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.

1 . A recombinant variant of a parent α-amylase comprising:
 a mutation at an amino acid residue corresponding to E187 or S241; and 
 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; 
 wherein the variant α-amylase or the parent α-amylase has at least 60% amino acid sequence identity relative to SEQ ID NO: 1, which is used for numbering; and 
 wherein the variant has increased thermostability, detergent stability, starch liquifaction activity, and/or cleaning performance compared to the parent α-amylase or a reference α-amylase differing from the variant α-amylase only by the absence of the mutations. 
 
     
     
         2 . The variant α-amylase of  claim 1 , comprising at least two mutations at amino acid residues corresponding N126, Y150, F153, L171, and, I203, using SEQ ID NO: 1 for numbering. 
     
     
         3 . The variant α-amylase of  claim 1 , 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. 
     
     
         4 . The variant α-amylase of  claim 1 , further comprising deletions of amino acid residues corresponding to R178 and G179, or T180 and G181. 
     
     
         5 . The variant α-amylase of  claim 1 , further comprising a mutation at an amino acid residue corresponding to G476 and/or G477, using SEQ ID NO: 1 for numbering. 
     
     
         6 . The variant α-amylase of  claim 1 , 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  claim 1 , 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  claim 1 , 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  claim 1 , 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  claim 1 , 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  claim 1 , 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  claim 1 , comprising a combinations of mutations corresponding to mutations selected from the group consisting of:
 E187P+I203Y+G476K, 
 E187P+I203Y+G476K+R458N+T459S+D460T, 
 T180D+E187P+I203Y+G476K, 
 N126Y+T180D+E187P+I203Y+G476K, 
 N126Y+T180D+E187P+I203Y+Y303D+G476T+G477E, 
 N126Y+T180D+E187P+I203Y+Y303D+N475E+G477Q, 
 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, 
 N126Y+E187P+G476K, 
 N126Y+F153W+E187P+G476K, 
 N126Y+F153W+E187P+G4726+G477R, 
 N126Y+E187P+I203Y, 
 N126Y+I203Y+S241Q, 
 N126Y+T180H+E187P+I203Y, 
 N126Y+T180H+I203Y+S241Q, 
 N126Y+F153W+T180H+E187P+I203Y, 
 N126Y+F153W+T180H+I203Y+S241Q, 
 N126Y+Y150H+F153W+L171N+E187P+I203Y, 
 N126Y+Y150H+F153W+L171N+I203Y+S241Q, 
 N126Y+Y150H+F153W+L171N+T180H+E187P+I203Y, 
 N126Y+Y150H+F153W+L171N+T180H+I203Y+S241Q, and 
 N126Y+F153W+T180D+I203Y+S241Q; 
 wherein the variant has increased thermostability, detergent stability, stability starch liquifaction activity, or cleaning performance compared to the parent; 
 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  claim 1 , comprising the combinations of mutations corresponding to N126Y+F153W+T180D+I203Y+S241Q and one or more mutations corresponding to mutations selected from the group consisting of E132H, Q167E, A277F, and T400K. 
     
     
         14 . The variant amylase of  claim 13 , comprising the combinations of mutations corresponding to mutations selected from the group consisting of:
 N126Y+E132H+F153W+T180D+I203Y+S241Q+A277F,   N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F, and   N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F+T400K.   
     
     
         15 . The variant amylase of  claim 1 , wherein the parental α-amylase is from a  Cytophaga  species. 
     
     
         16 . The variant amylase of  claim 1 , wherein the parental α-amylase is from a  Paenibacillus  species. 
     
     
         17 . The variant amylase of  claim 1 , wherein the parental α-amylase is not from a  Bacillus  species. 
     
     
         18 . The variant amylase of  claim 1 , 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. 
     
     
         19 . The variant amylase of  claim 1 , 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. 
     
     
         20 . The variant amylase of  claim 1 , 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. 
     
     
         21 . The variant amylase of  claim 1 , 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. 
     
     
         22 . A composition comprising the variant α-amylase of any of the preceding claims. 
     
     
         23 . The composition of  claim 22 , wherein the composition is effective for removing starchy stains from laundry, dishes, or textiles. 
     
     
         24 . The composition of  claim 22 , further comprising a surfactant. 
     
     
         25 . The composition of  claim 22 , wherein the composition is a detergent composition. 
     
     
         26 . The composition of  claim 22 , wherein the composition is a laundry detergent or a laundry detergent additive. 
     
     
         27 . The composition of  claim 22 , wherein the composition is a manual or automatic dishwashing detergent. 
     
     
         28 . The composition of  claim 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  claim 1 . 
     
     
         29 . The composition of  claim 21 , wherein the composition is for liquifying starch. 
     
     
         30 . The composition of  claim 21 , wherein the composition is for saccharifying a composition comprising starch, for SSF post liquefaction, or for direct SSF without prior liquefaction. 
     
     
         31 . The composition of  claim 21 , wherein the composition is for producing a fermented beverage. 
     
     
         32 . The composition of  claim 21 , wherein the composition is for producing a baked food product. 
     
     
         33 . The composition of  claim 21 , wherein the composition is for textile desizing. 
     
     
         34 - 56 . (canceled)