Alpha-amylase combinatorial variants

Abstract

Disclosed are compositions and methods relating to variant alpha-amylases. The variant alpha-amylases are useful, for example, for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing, and other applications, for textile processing (e.g., desizing), in animal feed for improving digestibility, and for baking and brewing.

What is claimed is: 
     
         1 . A recombinant variant of a parent α-amylase comprising pair-wise amino acid substitutions at amino acid residues corresponding to G476 and G477 in SEQ ID NO: 1, wherein each glycine residue present in the parent α-amylase is independently substituted with an amino acid residue other than glycine, and wherein the variant exhibits improved starch hydrolysis activity compared to the parent. 
     
     
         2 . The variant of  claim 1 , further comprising a mutation at an amino acid residue corresponding to E187 or S241, using SEQ ID NO: 1 for numbering. 
     
     
         3 . The variant α-amylase of any of the preceding claims, further comprising at least one mutation at an amino acid residue corresponding to an amino acid residue selected from the group consisting of N126, Y150, F153, L171, T180, and, I203. 
     
     
         4 . The variant α-amylase of any of the preceding claims, further comprising a deletion of at least one amino acid residue corresponding to R178, G179, T180, and G181, using SEQ ID NO: 1 for numbering. 
     
     
         5 . The variant α-amylase of any of the preceding claims, further comprising deletions of amino acid residues corresponding to R178 and G179, or T180 and G181. 
     
     
         6 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of E132, Q167, T180, and A277, using SEQ ID NO: 1 for numbering. 
     
     
         7 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of R458, T459, and D460, using SEQ ID NO: 1 for numbering. 
     
     
         8 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to T180, using SEQ ID NO: 1 for numbering. 
     
     
         9 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to N205, using SEQ ID NO: 3 for numbering. 
     
     
         10 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to an amino acid residue selected from the group consisting of T333G, A335S, and Q337E, using SEQ ID NO: 3 for numbering. 
     
     
         11 . The variant α-amylase of any of the preceding claims, further comprising a mutation in an amino acid residue corresponding to an amino acid residue position selected from the group consisting of 6, 7, 8, 11, 14, 15, 20, 21, 23, 26, 27, 28, 37, 38, 39, 40, 42, 45, 46, 48, 49, 50, 51, 52, 53, 54, 58, 61, 62, 68, 70, 71, 72, 73, 79, 80, 81, 82, 84, 85, 87, 88, 89, 92, 93, 94, 95, 96, 97, 98, 101, 108, 111, 112, 113, 114, 115, 116, 117, 118, 120, 122, 123, 124, 126, 127, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 142, 143, 144, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 165, 167, 168, 170, 171, 172, 175, 176, 177, 180, 181, 182, 187, 190, 191, 193, 199, 200, 201, 203, 206, 208, 210, 211, 212, 214, 215, 216, 219, 221, 223, 225, 226, 227, 235, 238, 239, 240, 241, 242, 243, 245, 246, 247, 248, 249, 250, 252, 253, 254, 256, 257, 258, 260, 261, 262, 266, 267, 268, 269, 270, 271, 273, 276, 277, 279, 280, 282, 284, 285, 286, 288, 296, 299, 300, 301, 302, 303, 304, 307, 308, 310, 311, 312, 313, 316, 317, 318, 320, 321, 325, 327, 335, 338, 342, 348, 349, 352, 356, 357, 360, 362, 363, 368, 369, 377, 381, 382, 383, 384, 385, 388, 390, 392, 394, 395, 396, 397, 398, 400, 401, 402, 403, 404, 405, 407, 408, 410, 414, 415, 416, 418, 419, 420, 421, 422, 423, 424, 426, 428, 429, 430, 431, 434, 435, 436, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 454, 455, 457, 460, 461, 462, 463, 464, 465, 466, 467, 469, 470, 471, 473, 474, 475, 476, 477, 479, 480, 481, 482, 483, and 484, using SEQ ID NO: 1 for numbering. 
     
     
         12 . The variant α-amylase of any of the preceding claims, comprising a combination of mutations corresponding to mutations selected from the group consisting of:
 N126Y+T180D+E187P+I203Y+Y303D+G476T+G477E, 
 N126Y+T180D+E187P+I203Y+Y303R+N475E+G476T+G477R, 
 T038N+N088H+N126Y+T129I+N134M+F153W+L171R+T180D+E187P+I203Y+G476K+G477E, 
 N126Y+E132H+T180D+E187P+I203Y+Y303D+G476T+G477E, and 
 N126Y+F153W+E187P+G4726+G477R; 
 wherein the variant has increased thermostability, detergent stability, stability starch liquifaction activity, or cleaning performance compared to the parent; and 
 wherein the variant or the parent has at least 60% amino acid sequence identity relative to SEQ ID NO: 1, which is used for numbering. 
 
     
     
         13 . The variant amylase of any of the preceding claims, wherein the parental α-amylase is from a  Cytophaga  species. 
     
     
         14 . The variant amylase of any of the preceding claims, wherein the parental α-amylase is from a  Paenibacillus  species. 
     
     
         15 . The variant amylase of any of the preceding claims, wherein the parental α-amylase is not from a  Bacillus  species. 
     
     
         16 . The variant amylase of any of the preceding claims, wherein the parental α-amylase or the variant α-amylase has at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. 
     
     
         17 . The variant amylase of any of the preceding claims, wherein the parental α-amylase or the variant α-amylase has at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 
     
     
         18 . The variant amylase of any of  claims 1 - 16 , wherein the parental α-amylase or the variant α-amylase has at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. 
     
     
         19 . The variant amylase of any of  claims 1 - 16 , wherein the parental α-amylase or the variant α-amylase has at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. 
     
     
         20 . A composition comprising the variant α-amylase of any of the preceding claims. 
     
     
         21 . The composition of  claim 20 , wherein the composition is effective for removing starchy stains from laundry, dishes, or textiles. 
     
     
         22 . The composition of  claim 20  or  21 , further comprising a surfactant. 
     
     
         23 . The composition of any of  claims 20 - 22 , wherein the composition is a detergent composition. 
     
     
         24 . The composition of any of  claims 20 - 22 , wherein the composition is a laundry detergent or a laundry detergent additive. 
     
     
         25 . The composition of any of  claims 20 - 22 , wherein the composition is a manual or automatic dishwashing detergent. 
     
     
         26 . The composition of any of  claims 20 - 22 , further comprising one or more additional enzymes selected from the group consisting of protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, metalloproteinase, amadoriase, glucoamylase, arabinofuranosidase, phytase, isomerase, transferase, and an amylase other than the amylase of any one of  claims 1 - 19 . 
     
     
         27 . The composition of  claim 19 , wherein the composition is for liquifying starch. 
     
     
         28 . The composition of  claim 19 , wherein the composition is for saccharifying a composition comprising starch, for SSF post liquefaction, or for direct SSF without prior liquefaction. 
     
     
         29 . The composition of  claim 19 , wherein the composition is for producing a fermented beverage. 
     
     
         30 . The composition of  claim 19 , wherein the composition is for producing a baked food product. 
     
     
         31 . The composition of  claim 19 , wherein the composition is for textile desizing. 
     
     
         32 . A method for removing a starchy stain or soil from a surface, comprising:
 contacting the surface in the presence of a composition comprising an effective amount of the variant amylase of any of the  claims 1 - 19 , and   allowing the polypeptide to hydrolyze starch components present in the starchy stain to produce smaller starch-derived molecules that dissolve in the aqueous composition,   thereby removing the starchy stain from the surface.   
     
     
         33 . The method of  claim 32 , wherein the aqueous composition further comprises a surfactant. 
     
     
         34 . The method of any of  claim 32  or  33 , wherein the surface is a textile surface or a surface on dishes. 
     
     
         35 . The method of any of  claims 32 - 34 , wherein the composition further comprises at least one additional enzymes selected from the group consisting of protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, metalloproteinase, amadoriase, glucoamylase, arabinofuranosidase, phytase, isomerase, transferase, and an amylase other than the amylase of any one of  claims 1 - 19 . 
     
     
         36 . A method for saccharifying a composition comprising starch to produce a composition comprising glucose, wherein the method comprises:
 (i) contacting the solution comprising starch with effective amount of the variant amylase of any of the  claims 1 - 19 ; and   (ii) saccharifying the solution comprising starch to produce the composition comprising glucose; wherein the variant amylase catalyzes the saccharification of the starch solution to glucose or other enriched carbohydrate syrups.   
     
     
         37 . The method of  claim 36 , wherein the composition comprising starch comprises liquefied starch, gelatinized starch, granular starch, or starch heat-treated below its gelatinization temperature. 
     
     
         38 . The method of  claim 36  or  37 , wherein the fermentation is a simultaneous saccharification and fermentation (SSF) reaction. 
     
     
         39 . The method of any of  claims 36 - 38 , wherein the method further comprises contacting a mash and/or a wort with an amylase. 
     
     
         40 . The method of any one of  claims 36 - 39 , further comprising adding glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, β-amylase, α-amylase that is not the variant α-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, alpha-glucosidase, beta-glucosidase, or a combination thereof, to the starch solution. 
     
     
         41 . The method of any one of  claims 36 - 40 , wherein the amylase is expressed and secreted by a host cell. 
     
     
         42 . The method of  claim 41 , wherein the composition comprising starch is contacted with the host cell. 
     
     
         43 . The method of  claim 41  or  42 , wherein the host cell further expresses and secretes one or more enzymes selected from the group consisting of glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, β-amylase, α-amylase that is not the variant α-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, alpha-glucosidase, and beta-glucosidase. 
     
     
         44 . The method of any one of  claims 41 - 43 , wherein the host cell further expresses and secretes a glucoamylase. 
     
     
         45 . The method of any one of  claims 41 - 44 , wherein the host cell is capable of fermenting the composition. 
     
     
         46 . A composition comprising glucose produced by the method of any one of  claims 36 - 45 . 
     
     
         47 . A liquefied starch produced by the method of any one of  claims 36 - 45 . 
     
     
         48 . A fermented beverage produced by the method of any one of  claims 36 - 45 . 
     
     
         49 . Use of an amylase of any of  claims 1 - 19  in the production of a composition comprising glucose, in the production of a liquefied starch, in the production of a fermented beverage, in cleaning starchy stains, or in textile desizing. 
     
     
         50 . A method of desizing a textile comprising contacting a desizing composition with a sized textile for a time sufficient to desize the textile, wherein the desizing composition comprises a variant α-amylase of any one of  claims 1 - 19 . 
     
     
         51 . An isolated polynucleotide encoding a polypeptide of any of  claims 1 - 19 . 
     
     
         52 . An expression vector comprising the polynucleotide of  claim 51 . 
     
     
         53 . A host cell comprising the expression vector of  claim 52 . 
     
     
         54 . A polypeptide according to any one of  claims 1 - 19  encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide complementary to the full-length of the polynucleotide of SEQ ID NO: 7, SEQ ID NO: 33, or SEQ ID NO: 38.