q319R-Q358R +4
  • the AmyS-S242Q plasmid DNA was isolated from a transformed B. subtilis strain (genotype: ⁇ aprE, ⁇ nprE, amyE::xylRPxylAcomK-phleo) and sent to DNA2.0 Inc. as the template for CCL construction.
  • a request was made to DNA2.0 Inc. (Mountain View, Calif.) for the generation of positional libraries at each of the four sites in AmyS-S242Q (S242Q) amylase that are shown in Table 9-2.
  • Variants were supplied as glycerol stocks in 96-well plates.
  • the AmyS S242Q combinatorial charge library was designed by identifying the following four residues: Gln97, Gln319, Gln358, and Gln 443.
  • a four site, 81-member CCL was created by making all combinations of three possibilities at each site: wild-type, arginine, or aspartic acid.
  • Example 6 describes the testing of S242Q variant in a microswatch assay 1.0 ⁇ g/mL in AATCC HDL detergent or 5 mM HEPES buffer under varying ionic strength. The methods provided in Example 6 were used (See, “Rice Starch Microswatch Assay for testing Amylase Performance” and “Corn Four Hydrolysis”).
  • electrostatic interactions are governed primarily by the strength of double-layer forces between interacting species at constant potential or constant charge (enzymes, substrates, fabric, and detergent), their size, and the dielectric constant of the surrounding medium.
  • a complex medium such as a detergent formulation
  • FIG. 10 shows that positive charge S242Q charge variants are superior for cleaning of rice starch microswatch under North American laundry conditions. Likewise, negative charge TS23t variants are superior for cleaning of rice starch microswatches in Western European laundry conditions ( FIG. 11 ).
  • FIG. 12 demonstrates that positive S242Q variants exhibit higher specific activity for granular corn starch substrates hydrolysis.
  • Example 6 describes determining the relationship between protein charge and thermal stability.
  • Alpha-amylase assays were based on BODIPY starch hydrolysis before and after heating the culture supernatant. The same chemical and reagent solutions used are as described in Example 6.
  • the residual activity of a sample was expressed as the ratio of the final absorbance and the initial absorbance, both corrected for blanks. A higher index indicates a more thermally-stable variant. This is an example of optimizing a protein physical property, in this case net charge, for improving enzyme thermal stability for a liquid laundry application.
  • Thermostability Assay Thermostability of the variants was assessed as described above. Thermostability winners are listed in Table 11-1. Winners were defined as those having a ratio of mutant residual activity to parent (i.e., S242Q) residual activity greater than 1.
  • PI Performance Index
  • This example illustrates that two separate enzyme properties can be simultaneously optimized by the introduction of multiple amino acid substitutions, even where the properties are negatively correlated due, for example, to oppositely linked to charge characteristics of the protein.
  • the strategy described herein was successfully used to produce and select multiply-substituted AmyS-242Q variants having improvements in a first property (e.g., expression as the primary property), while improving or not sacrificing a second property (e.g., starch hydrolysis as the secondary property).
  • a first property e.g., expression as the primary property
  • a second property e.g., starch hydrolysis as the secondary property
  • AmyS-S242Q charge combinatorial library comprising variants having combinations of from one to four substitutions of charged residues was tested for shake tube expression, BODIPY-starch hydrolysis, and rice starch cleaning activity.
  • AmyS-S242Q winners are shown in Tables 13-1 and 13-2.
  • the multiply-substituted variants of Table 13-1 have equal or improved expression and equal or improved BODIPY-starch hydrolysis as compared to the parent enzyme.
  • the multiply-substituted variants of Table 13-2 have equal or improved expression and equal or improved rice starch cleaning activity as compared to the parent enzyme.
  • amylases Although demonstrated with amylases, this method is applicable to other enzyme classes such as proteases, lipases, cellulases, transferases and pectinases. Moreover any combination of two or more properties can be analyzed simultaneously such as expression, activity, binding, thermal stability, stability in the presence of one or detergents, and chelant stability.
  • CaCl 2 concentration was varied from 0-20 ppm per test by adding various amounts of stock CaCl 2 solution to Milli Q water, pH ⁇ 6.5. Ethyl, Xtra and variant S242Q were used at 0.01 ppm active protein per test.
  • the assay was performed in a LAUNDER-O-METER using a liquor ratio of 50:1. Performance tests were conducted on rice starch-stained fabric swatches with an indicator dye bound to the starch (TestFabrics Cat. No. CS-28; TestFabrics Inc.). Three CS-28 swatches (6 cm ⁇ 8 cm) and 4 greige print cloth swatches (Testfabrics, Style 400R; 3 inches ⁇ 4 inches) were used as substrates per experiment.
  • the temperature of the LAUNDER-O-METER with Milli Q water/Ca was pre-adjusted to 85° C. or 97° C., after which the enzymes and swatches were added. The reaction was carried out for 30 min, after which the swatches were rinsed in water and dried before reading.
  • Measurements are made by reflectometry using the CIE L*a*b* color space. Every perceivable color can be represented by L*a*b* coordinate in the color space.
  • L* represents the lightness or grey scale value on a scale of 0 to 100, pure black to pure white.
  • a* represents the magenta to green shift, wherein large positive values represent a very magenta hue and large negative values represent a very green hue.
  • b* represents the yellow to blue shift where large positive values represent a very yellow hue and large negative values represent a very blue hue. When both a* and b* values are 0, there is an absence of color, leaving pure grey colors with their lightness defined by the L* value.
  • a Minolta Chromameter CR 200 in the CIE Lab color space with a D 65 light source was utilized for measuring desizing performance.
  • CIE L* readings i.e., 2 readings each from the front and the back of the swatch
  • Higher CIE L* values indicate better desizing performance.
  • the S242Q variant showed significantly lower calcium dependency for desizing performance compared to both Ethyl and Xtra under the conditions tested.

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