Enzyme variants and polynucleotides encoding same
US-11407986-B2 · Aug 9, 2022 · US
US11859221B2 · US · B2
| Field | Value |
|---|---|
| Publication number | US-11859221-B2 |
| Application number | US-202217854774-A |
| Country | US |
| Kind code | B2 |
| Filing date | Jun 30, 2022 |
| Priority date | Dec 30, 2015 |
| Publication date | Jan 2, 2024 |
| Grant date | Jan 2, 2024 |
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The present invention relates to polypeptide having alpha-amylase activity. The present invention also relates to polynucleotides encoding the polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the polypeptides.
Opening claim text (preview).
The invention claimed is: 1. A polypeptide comprising a variant amino acid sequence of SEQ ID NO: 1, wherein the polypeptide has alpha-amylase activity, has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, comprises a mutation corresponding to position 339 wherein numbering is according to the amino acid sequence set forth in SEQ ID NO: 2, and exhibits an enhanced wash performance compared to the polypeptide of SEQ ID NO: 1. 2. A polypeptide according to claim 1 wherein the enhanced wash performance is assessed using an Automatic Mechanical Stress Assay (AMSA) assay. 3. A polypeptide according to claim 1 wherein the enhanced wash performance is exhibited during high temperature washes. 4. A polypeptide according to claim 3 , wherein said high temperature is at least 40° C. 5. A polypeptide according to claim 2 , wherein said polypeptide exhibits an enhanced wash performance in one or more of the conditions selected from the group consisting of: (a) Model Automatic Dish Wash (ADW) detergent with bleach at 40° C. and a wash cycle of 10 min; (b) Model ADW detergent without bleach at 40° C. and a wash cycle of 10 min; (c) Model ADW detergent with bleach at 50° C. and a wash cycle of 20 min; and (d) Model ADW detergent without bleach at 50° C. and a wash cycle of 20 min; wherein said model ADW detergent comprises trisodium salt of methylglycinediacetic acid, sodium citrate, sodium carbonate, sodium silicate, sodium sulphate, polyphosphate and silicate scale inhibitor. 6. A polypeptide according to claim 1 further comprising a mutation at one or more positions corresponding to positions 10, 25, 30, 37, 40, 48, 51, 54, 64, 81, 86, 93, 98, 105, 108, 109, 113, 116, 118, 121, 130, 135, 138, 142, 167, 174, 175, 178, 182, 186, 187, 189, 195, 198, 202, 203, 206, 208, 210, 214, 218, 235, 238, 242, 243, 246, 247, 250, 255, 257, 259, 260, 261, 265, 267, 269, 270, 274, 275, 276, 281, 295, 298, 299, 311, 319, 320, 334, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474, and 482, wherein numbering is according to the amino acid sequence set forth in SEQ ID NO: 2. 7. A polypeptide according to claim 1 , wherein said mutation is a substitution, deletion, or insertion. 8. A polypeptide according to claim 1 , wherein said polypeptide further comprises one or more of the following substitutions relative to the amino acid sequence of SEQ ID NO: 2; M10L, N25K, D30N, K37H, K37L, K37M, K37R, K37V, S40T, W48F, A51Q, A51T, N54S, Y64W, T81S, Q86H, Q86I, Q86L, K93H, K93R, Q98R, M105Y, M105F, M105I, M105L, K108R, G109A, G109M, A113E, M116I, M116L, M116A, M116V, M116F, K118Q, K118H, K118N, K118R, E121H, E130H, E130Q, Y135H, E138Q, K142R, K142Q, W167Y, W167H, N174Q, N174*, N175Q, Y178W, T182G, A186D, A186G, W187Y, W189H, F195N, Y198F, L202M, Y203H, Y203N, Y203G, Y203F, I206L, M208F, M208L, M208V, H210N, V214R, V214T, V214I, R218N, I235V, I235L, I235M, V238T, V238A, K242P, Y243F, Y243M, T246V, T246I, T246L, T246M, R247K, I250L, I250V, S255K, I257A, K259N, N260D, M261L, M261A, A265G, F267Y, K269S, K269N, N270G, A274K, I275L, E276Q, K281H, F295Y, F295W, Y298W, Y298F, Y299W, Y299F, Q311T, Q311H, Q311R, Q319H, Q319R, K320H, K320R, S334T, E360F, S365M, S365C, V366I, K383Q, K383R, S384E, K385H, K385Q, K385R, Q394K, Y398W, N402Y, Y404W, E416L, A434D, G460E, W469F, V474C, and W482Y, wherein numbering is according to the amino acid sequence set forth in SEQ ID NO: 2. 9. A polypeptide according to claim 1 , wherein said polypeptide further comprises mutations selected from the group consisting of: K37H+L202M, E360F+S365C, M261L, H210N, Y243F, K108R, V474C, G460E, T81S, K269S+N270G+A274K, K37V+L202M, L202M+Q311R, N174Q+L202M, K385H, K385Q, K385R, K383Q, K320H, K320R, E276Q, K93R, K93H, Q98R, K118Q, K118H, K118N, E130H, E130Q, E138Q, K142R, K142Q, E416L, Q394K, S384E, Y64W, K37R, D30N, F295Y, Y243M, Y178W, K281H, K269N, Y198F, Q311T, F195N, I257A, S255K, R247K, Y404W, Y398W, Q319H, Q319R, Q311H, Q311R, Y299W, N260D, K259N, Y299F, A434D, V366I, S365M, N174*, I235V, I250L, I250V, K37V, K37M, K37L, Y203H, S40T, W187Y, V238T, M105Y, M105F, M105I, I206L, T182G, M116I, Y135H, Y203N, Y203G, N25K, M116L, A265G, F295Y, A265G, K242P, V214R, M208F, Y198F, W482Y, V214T, V214I, M261A, M105F, M208L, M116A, N174Q, N174*+N175Q, I235L, A265G, M105L, K37H, Q311R, W469F, Y203F, G109A, N175Q, W48F, M116V, M116F, F295W, Y298W, M208V, M208F, M10L+M261L, W187Y+M208L, Y298F, W167Y, W167H, W189H, F295Y, I235M, Y243F+F267Y, K37V+P45R+K383R, D30N+N33D+K37V+K383R, W167Y+H210N+V366I, W167Y+H210N W167Y+L202M+H210N+Y299F+V366I, W167Y+H210N+Y243F+V366I, W167Y+H210N+V366I+W482Y, M116F+W167Y+H210N+V366I, W48F+W167Y+H210N+V366I, W167Y+H210N+Y299F+V366I, W167Y+L202M+H210N+V366I, W167Y+L202M+H210N+Y299F, W167Y+H210N+W482Y, M116F+W167Y+H210N, W48F+W167Y+H210N, W167Y+H210N+Y299F, W167Y+L202M+H210N, W167Y+L202M+Y299F, W167Y+Y243F, M116F+W167Y, W48F+W167Y, W167Y+Y299F, W167Y+L202M, W167Y+H210N+V366I, W167Y+V366I, H210N+V366I, D30N+N33D+K37V+L202M+K383R, D30N+N33D+K37V+W48F+K383R, D30N+N33D+K37V+M116F+K383R, D30N+N33D+K37V+K383R+W482Y, A51T, A51T+N54S, S334T, T246M, T246L, T246V, T246I, A186G, A51Q+G109M+Y203G, V238A+S334T, A51T+L202M, L202M+T246L, A51T+N174Q+L202M+T246I+S334T, A51T+N174Q+L202M+Q311R+S334T, I235L+T246M+I250L, A186D+L202M, A186D+L202M+N270G+N402Y, A186D+F195N+L202M, K37H+A51T+L202M, K37V+, A51T+L202M, A51T+L202M+S365C, A51T+L202M+Q311T, A51T+L202M+M261L, A51T+L202M+H210N, A51T+L202M+N270G, A51T+F195N+L202M, A51T+L202M+Q319H, A51T+L202M+Q319R, A51T+L202M+Q311H, A51T+L202M+R247K, A51T+L202M+Q311R, A51T+L202M+Y398W, A51T+L202M+Y299W, A51T+K108R+L202M, A51T+L202M+Y243F, A51T+L202M+V474C, A51T+L202M+G460E, N174Q+L202M+A265G+Q311R+S334T, A51T+L202M+T246V+A265G+Q311R, A51T+L202M+A265G+Q311R, K37H+L202M+T246V+S334T, L202M+T246V+S334T+E416L, L202M+T246V+S334T+N402Y, L202M+T246V+S334T+V366I, L202M+T246V+S334T+S365M, L202M+T246V+S334T+S365C, L202M+T246V+M261L+S334T, D30N+L202M+H210N+T246V+S334T, L202M+T246V+N270G+S334T, F195N+L202M+T246V+S334T, L202M+T246V+Q319H+S334T, L202M+T246V+Q319R+S334T, L202M+T246V+Q311H+S334T, L202M+T246V+Q311R+S334T, L202M+T246V+S334T+Y398W, L202M+T246V+K320H+S334T, L202M+T246V+Y299W+S334T, L202M+T246V+K320R+S334T, L202M+Y243F+S334T, L202M+T246V+S334T+V474C, L202M+T246V+S334T+G460E, L202M+I235M+T246V+S334T, K108R+L202M+T246V+S334T, A51T+A186D+L202M+N270G+N402Y, A51T+A186D+L202M+N270G, A51T+L202M+T246V+N270G, K37H+A51T+L202M+N270G, A51T+L202M+T246L+N270G, A51T+N174Q+L202M+N270G, A51T+L202M+V238A+N270G, A51T+L202M+A265G, A51T+L202M+M261L+N270G, A51T+L202M+F267Y, A51T+L202M+I275L, A51T+L202M+N270G+S365M, A51T+L202M+N270G+S365C, A51T+L202M+N270G+Q311T, A51T+L202M+N270G+E416L, A51T+L202M+N270G+N402Y, A51T+L202M+N270G+S365C, A51T+L202M+N270G+K383R, A51T+L202M+N270G+V474C, A51T+L202M+N270G+G460E, A51T+Q86L+L202M+N270G, A51T+Q86I+L202M+N270G, A51T+A113E+L202M+N270G, A51T+K93H+L202M+N270G, A51T+K108R+L202M+N270G, A51T+L202M+K269N, A51T+L202M+Y243F+N270G, A51T+F195N+L202M+N270G, A51T+L202M+R247K+N270G, A51T+L202M+R218N+N270G, A51T+L202M+S255K+N270G, A51T+L202M+I257A+N270G, A51T+L202M+V214I+R218N+N270G, A51T+L202M+N270G+Q311H, A51T+L202M+N270G+K320H, A51T+L202M+N270G+Y299W, A51T+L202M+N270G+K320R, A51T+L202M+N270G+K383Q, A51T+K142R+L202M+N270G, A51T+E130Q+L202M+N270G, A51T+K118N+L202M+N270G, A51T+E138Q+L202M+N270G, A51T+K118H+L202M+N270G, A51T+K118Q+L202M+N270G, A51T+Q86H+L202M+N270G, A51T+E121H+L202M+N270G, A51T+K118R+L202M+N270G, K37H+A51T+N174Q+L202M+A265G+Q311R+S334T, A51T+N174Q+L202M+T246I+Q311R+S334T, K37V+A51T+L202M+A265G+F267Y+Q311R+S334T, A51T+N174Q+L202M+A265G+F267Y+Q311R+S334T, A51G+L202M+Q311R+S334T, L202M+T246I+A265G+F267Y+Q311R+S334T, A51T+L202M+F267Y+Q311R+S334T+S365L, A51T+L202M+S365C, A51T+K108R+L202M, A51T+S365C, A51T+K108R, L202M+V238A+S334T,
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