Enzyme variants and polynucleotides encoding same

Abstract

Enzyme variants and polynucleotides encoding them are provided. The present invention relates to polypeptides having alpha-amylase activity. The invention also relates to polynucleotides encoding the polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the polypeptides.

Description

[0001] This application is a divisional application based on a patent application with an application date of December 29, 2016, a priority date of December 30, 2015, an application number of 202210558733.1, and an invention name of “Enzyme variants and polynucleotides encoding them”.

[0002] References to sequence listings

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0004] The present invention relates to alpha-amylase variants (ie, polypeptides having alpha-amylase activity), nucleic acids encoding alpha-amylases, methods of producing alpha-amylases, compositions comprising alpha-amylases, and methods of using alpha-amylases. Background Art

[0005] α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligo- and polysaccharides.

[0006] The use of alpha-amylases in industry for several known applications has a long history, such as detergents, baking, brewing, starch liquefaction and saccharification, for example in the preparation of high fructose syrup or as part of the production of ethanol from starch. These and other applications of alpha-amylases are known and utilize alpha-amylases derived from microorganisms, particularly bacterial alpha-amylases.

[0007] Bacillus alpha-amylases such as indelible amylase, AA560 (SEQ ID NO: 2; see also WO 2000 / 060060) and SP707 (SEQ ID NO: 8; see also Tsukamoto et al., 1988, Biochem. Biophys. Res. Comm. [Biochemistry and Biophysics Research Communications] 151: 25-31) form a specific group of alpha-amylases that have been used in detergents. These amylases have been modified to improve stability in detergents. For example, WO 96 / 23873 discloses deletion mutants of alpha-amylases SP690, SP722 (SEQ ID NO: 5) and SP707 (SEQ ID NO: 8) (see also SEQ ID NOs: 1, 2 and 7 of WO 96 / 23873) to improve the stability of these amylases. WO 96 / 23873 further discloses substitution mutants to stabilize the amylases against oxidation. Further alpha-amylase mutants with improved properties are disclosed in WO 2006 / 002643 and WO 01 / 66712.

[0008] Thus, it is known to modify naturally occurring amylases to improve certain properties.

[0009] The object of the present invention is to provide polypeptides having α-amylase activity (α-amylases) having enhanced washing performance, especially at high temperatures (eg at 40° C. or higher). Summary of the invention

[0010] The present invention relates to a polypeptide having α-amylase activity corresponding to a variant of an α-amylase derived from Bacillus. In particular, the polypeptide of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 1, wherein one or more of these amino acids are mutated (e.g., by substitution or deletion) so that the polypeptide exhibits enhanced washing performance (especially at a temperature of 40° C. or higher).

[0011] The present invention also relates to polynucleotides encoding variant α-amylase polypeptides; nucleic acid constructs, vectors, and host cells comprising these polynucleotides; and methods of producing these variant α-amylase polypeptides.

[0012] The present invention also relates to detergent compositions comprising these variant α-amylase polypeptides, and the use of these detergent compositions in domestic and industrial cleaning processes such as laundry and dishwashing.

[0013] The present invention further relates to the following embodiments:

[0014] 1. A polypeptide comprising a variant amino acid sequence of SEQ ID NO: 1, wherein the polypeptide has α-amylase activity and exhibits enhanced washing performance compared to the polypeptide of SEQ ID NO: 1.

[0015] 2. The polypeptide according to embodiment 1, wherein the variant amino acid sequence comprises a mutation at amino acid position 167 of SEQ ID NO: 1.

[0016] 3. A polypeptide according to embodiment 2, wherein the variant amino acid sequence comprises a substitution at amino acid position 167 of SEQ ID NO: 1, such as W167Y.

[0017] 4. The polypeptide according to any one of the preceding embodiments, wherein the enhanced wash performance is assessed using an automated mechanical stress assay (AMSA).

[0018] 5. The polypeptide according to any one of the preceding embodiments, wherein enhanced wash performance is exhibited during high temperature washing.

[0019] 6. A polypeptide according to embodiment 5, wherein the elevated temperature is at least 40°C, such as at least 45°C, such as at least 50°C, such as at least 55°C, and such as at least 60°C.

[0020] 7. The polypeptide according to any one of embodiments 4 to 6, wherein the polypeptide exhibits enhanced wash performance under one or more conditions selected from the group consisting of:

[0021] (a) Standard ADW detergent, with bleach, at 40°C, and a wash cycle of 10 min;

[0022] (b) standard ADW detergent, no bleach, at 40 °C, and a 10 min wash cycle;

[0023] (c) standard ADW detergent, with bleach, at 50°C, and a wash cycle of 20 min; and

[0024] (d) standard ADW detergent, no bleach, at 50°C, and a 20 min wash cycle;

[0025] The standard ADW detergent comprises trisodium salt of methylglycine diacetic acid (such as Trilon M granular SG), sodium citrate, sodium carbonate, sodium silicate, sodium sulfate, polyphosphate and silicate antiscalant (such as Acusol 588G) and Surfac 23-6.5.

[0026] 8. A polypeptide according to any of the preceding embodiments, wherein the polypeptide is 1000 or fewer amino acids in length, such as 900, 800, 700, 600, 500, 400, 300, 200, 175, 150, 125, 100 or fewer amino acids in length.

[0027] 9. A polypeptide according to embodiment 8, wherein the polypeptide is between 400 and 600 amino acids in length, such as between 450 and 500, between 460 and 500, or between 470 and 490 amino acids in length.

[0028] 10. A polypeptide according to any of the preceding embodiments, comprising a mutation at one or more positions corresponding to the following 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, 460, 469, 474, and 482, wherein the numbering is based on the amino acid sequence set forth in SEQ ID NO:2.

[0029] 11. A polypeptide according to any of the preceding embodiments, wherein the mutation at one or more positions is a substitution, deletion, and / or insertion.

[0030] 12. The polypeptide according to any of the preceding embodiments, wherein the polypeptide 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, K116 18Q, 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, V2 14T, V214I, R218N, I235V, I235L, I235M, V238T, V238A, K242P, Y243F, Y243M, T246V, T246I, T246L, T246M, R247K, I250L, I250 V, S255K, I257A, K259N, N260D, M261L, M261A, A265G, F267Y, K269S, K269N, N270G, A274K, I275L, E276Q, K281H, F295Y, F295W, Y 402Y, Y404W, E416L, A434D, G460E, W469F, V474C, and W482Y, wherein the numbering is according to the amino acid sequence listed in SEQ ID NO:2.

[0031] 13. A polypeptide according to any of the preceding embodiments, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 with mutations, or a fragment thereof having alpha amylase activity, 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, E1 30H, 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, S339A, N174*, I235V, I250L, I250V, K37V, K37M, K37L, Y203H, S40T, W187Y, V238T, M105Y, M105F, M105I, I206L, T18 2G, M116I, Y135H, Y203N, Y203G, N25K, M116L, A265G, F295Y, A265G, K242 P, V214R, M208F, Y198F, W482Y, V214T, V214I, M261A, M105F, M208L, M116 A. N174Q, N174*+N175Q, I235L, A265G, M105L, K37H, Q311R, W469F, Y203F , G109A, N175Q, W48F, M116V, M116F, F295W, Y298W, M208V, M208F, M10L+M 261L, W187Y+M208L, Y298F, W167Y, W167H, W189H, F295Y, I235M, Y243F+F 267Y, K37V+P45R+K383R, D30N+N33D+K37V+K383R, W167Y+H210N+S339A+ V366I, S339A+V366I, W167Y+H210N+S339A, H210N+S339A, W167Y+H210N,<h2 style=";text-align:left;direction:ltr">W167Y+L202M+H210N+Y299F+S339A+V366I 、W167Y+H210N+Y243F+S339A+V366I 、W167Y+H210N+S339A+V366I+W482Y 、M116F+W167Y+H210N+S339A+V366I 、W48F+W167Y+H210N+S339A+V366I 、W167Y+H210N+Y299F+S339A+V366I 、W167Y+L202M+H210N+S339A+V366I 、W167Y+L202M+H210N+Y299F+S339A、W167Y+H210N+S339A+W482Y、M116F+W167Y+H210N+S339A、W48F+W167Y+H210N+S 339A、W167Y+H210N+Y299F+S339A、W167Y+L202M+H210N+S339A、W167Y+L202M+Y299F、W167Y+Y243F、M116F+W167Y、W48 F+W167Y, W167Y+Y299F, W167Y+L202M, W167Y+H210N+V366I, W167Y+V366I, H210N+V366I, W167Y+S339A+V366I, D30N+N33D+K37V+L202M+K383R, D30N+N33D+K37V+W48F+K383R, D30N+N33D+K37V+M116F+K383R, D30N+N33D+K37V+K383R+W4 82Y、A51T、A51T+N54S、S334T、T246M、T246L、T246V、T246I 、A186G、A51Q+G109M+Y203G、V238A+S334T、A51T+L202M、L2 02M+T246L、A51T+N174Q+L202M+T246I+S334T、A51T+N174Q+L202M+Q311R+S334T、I235L+T246M+I250L、A186D+L202M、 A186D+L202M+N270G+N402Y、A186D+L202M+S339A、A186D+F195N+L202M、K37H+A51T+L202M、K37V+、A51T+L202M、A51T +L202M+S365C、A51T+L202M+S339A、A51T+L202M+Q311T、A51T+L202M+M261L、A51T+L202M+H210N、A51T+L202M+N270G、<h2 style=";text-align:left;direction:ltr">A51T+F195N+L202M、A51T+L202M+Q319H、A51T+L202M+Q319R、A51T+L202M+Q311H、A51T+L202 M+R247K、A51T+L202M+Q311R、A51T+L202M+Y398W、A51T+L202M+Y299W、A51T+K108R+L202M、A 51T+L202M+Y243F、A51T+L202M+V474C、A51T+L202M+G460E、N174Q+L202M+A265G+Q311R+S33 4T、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 、A186D+L202M+N270G+S339A+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+K 108R, L202M+V238A+S334T, W48F+V238A+S334T, M116F+V238A+S334T, V238A+S334T+W482Y, Y243F+S334T , L202M+V238A+Y299F+S334T, L202M+T246V+N270G+S334T, W48F+K118H+V238A+S334T, W48F+L202M+V238A+S334T, A51T+A186N+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299F+S339A+V366I, L202M+T246V+N270G+S334T+S365C, or A186N+L202M+T246V+N270G+S334T.

[0032] 14. The polypeptide according to any one of the preceding embodiments, wherein the enhanced wash performance corresponds to an Improvement Factor (IF) of at least 1.2 when the polypeptide is evaluated in an ADW assay of a detergent comprising bleach and at 40°C for 10 min.

[0033] 15. The polypeptide of embodiment 14, wherein the polypeptide comprises a mutation at one or more positions corresponding to the following positions: 37, 51, 93, 98, 108, 118, 167, 186, 202, 210, 235, 243, 246, 247, 250, 255, 259, 260, 261, 270, 299, 311, 319, 334, 339, 365, 385, 398, and 404, wherein the numbering is according to SEQ ID NO:2.

[0034] 16. The polypeptide according to embodiment 14 or 15, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 with mutations, or a fragment thereof having alpha amylase activity, selected from the group consisting of M261L, Y243F, K385H, K385R, K93H, Q98R, K118Q, K118H, S255K, Y404W, Y398W, Q319R, Y299W, N260D, K259N, Y299F, S339A, S334 T, A51T+L202M, I235L+T246M+I250L, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M +Q311T, A51T+L202M+M261L, A51T+L202M+R247K, A51T+L202M+Y299W, A51T+K108R+L202M , A51T+L202M+Y243F, A51T+A186D+L202M+N270G, A51T+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, and W167Y+A186N+H210N+Y299F+S339A+V366I.

[0035] 17. The polypeptide according to any one of embodiments 1 to 13, wherein the enhanced wash performance corresponds to an IF of at least 1.2 when the polypeptide is evaluated in an ADW assay in a detergent comprising bleach and at 50°C for 20 min.

[0036] 18. The polypeptide of embodiment 17, wherein the polypeptide comprises a mutation at one or more positions corresponding to the following positions: 30, 33, 37, 40, 48, 51, 81, 86, 93, 105, 108, 116, 118, 130, 138, 142, 167, 174, 175, 182, 186, 195, 198, 202, 206, 2 10, 235, 238, 243, 246, 247, 250, 255, 257, 259, 260, 261, 265, 266, 267, 269, 270, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 385, 402, 416, 474, and 482, wherein the numbering is according to SEQ ID NO:2.

[0037] 19. The polypeptide according to embodiment 17 or 18, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 with mutations, or a fragment thereof having alpha amylase activity, selected from the group consisting of K37H+L202M, E360F+S365C, M261L, H210N, Y243F, K108R, V474C, T81S, K385H, K385Q, K385R, K93R, K118Q, K118N , E130H, E130Q, E138Q, K37R, D30N, Y243M, K269N, Y198F, Q311T, F195N, I257A, S25 5K, R247K, Y404W, Y398W, Q319H, Q319R, Q311H, Q311R, Y299W, N260D, K259N, Y299F , V366I, S365M, S339A, I250L, I250V, K37M, S40T, V238T, M105F, M105I, I206L, T18 2G, M116I, M116L, N174Q, I235L, A265G, K37H, Q311R, N175Q, W48F, M116F, W167Y+H 210N+S339A, H210N+S339A, W48F+W167Y+H210N+S339A, W48F+W167Y, W167Y+H210N +V366I, D30N+N33D+K37V+K383R+W482Y, A51T, S334T, T246M, T246L, T246V, T246I, A186G, V238A+S334T, A51T+L202M, A51T+N174Q+L202M+Q311R+S334T, I235L+T246M+I250L, L202M+T24 6V+S334T+E416L, D30N+L202M+H210N+T246V+S334T, L202M+T246V+N270G+S334T, F195N+L202M+T246V+S334T, L202M+T246V+K320R+S334T, L202M+Y243F+S334T , A51T+A186D+L202M+N270G+N402Y, A186D+L202M+N270G+S339A+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+N270G+S365C, A51T+L202M+N270G+S365C, A51T+L202M+N270G+K383R, A51T+Q86I+L202M+N270G, A51T+K93H+L202M+N270G, A51T+K108R+L202M+N270G, A51T+L202M+N270G+K320H, A51T+L202M+N270G+K383Q, A51T+K142R+L202M+N270G, A51T+E130Q+L202M+N270G, A51T+E138Q+L202M+N270G, A51T+L202M+S365C, A51T+S 365C, W48F+V238A+S334T, M116F+V238A+S334T, V238A+S334T+W482Y, Y243F+S334T , W48F+K118H+V238A+S334T, A51T+A186N+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299F+S339A+V366I, L202M+T246V+N270G+S334T+S365C, and A186N+L202M+T246V+N270G+S334T.

[0038] 20. The polypeptide of any one of embodiments 1 to 13, wherein said enhanced wash performance corresponds to an Improvement Factor (IF) of at least 1.2 when the polypeptide is evaluated in an ADW assay in a detergent without bleach and at 40°C for 10 min.

[0039] 21. The polypeptide of embodiment 20, wherein the polypeptide comprises a mutation at one or more positions corresponding to the following positions: 37, 48, 51, 64, 81, 108, 116, 167, 174, 186, 187, 189, 195, 198, 202, 208, 210, 235, 238, 243, 246, 250, 261, 265, 267, 269, 270, 275, 311, 319, 334, 339, 365, 366, 385, 460, and 474, wherein the numbering is according to SEQ ID NO:2.

[0040] 22. The polypeptide according to embodiment 20 or 21, wherein the polypeptide is composed of SEQ ID NO: NO:2, or a fragment thereof having alpha-amylase activity, wherein the mutations are selected from the group consisting of K37H+L202M, M261L, H210N, Y243F, K108R, G460E, T81S, N174Q+L202M, K385R, Y64W, Y243M, K269N, Y198F, S365M, S339A, W48F, M116V, M116F, W187Y+M208L, W167Y, W189H, W167Y+L202M+H210N+Y299F+S339A, W167Y+L202M+H210N+S339A, W167Y+L202M, V238A+S334T, A51T+L202M, L202M+T246L, A51T+N174Q+L202M+T246I+S334 T、I235L+T246M+I250L、A186D+F195N+L202M、K37H+A51T+L202M、K37V+A51T +L202M, A51T+L202M+S365C, A51T+L202M+Q311T, A51T+L202M+M261L, A51T +L202M+Q319R, A51T+L202M+Q311H, A51T+K108R+L202M, A51T+L202M+V474C , A51T+A186D+L202M+N270G, K37H+A51T+L202M+N270G, A51T+L202M+A265G, A51T+L202M+M261L+N270G, A51T+L202M+F267Y, A51T+L202M+I275L, A51T+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, and W167Y+A186N+H210N+Y299F+S339A+V366I.

[0041] 23. The polypeptide of any one of embodiments 1 to 13, wherein the enhanced wash performance corresponds to an IF of at least 1.2 when the polypeptide is evaluated in an ADW assay in a detergent without bleach and at 50°C for 20 min.

[0042] 24. The polypeptide of embodiment 23, wherein the polypeptide comprises a mutation at one or more positions corresponding to the following positions: 30, 33, 37, 40, 48, 51, 86, 93, 108, 109, 113, 116, 118, 121, 130, 138, 142, 167, 174, 175, 182, 186, 195, 198, 202, 203, 210, 218 , 235, 238, 243, 246, 247, 250, 255, 257, 259, 260, 261, 265, 267, 269, 270, 274, 275, 299, 311, 319, 320, 334, 339, 365, 366, 383, 385, 398, 402, 416, 404, 460, 474, and 482, wherein the numbering is according to SEQ ID NO:2.

[0043] 25. The polypeptide according to embodiment 23 or 24, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 with mutations, or a fragment thereof having alpha amylase activity, selected from the group consisting of K269S+N270G+A274K, K37V+L202M, N174Q+L202M, K385H, K385R, K93H, K118Q, E130Q, E138Q, K37R, D30N, Y243M, K269N, Y198F, F195N, I257A, S255K, R247K, Y404W, Y398W, Q319H, Q319R, Q311H, Q311R, Y299W, N260D, K259N, Y299F, V366I, S365M, S339A, N174*, K37L, S40T, T182G, A265G, W482Y, M116A, N174Q, I 235L, A265G, K37H, Q311R, N175Q, W48F, M116V, M116F, W167Y+L202M+H21 0N+S339A+V366I, W167Y+L202M+H210N+Y299F+S339A, W167Y+L202M+H2 10N+S339A, W167Y+L202M+Y299F, W48F+W167Y, W167Y+Y299F, W167Y+L20 2M, D30N+N33D+K37V+L202M+K383R, D30N+N33D+K37V+W48F+K383R, A51 T, S334T, T246M, T246L, T246I, A186G, A51Q+G109M+Y203G, V238A+S334T , A51T+L202M, L202M+T246L, A51T+N174Q+L202M+T246I+S334T, A51T+N 174Q+L202M+Q311R+S334T, I235L+T246M+I250L, A186D+L202M, A186D+L 202M+N270G+N402Y, A186D+L202M+S339A, A186D+F195N+L202M, K37H+A 51T+L202M, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M+S339A, A51T+L202M+Q311T, A51T+L202M+M261L, A51T+L202M+H210N, A51T+L20 2M+N270G, A51T+F195N+L202M, A51T+L202M+Q319H, A51T+L202M+Q319R,<h2 style=";text-align:left;direction:ltr">A51T+L202M+Q311H、A51T+L202M+R247K、A51T+L202M+Q311R、A51T+L202M+Y3 98W、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、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+S33 4T、L202M+T246V+Q311H+S334T、L202M+T246V+Q311R+S334T、L202M+T246V+S334T+Y398 W、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 、A186D+L202M+N270G+S339A+N402Y、A51T+A186D+L202M+N270G、A51T+L202M+T246V+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+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+E121H+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、L202M+T246I+A265G+F267Y+Q311R+S334T 、A51T+L202M+F267Y+Q311R+S334T+S365L, A51T+L202M+S365C, A51T+K108R+L202M, L202M+V238A+S334T, W48F+V238A+S334T, M116F+V 238A+S334T, V238A+S334T+W482Y, Y243F+S334T, L202M+V238A+Y299F+S334T, L202M+T246V+N270G+S334T, W48F+K118H+V238A+S334T, W48F+L202M+V238A+S334T, A51T+A186N+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299F+S339A+V366I, L202M+T246V+N270G+S334T+S365C, and A186N+L202M+T246V+N270G+S334T.

[0044] 26. A polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises a mutation at one or more positions corresponding to the following positions: 48, 167, 210, 299, 339, 366, wherein numbering is according to SEQ ID NO: 2.

[0045] 27. A polypeptide according to embodiment 26, wherein the polypeptide consists of the amino acid sequence of SEQ IDNO:2 having one or more mutations, or a fragment thereof having alpha amylase activity, and these mutations are selected from the group consisting of: W48F, W167Y, H210N, Y299F, S339A, and V366I.

[0046] 28. The polypeptide according to embodiment 26 or 27, wherein the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of:

[0047] (a) SEQ ID NO: 1 having mutations W167Y, H210N and S339A;

[0048] (b) SEQ ID NO: 1 having mutations W167Y, H210N, S339A and V366I;

[0049] (c) SEQ ID NO: 1 having mutations W167Y, H210N, Y299F, S339A and V366I;

[0050] (d) SEQ ID NO: 1 with mutations W48F and W167Y;

[0051] (e) SEQ ID NO: 1 having mutations W48F, W167Y, H210N and S339A;

[0052] (f) SEQ ID NO: 1 having mutations W48F, W167Y, H210N, Y299F, S339A and V366I;

[0053] (g) SEQ ID NO: 1 having mutations V238A, S334T, and W482Y;

[0054] (h) SEQ ID NO: 1 having mutations Y243F and S334T;

[0055] (i) SEQ ID NO: 1 with mutation W48F;

[0056] (j) SEQ ID NO: 1 with mutation K118H;

[0057] (k) SEQ ID NO: 1 with mutation W167Y;

[0058] (l) SEQ ID NO: 1 having mutations W48F, V238A, and S334T;

[0059] (m) SEQ ID NO: 1 having mutations I234L, T246M, and I250L;

[0060] (n) SEQ ID NO: 1 having mutation S339A; and

[0061] (o) SEQ ID NO: 1 having mutation S334T;

[0062] The numbering is based on SEQ ID NO:2.

[0063] 29. A polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1.

[0064] 30. A polypeptide according to any of the preceding embodiments, wherein the number of mutations relative to SEQ ID NO: 1 is 1-20, such as 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations.

[0065] 31. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide is selected from the group of polypeptides of Table 1.

[0066] 32. A polynucleotide encoding the polypeptide according to any one of embodiments 1 to 31.

[0067] 33. A nucleic acid construct comprising the polynucleotide according to embodiment 32.

[0068] 34. An expression vector comprising the polynucleotide according to embodiment 32.

[0069] 35. A host cell comprising the polynucleotide according to embodiment 32.

[0070] 36. A method for producing an alpha-amylase polypeptide, the method comprising:

[0071] (a) culturing the host cell according to embodiment 35 under conditions suitable for expression of the polypeptide; and

[0072] (b) recovering the polypeptide.

[0073] 37. The method of embodiment 36, wherein the polypeptide comprises a mutation at one or more positions corresponding to the following 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, 460, 469, 474, and 482, wherein the numbering is based on the amino acid sequence set forth in SEQ ID NO:2.

[0074] 38. A detergent composition comprising a polypeptide according to any one of embodiments 1 to 31 and a surfactant, or a concentrate or an additive for preparing the detergent composition.

[0075] 39. The composition according to embodiment 38, wherein the surfactant is selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants and zwitterionic surfactants.

[0076] 40. A composition according to embodiment 38 or 39, further comprising one or more additional components selected from the group consisting of oxidants, bleach activators, chelants, fillers, builders, buffers, structurants, chelants, fluorescent whitening agents, defoamers, enzymes, fragrances, anti-redeposition agents, skin conditioning agents, softeners, emulsifiers, and colorants.

[0077] 41. The composition of embodiments 38 to 40, wherein the composition is a liquid or powder laundry detergent composition.

[0078] 42. The composition of embodiments 38 to 40, wherein the composition is a liquid or powder automatic dishwashing (ADW) detergent composition.

[0079] 43. The composition of embodiments 38 to 40, wherein the composition is a liquid manual dishwashing detergent composition.

[0080] 44. Use of a variant according to any one of embodiments 1 to 31, or a composition according to any one of embodiments 38 to 43, in a domestic or industrial cleaning process.

[0081] 45. The use according to embodiment 44 for cleaning fabrics, such as laundry.

[0082] 46. ​​The use according to embodiment 44 for cleaning of ceramic, plastic or glass materials, such as dishwashing.

[0083] definition

[0084] Alpha-amylase: The term "alpha-amylase" (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligo- and polysaccharides. For the purposes of the present invention, alpha-amylase activity is determined according to the procedure described in Example 1. In one aspect, a variant of the present invention has at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the alpha-amylase activity of the mature polypeptide of SEQ ID NO: 1.

[0085] Amino Acid: As used herein, the term 'amino acid' includes the standard twenty genetically encoded amino acids and their corresponding stereoisomers in the 'd' form (compared to the natural 'l' form), ω-amino acids, other naturally occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derived amino acids. Chemical derivatives of one or more amino acids can be achieved by reaction with functional side groups. Such derived molecules include, for example, those molecules in which the free amino group has been derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a carboxyphenoxy group, a tert-butoxycarbonyl group, a chloroacetyl group, or a formyl group. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are peptides that contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine and ornithine can replace lysine. Derivatives also include peptides containing one or more additions or deletions as long as the necessary activity is maintained. Other modifications included are amidation, amino terminal acylation (e.g., acetylation or thioglycollic acid amidation), terminal carboxyl amidation (e.g., with ammonia or methylamine), and similar terminal modifications.

[0086] When an amino acid is explicitly listed, such as 'alanine' or 'Ala' or 'A', the term refers to L-alanine and D-alanine unless otherwise expressly stated. Other unconventional amino acids may also be suitable components of the polypeptides of the invention, as long as the desired functional properties are retained by the polypeptide. For the peptides shown, each encoded amino acid residue is represented by a single letter code, corresponding to the common name of the conventional amino acid, as appropriate. In one embodiment, the polypeptide of the invention comprises or consists of an l-amino acid.

[0087] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The early, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature, spliced ​​mRNA.

[0088] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0089] Control sequences: The term "control sequences" means nucleic acid sequences required for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) to the polynucleotide encoding the variant, or native or exogenous to each other. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, and transcription and translation termination signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites that facilitate the connection of control sequences to the coding region of the polynucleotide encoding the variant.

[0090] Enhanced wash performance: The term "enhanced wash performance" or "improved wash performance" means that the ability of the polypeptide of the present invention to provide a cleaning effect (e.g., stain removal) in a washing process (e.g., laundry or dishwashing) is improved compared to the parent alpha amylase of SEQ ID NO: 1. Wash performance can be determined using methods well known in the art, such as using an automatic mechanical stress assay (AMSA) or automatic dishwashing (ADW) (see Examples 2 and 3). Those skilled in the art will appreciate that enhanced wash performance can be achieved only under some or possibly all washing conditions (e.g., at a washing temperature of 40°C or higher (e.g., at 40°C and / or at 50°C), and / or with or without the presence of bleach).

[0091] Expression: The term "expression" includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0092] Expression vector: The term "expression vector" means a linear or circular DNA molecule that includes a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.

[0093] Fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids that are not present at the amino and / or carboxyl terminus of the mature polypeptide of SEQ ID NO: 1; wherein the fragment has α-amylase activity. In one aspect, the fragment comprises at least 200 consecutive amino acid residues of SEQ ID NO: 1, such as at least 300 consecutive amino acid residues, or at least 350 consecutive amino acid residues, or at least 400 consecutive amino acid residues, or at least 450 consecutive amino acid residues of SEQ ID NO: 1.

[0094] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0095] Improvement factor: The term "improvement factor" means a quantitative way of calculating the improvement of a specific property of a variant according to the invention. The improvement factor can be determined according to the following formula:

[0096]

[0097] Other formulas may be used to determine the improvement factor. Those skilled in the art are aware of the presently proposed formula and alternative methods of calculating the improvement factor.

[0098] According to the present invention, a value of 1.0 corresponds to the specific activity observed for the parent α-amylase. Values ​​above 1.0 indicate an improvement in the specific activity of the tested variant compared to the parent α-amylase. Thus, > Any value of 1.0 indicates an improvement in a property (such as specific activity) of the variant compared to the parent alpha-amylase. A value of 1.0 indicates that the property of the variant is at least equivalent to that of the parent alpha-amylase.

[0099] Isolated: The term "isolated" means a substance in a form or environment that does not exist in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including but not limited to any enzyme, variant, nucleic acid, protein, peptide or cofactor that is at least partially removed from one or more or all of the naturally occurring components with which it is essentially associated; (3) any substance that has been artificially modified relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). The isolated substance may be present in a fermentation broth sample.

[0100] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is well known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0101] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having alpha-amylase activity.

[0102] Mutation: In the context of the polypeptides of the present invention, the term "mutation" means that one or more amino acids in a reference amino acid sequence (i.e., SEQ ID NO: 1) are changed by substitution with a different amino acid or by deletion. In addition, a mutation may correspond to the insertion of one or more additional amino acids in a reference amino acid sequence.

[0103] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or is modified to contain a segment of nucleic acid in a manner that does not exist in nature, or is synthetic, and includes one or more control sequences.

[0104] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0105] Parent or parent alpha-amylase: The term "parent" or "parent alpha-amylase" means the alpha-amylase of SEQ ID NO: 1 (available from Novozymes A / S under the trade name " Plus"; see WO2006 / 002643).

[0106] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0107] For purposes of the present invention, use the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [J.Molecular Biology] 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Genetics Trend] 16:276-277) (preferred 5.0.0 version or updated version) to determine the sequence identity between two amino acid sequences. The parameter used can be a gap open penalty of 10, a gap extension penalty of 0.5 and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the Needle labeled "longest consistency" (using-non-simplified (-nobrief) option to obtain) is used as a percentage consistency and is calculated as follows:

[0108] (identical residues x 100) / (alignment length - total number of gaps in the alignment)

[0109] Alternatively, the parameters used can be a gap open penalty of 10, a gap extension penalty of 0.5 and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0110] (number of identical deoxyribonucleotides x 100) / (alignment length - total number of gaps in the alignment)

[0111] Subsequence: The term "subsequence" means a polynucleotide having one or more (eg, several) nucleotides deleted from the 5' end and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having α-amylase activity.

[0112] Variant: The term "variant" means a polypeptide having alpha-amylase activity comprising a mutation (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to the position of the "parent" alpha-amylase of SEQ ID NO: 1. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. The variant of the present invention has at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the alpha-amylase activity of the mature polypeptide of SEQ ID NO: 1.

[0113] Wild-type α-amylase: The term "wild-type α-amylase" means an α-amylase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.

[0114] Variant naming conventions

[0115] The polypeptide of the present invention having alpha-amylase activity corresponds to a variant of an alpha-amylase derived from Bacillus (as shown in SEQ ID NO: 1) (which is marketed by Novozymes A / S under the trade name Stainzyme Plus TM down sales).

[0116]

[0117] Variants (ie mutated amino acids) in the polypeptides of the invention are defined by reference to the amino acid numbering of SEQ ID NO: 2 (which corresponds to the mature protein AA560 of Bacillus subtilis). The amino acid sequence differences relative to SEQ ID NO: 1 are shown in bold and underlined below.

[0118]

[0119]

[0120] Accordingly, amino acid positions 1 to 182 in SEQ ID NO:2 correspond to the same numbers in SEQ ID NO:1, amino acid positions 183 to 184 of SEQ ID NO:2 are not present in SEQ ID NO:1, and amino acid positions 185 to 485 of SEQ ID NO:2 correspond to positions 183 to 483 of SEQ ID NO:1, respectively.

[0121] Therefore, for the purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another alpha-amylase polypeptide. The amino acid sequence of another alpha-amylase is aligned with the mature polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Genetics Trends] 16: 276-277) (preferably version 5.0.0 or later) is used to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0122] The identification of the corresponding amino acid residue in another alpha-amylase can be determined by aligning multiple polypeptide sequences using several computer programs using their corresponding default parameters, including but not limited to MUSCLE (Multiple Sequence Comparison by Logarithmic Expectations; Version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (Version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 23:372-374. Biology [Molecular Biology Methods] 537:_39-64; Katoh and Toh, 2010, Bioinformatics [Bioinformatics] 26:_1899-1900), and EMBOSS EMMA using ClustalW (version 1.83 or later; Thompson et al., 1994, Nucleic Acids Research [Nucleic Acids Research] 22:4673-4680).

[0123] When other α-amylases deviate from the mature polypeptide of SEQ ID NO:2 so that traditional sequence-based comparison methods cannot detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. [Journal of Molecular Biology] 295: 613-615), other pairwise sequence comparison algorithms can be applied. Higher sensitivity in sequence-based searches can be obtained using search programs that use probabilistic representations (profiles) of polypeptide families to search databases. For example, the PSI-BLAST program generates multiple profiles through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. [Nucleic Acids Research] 25: 3389-3402). Even higher sensitivity can be achieved if the family or superfamily of polypeptides has one or more representatives in the protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure predictions, structural alignment spectra, and solvation potential) as input to a neural network that predicts the structural fold of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments, in turn, can be used to generate homology models for polypeptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.

[0124] For proteins of known structure, several tools and resources can be used to retrieve and generate structural alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and those alignments are accessible and downloadable. Multiple algorithms such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747) can be used to align two or more protein structures, and the implementation of these algorithms can be used in addition to query structural databases with structures of interest to find possible structural homologues (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0125] In describing the alpha-amylase variants of the present invention, the nomenclature described below is adapted for ease of reference. Accepted IUPAC single-letter and three-letter amino acid abbreviations are employed.

[0126] replace: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of threonine at position 226 by alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" indicates that glycine (G) and serine (S) at position 205 and position 411 are replaced by arginine (R) and phenylalanine (F), respectively.

[0127] Missing: For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, the deletion of glycine at position 195 is represented as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*".

[0128] insert: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0129] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position number of the amino acid residue preceding the inserted amino acid residue. In the above example, the sequence would therefore be:

[0130] <![CDATA[ Parents: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G GKA

[0131] Various changes: Variants containing multiple alterations are separated by plus signs ("+"), for example "Arg170Tyr+Gly195Glu" or "R170Y+G195E" indicate that the arginine and glycine at positions 170 and 195 are substituted with tyrosine and glutamic acid, respectively.

[0132] Different changes:Where different changes can be introduced at a certain position, the different changes are separated by commas, for example "Arg170Tyr,Glu" means that the arginine at position 170 is replaced by tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" means the following variants:

[0133] "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly" and "Tyr167Ala+Arg170Ala". DETAILED DESCRIPTION

[0134] Peptides with α-amylase activity

[0135] The present invention relates to variant α-amylase polypeptides comprising mutations at one or more (e.g., several) positions within the amino acid sequence of the mature polypeptide of SEQ ID NO: 1, wherein the variant exhibits enhanced wash performance compared to the polypeptide of SEQ ID NO: 1.

[0136] In one embodiment, the present invention relates to a polypeptide comprising a variant amino acid sequence of SEQ ID NO: 1, wherein the polypeptide has alpha-amylase activity and exhibits enhanced wash performance compared to the polypeptide of SEQ ID NO: 1.

[0137] In one embodiment, the variant amino acid sequence comprises a mutation at amino acid position 167 of SEQ ID NO:1.

[0138] Thus, in one embodiment, the variant amino acid sequence comprises a substitution at amino acid position 167 of SEQ ID NO: 1, such as W167Y.

[0139] The amino acid sequence of the parent α-amylase is shown below:

[0140]

[0141] The polypeptides of the present invention represent variants of the parent alpha amylase of SEQ ID NO: 1, which exhibit enhanced washing performance in household and / or industrial cleaning processes (such as laundry and dishwashing). Washing performance can be determined using methods well known in the art, such as using automatic dishwashing (ADW) (see Example 2) or automatic mechanical stress determination (AMSA) (see Example 3).

[0142] In one embodiment the polypeptide exhibits enhanced wash performance in a high temperature wash process, for example wherein the temperature is at least 40°C, such as at least 45°C, such as at least 50°C, such as at least 55°C, and such as at least 60°C.

[0143] In one embodiment, the enhanced wash performance is evaluated in an automatic dishwashing (ADW) assay using starch-stained melamine tiles. Exemplary wash conditions for determining such enhanced wash performance of the polypeptides of the present invention include:

[0144] a. Standard ADW detergent, with bleach, at 40°C, and a wash cycle of 10 min;

[0145] b. Standard ADW detergent, no bleach, at 40°C, and a 10 min wash cycle;

[0146] c. Standard ADW detergent, with bleach, at 50°C, and a 20 min wash cycle; and

[0147] d. Standard ADW detergent, no bleach, at 50°C, and a 20 min wash cycle;

[0148] The standard ADW detergent comprises trisodium salt of methylglycine diacetic acid (such as Trilon M granular SG), sodium citrate, sodium carbonate, sodium silicate, sodium sulfate, polyphosphate and silicate antiscalant (such as Acusol 588G) and Surfac 23-6.5.

[0149] In addition to enhanced wash performance, those skilled in the art will appreciate that the polypeptides of the present invention may also exhibit improvements in one or more of the following properties relative to the parent alpha amylase of SEQ ID NO: 1:

[0150] (i) Substrate specificity;

[0151] (ii) substrate binding;

[0152] (iii) specific activity;

[0153] (iv) Thermal stability

[0154] (v) pH stability curve;

[0155] (vi)Ca 2+ Dependency;

[0156] (vii) oxidative stability;

[0157] (viii) increased / decreased pi; and / or

[0158] (ix) Sensitivity to surfactants.

[0159] Assays for determining the above properties of polypeptides are described in WO 2006 / 002643, WO 2001 / 066712 and EP 2 264 460 A.

[0160] The polypeptides of the invention may be longer or shorter than the parent alpha-amylase of SEQ ID NO: 1. Thus, the length of the polypeptide may be 1000 or fewer amino acids, such as 900, 800, 700, 600, 500, 400, 300, 200, 175, 150, 125, 100 or fewer amino acids. In one embodiment, the length of the polypeptide is between 400 and 600 amino acids, such as between 450 and 500, between 460 and 500, or between 470 and 490 amino acids in length.

[0161] Variant polypeptides of the present invention comprise mutations (ie, substitutions, insertions, and / or deletions) at one or more amino acid positions relative to the "parent" alpha-amylase of SEQ ID NO:1.

[0162] These amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine stretch, an antigenic epitope, or a binding domain).

[0163] Examples of conservative substitutions are within the group consisting of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0164] Alternatively, these amino acid changes have such properties that the physicochemical properties of the polypeptide are altered. For example, amino acid changes can increase the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0165] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for α-amylase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from alignments with related polypeptides.

[0166] In an alternative or additional embodiment, the polypeptide of the invention is a variant of the alpha-amylase of SEQ ID NO: 1 having a mutation (e.g., substitution, deletion, and / or insertion) at one or more positions corresponding to the following 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, 460, 469, 474, and 482, wherein the numbering is based on the amino acid sequence set forth in SEQ ID NO:2.

[0167] Thus, relative to the amino acid sequence of SEQ ID NO: 2, the polypeptide may comprise one or more of the following substitutions: 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, M 116F, K118Q, K118H, K118N, K118R, E121H, E130H, E130Q, Y135H, E138Q, K142R, K142Q, W167Y, W167H, N174Q, N174*, N175Q, Y17 8W, 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, I 250L, I250V, S255K, I257A, K259N, N260D, M261L, M261A, A265G, F267Y, K269S, K269N, N270G, A274K, I275L, E276Q, K281H, F29 5Y, F295W, Y298W, Y298F, Y299W, Y299F, Q311T, Q311H, Q311R, Q319H, Q319R, K320H, K320R, S334T, S339A, E360F, S365M, S365C, V366I, K383Q, K383R, S384E, K385H, K385Q, K385R, Q394K, Y398W, N402Y, Y404W, E416L, A434D, G460E, W469F, V474C, and W482Y.

[0168] For example, the polypeptide of the present invention can be selected from the group consisting of: a polypeptide having the amino acid sequence of SEQ ID NO: 1 with the following mutations; E360F+S365C (i.e., having a polypeptide based on SEQ ID NO: NO:1, but with mutations E360F and S365C), K37H+L202M, 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, Y40 4W, Y398W, Q319H, Q319R, Q311H, Q311R, Y299W, N260D, K259N, Y299F, A 434D, V366I, S365M, S339A, N174*, I235V, I250L, I250V, K37V, K37M, K 37L, 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, Y 203F, G109A, N175Q, W48F, M116V, M116F, F295W, Y298W, M208V, M208F, M10L+M261L, W187Y+M208L, Y298F, W167Y, W167H, W189H, F295Y, I235 M、Y243F+F267Y、K37V+P45R+K383R、D30N+N33D+K37V+K383R、W167Y+H210N+S339A+V366I、S339A+V366I、W167Y+H210N+S339A、H210N+S339A ,<h2 style=";text-align:left;direction:ltr">W167Y+H210N, W167Y+L202M+H210N+Y299F+S339A+V366I, 、M116F+W167Y+H210N+S339A+V366I 、W48F+W167Y+H210N+S339A+V366I 、W167Y+H210N+Y299F+S339A+V366I 、W167Y+L202M+H210N+S339A+V366I 、W167Y+L202M+H210N+Y299F+S339A、W167Y+H210N+S339A+W482Y、M116F+W167Y+H210N+S339A、W48F+W167Y+H210N +S339A、W167Y+H210N+Y299F+S339A、W167Y+L202M+H210N+S339A、W167Y+L202M+Y299F、W167Y+Y243F、M116F+W167 Y, W48F+W167Y, W167Y+Y299F, W167Y+L202M, W167Y+H210N+V366I, W167Y+V366I, H210N+V366I, W167Y+S339A+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、T 246V、T246I、A186G、A51Q+G109M+Y203G、V238A+S334T、A5 1T+L202M、L202M+T246L、A51T+N174Q+L202M+T246I+S334T、A51T+N174Q+L202M+Q311R+S334T、I235L+T246M+I250L 、A186D+L202M、A186D+L202M+N270G+N402Y、A186D+L202M+S339A、A186D+F195N+L202M、K37H+A51T+L202M、K37V+、 A51T+L202M、A51T+L202M+S365C、A51T+L202M+S339A、A51T+L202M+Q311T、A51T+L202M+M261L、A51T+L202M+H210N、<h2 style=";text-align:left;direction:ltr">A51T+L202M+N270G、A51T+F195N+L202M、A51T+L202M+Q319H、A51T+L202M+Q319R、A51T+L202M+Q3 11H、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 、A186D+L202M+N270G+S339A+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+K 108R, L202M+V238A+S334T, W48F+V238A+S334T, M116F+V238A+S334T, V238A+S334T+W482Y, Y243F+S334T, L202M+V238A+Y299F+S334T, L202M+T246V+N270G+S334T, W48F+K118H+V238A+S334T, W48F+L202M+V238A+S334T, A51T+A186N+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I , W48F+W167Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299F+S339A+V366I, L202M+T246V+N270G+S334T+S365C , A186N+L202M+T246V+N270G+S334T, and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is based on the amino acid sequence listed in SEQ ID NO: 2. ,

[0169] In one embodiment, the polypeptide exhibits enhanced wash performance corresponding to an improvement factor (IF) of at least 1.2 when the polypeptide is evaluated in an ADW assay of a detergent containing bleach and at 40°C for 10 min (e.g., see Example 2). A suitable polypeptide may be a variant of SEQ ID NO: 1 comprising a mutation at one or more positions corresponding to the following positions: 37, 51, 93, 98, 108, 118, 167, 186, 202, 210, 235, 243, 246, 247, 250, 255, 259, 260, 261, 270, 299, 311, 319, 334, 339, 365, 385, 398, and 404, wherein the numbering of the amino acid positions is according to the amino acid sequence listed in SEQ ID NO: 2. For example, the polypeptide may be selected from the group consisting of: a SEQ ID NO: 1 having the following mutations: Peptide of amino acid sequence of NO:1: K37H+L202M, M261L, Y243F, K385H, K385R, K93H, Q98R, K118Q, K118H, S255K, Y404W, Y398W, Q319R, Y299W, N260D, K259N, Y299F, S339A, S334T, A51T+L2 02M, I235L+T246M+I250L, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M+Q31 1T, A51T+L202M+M261L, A51T+L202M+R247K, A51T+L202M+Y299W, A51T+K108R+L202M , A51T+L202M+Y243F, A51T+A186D+L202M+N270G, A51T+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, or W167Y+A186N+H210N+Y299F+S339A+V366, and fragments thereof having alpha-amylase activity, wherein the numbering of amino acid positions is according to the amino acid sequence listed in SEQ ID NO:2.

[0170] In another embodiment, the polypeptide exhibits enhanced wash performance corresponding to an IF of at least 1.2 when the polypeptide is evaluated in an ADW assay in a detergent containing bleach and at 50°C for 20 min (e.g., see Example 2). A suitable polypeptide may be a variant of SEQ ID NO: 1 comprising a mutation at one or more positions corresponding to the following positions: 30, 33, 37, 40, 48, 51, 81, 86, 93, 105, 108, 116, 118, 130, 138, 142, 167, 174, 175, 182, 186, 195, 198, 202, 206, 210, 235, 238 , 243, 246, 247, 250, 255, 257, 259, 260, 261, 265, 266, 267, 269, 270, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 385, 402, 416, 474, and 482, wherein the numbering of amino acid positions is based on the amino acid sequence listed in SEQ ID NO: 2. For example, the polypeptide can be selected from the group consisting of: SEQ ID NO: 2 having the following mutations: NO:1 amino acid sequence polypeptide: K37H+L202M, E360F+S365C, M261L, H210N, Y243F, K108R, V474C, T81S, K385H, K385Q, K385R, K93R, K118Q, K118N, E130H, E130Q, E138Q, K37 R, D30N, Y243M, K269N, Y198F, Q311T, F195N, I257A, S255K, R247K, Y404W, Y39 8W, Q319H, Q319R, Q311H, Q311R, Y299W, N260D, K259N, Y299F, V366I, S365M, S3 39A, I250L, I250V, K37M, S40T, V238T, M105F, M105I, I206L, T182G, M116I, M1 16L, N174Q, I235L, A265G, K37H, Q311R, N175Q, W48F, M116F, W167Y+H210N+S33 9A, H210N+S339A, W48F+W167Y+H210N+S339A, W48F+W167Y, W167Y+H210N+V36 6I, D30N+N33D+K37V+K383R+W482Y, A51T, S334T, T246M, T246L, T246V, T246I, A186G, V238A+S334T, A51T+L202M, A51T+N174Q+L202M+Q311R+S334T,I235L+T246M+I250L、L202M+T246V+S334T+E416L、D30N+L202M+H210N+T246V+S334T、L202M+T246V+N270G+S334T、 F195N+L202M+T246V+S334T、L202M+T246V+K320R+S334T 、 L202M+Y243F+S334T 、A51T+A186D+L202M+N270G+N402Y 、A186D+L202M+N270G+S339A+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+N270G+S365C、 A51T+L202M+N270G+S365C、A51T+L202M+N270G+K383R、A51T+Q86I+L202M+N270G、A51T+K93H+L202M+N270G、 A51T+K108R+L202M+N270G、A51T+L202M+N270G+K320H、 A51T+L202M+N270G+K383Q、A51T+K142R+L202M+N270G、 A51T+E130Q+L202M+N270G、A51T+E138Q+L202M+N270G、A51T+L202M+S365C、A51T+S365C、W48F+V238A+S334T、M116F+V238A+S334T、V238A+S334T+W482Y、Y243F+S334T 、 W48F+K118H+V238A+S334T 、A51T+A186N+L202M+N270G+S365C 、W167Y+A186N+H210N+S339A+V366I 、W48F+W167Y+A186N+H210N+S339A 、W167Y+A186N+H210N+Y299F+S339A+V366I 、L202M+T246V+N270G+S334T+S365C 、or A186N+L202M+T246V+N270G+S334T, and fragments thereof having alpha amylase activity, wherein the numbering of amino acid positions is according to the amino acid sequence listed in SEQ ID NO:2.

[0171] In further embodiments, the polypeptide exhibits enhanced wash performance corresponding to an Improvement Factor (IF) of at least 1.2 when the polypeptide is evaluated in an ADW assay in a detergent without bleach and at 40°C for 10 min (eg, see Example 2). A suitable polypeptide may be a variant of SEQ ID NO: 1 comprising a mutation at one or more positions corresponding to the following positions: 37, 48, 51, 64, 81, 108, 116, 167, 174, 186, 187, 189, 195, 198, 202, 208, 210, 235, 238, 243, 246, 250, 261, 265, 267, 269, 270, 275, 311, 319, 334, 339, 365, 366, 385, 460, and 474, wherein the numbering of amino acid positions is according to the amino acid sequence listed in SEQ ID NO: 2.For example, the polypeptide can be selected from the group consisting of: SEQ ID NO: Peptide of the amino acid sequence of NO:1: K37H+L202M, M261L, H210N, Y243F, K108R, G460E, T81S, N174Q+L202M, K385R, Y64W, Y243M, K269N, Y198F, S365M, S339A, W48F, M116V, M116F, W187Y+M208L, W167Y, W189H, W167Y+L202M+H210N+Y299F+S339A, W167Y+L202M+H210N+S339A, W167Y+L202M, V238A+S334T, A51 T+L202M, L202M+T246L, A51T+N174Q+L202M+T246I+S334T, I235L+T24 6M+I250L, A186D+F195N+L202M, K37H+A51T+L202M, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M+Q311T, A51T+L202M+M261L, A51T+L20 2M+Q319R, A51T+L202M+Q311H, A51T+K108R+L202M, A51T+L202M+V474C , A51T+A186D+L202M+N270G, K37H+A51T+L202M+N270G, A51T+L202M+A265G , A51T+L202M+M261L+N270G, A51T+L202M+F267Y, A51T+L202M+I275L, A51T+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, or W167Y+A186N+H210N+Y299F+S339A+V366I, and fragments thereof having alpha amylase activity, wherein the amino acid positions are numbered according to the amino acid sequence listed in SEQ ID NO:2.

[0172] In still further embodiments, the polypeptide exhibits enhanced wash performance corresponding to an IF of at least 1.2 when the polypeptide is evaluated in an ADW assay in a detergent without bleach and at 50°C for 20 min (e.g., see Example 2). A suitable polypeptide may be a variant of SEQ ID NO: 1 comprising a mutation at one or more positions corresponding to the following positions: 30, 33, 37, 40, 48, 51, 86, 93, 108, 109, 113, 116, 118, 121, 130, 138, 142, 167, 174, 175, 182, 186, 195, 198, 202, 203, 210, 218, 235, 238, 2 43, 246, 247, 250, 255, 257, 259, 260, 261, 265, 267, 269, 270, 274, 275, 299, 311, 319, 320, 334, 339, 365, 366, 383, 385, 398, 402, 416, 404, 460, 474, and 482, wherein the numbering of amino acid positions is based on the amino acid sequence listed in SEQ ID NO: 2. For example, the polypeptide can be selected from the group consisting of: SEQ ID NO: 2 having the following mutations: Peptide with amino acid sequence of NO:1: K269S+N270G+A274K, K37V+L202M, N174Q+L202M, K385H, K385R, K93H, K118Q, E130Q, E138Q, K37R, D30N, Y243M, K269N, Y198F, F195N, I257A, S255K, R2 47K, Y404W, Y398W, Q319H, Q319R, Q311H, Q311R, Y299W, N260D, K259N, Y299F, V366I , S365M, S339A, N174*, K37L, S40T, T182G, A265G, W482Y, M116A, N174Q, I235L, A265G , K37H, Q311R, N175Q, W48F, M116V, M116F, W167Y+L202M+H210N+S339A+V366I, W167 Y+L202M+H210N+Y299F+S339A, W167Y+L202M+H210N+S339A, W167Y+L202M+Y299F, W 48F+W167Y, W167Y+Y299F, W167Y+L202M, D30N+N33D+K37V+L202M+K383R, D30N+N33 D+K37V+W48F+K383R, A51T, S334T, T246M, T246L, T246I, A186G, A51Q+G109M+Y203G,<h2 style=";text-align:left;direction:ltr">V238A+S334T、A51T+L202M、L202M+T246L、A51T+N174Q+L202M+T246I+S334T、A51T+N174Q+L20 2M+Q311R+S334T、I235L+T246M+I250L、A186D+L202M、A186D+L202M+N270G+N402Y、A186D+L20 2M+S339A, A186D+F195N+L202M, K37H+A51T+L202M, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M+S339A, A51T+L202M+Q311T, A51T+L202M+M261L, A51T+L202M+H210N, A51T+L202M+N2 70G、A51T+F195N+L202M、A51T+L202M+Q319H、A51T+L202M+Q319R、A51T+L202M+Q311H、A51T+L 202M+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+S33 4T、A51T+L202M+T246V+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+N2 70G+S334T、F195N+L202M+T246V+S334T、L202M+T246V+Q319H+S334T、L202M+T24 6V+Q319R+S334T、L202M+T246V+Q311H+S334T、L202M+T246V+Q311R+S334T、L202 M+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 、A186D+L202M+N270G+S339A+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+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+E121H+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 , L202M+T246I+A265G+F267Y+Q311R+S334T , A51T+L202M+F267Y+Q311R+S334T+S365L, A51T+L202M+S365C, A51T+K108R+L202M, L202M+V238A+S334T, W48F+V238A+S334T, M116F+V 238A+S334T, V238A+S334T+W482Y, Y243F+S334T, L202M+V238A+Y299F+S334T, L202M+T246V+N270G+S334T, W48F+K118H+V238A+S334T, W48F+L202M+V238A+S334T, A51T+A186N+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299F+S339A+V366I, L202M+T246V+N270G+S334T+S365C, or A186N+L202M+T246V+N270G+S334T, and fragments thereof having alpha amylase activity, wherein the numbering of amino acid positions is based on the amino acid sequence listed in SEQ ID NO: 2.

[0173] In a specific subset of the polypeptides of the invention, the polypeptide is a variant of SEQ ID NO: 1 comprising a mutation at one or more positions corresponding to the following positions: 48, 118, 167, 186, 210, 235, 238, 243, 246, 250, 299, 334, 339, 366, and 482, wherein the numbering is according to SEQ ID NO: 2. Thus, the polypeptide may consist of the amino acid sequence of SEQ ID NO: 1, or a fragment thereof having alpha amylase activity, having one or more mutations selected from the group consisting of W48F, K118H, W167Y, H210N, I235L, V238A, Y243F, T246M, I250L, Y299F, S334T, S339A, V366I, and W482Y, wherein the numbering of the amino acid positions is according to the amino acid sequence listed in SEQ ID NO: 2. For example, the polypeptide may comprise or consist of an amino acid sequence selected from the group consisting of:

[0174] (a) SEQ ID NO: 1 having mutations W167Y, H210N and S339A;

[0175] (b) SEQ ID NO: 1 having mutations W167Y, H210N, S339A and V366I;

[0176] (c) SEQ ID NO: 1 having mutations W167Y, H210N, Y299F, S339A and V366I;

[0177] (d) SEQ ID NO: 1 with mutations W48F and W167Y;

[0178] (e) SEQ ID NO: 1 having mutations W48F, W167Y, H210N and S339A;

[0179] (f) SEQ ID NO: 1 having mutations W48F, W167Y, H210N, Y299F, S339A and V366I;

[0180] (g) SEQ ID NO: 1 having mutations V238A, S334T, and W482Y;

[0181] (h) SEQ ID NO: 1 having mutations Y243F and S334T;

[0182] (i) SEQ ID NO: 1 with mutation W48F;

[0183] (j) SEQ ID NO: 1 with mutation K118H;

[0184] (k) SEQ ID NO: 1 with mutation W167Y;

[0185] (l) SEQ ID NO: 1 having mutations W48F, V238A, and S334T;

[0186] (m) SEQ ID NO: 1 having mutations I234L, T246M, and I250L;

[0187] (n) SEQ ID NO: 1 having mutation S339A; and

[0188] (o) SEQ ID NO: 1 having mutation S334T;

[0189] The numbering of the amino acid positions is based on the amino acid sequence listed in SEQ ID NO:2.

[0190] The polypeptides of the present invention represent variants of the amino acid sequence of the parent alpha-amylase (SEQ ID NO: 1), which variants exhibit enhanced washing performance (e.g., at a temperature of 40°C or higher). It will be appreciated by those skilled in the art that different examples of the polypeptides of the present invention will have different degrees of amino acid sequence identity with the sequence of SEQ ID NO: 1. Thus, the polypeptide may comprise or consist of an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1. In one embodiment, relative to the amino acid sequence of SEQ ID NO: 1, the number of variants within the polypeptide is between 1 and 20, such as between 1 and 10 mutations or between 1 and 5 mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations.

[0191] Examples of alpha-amylase polypeptides of the invention are shown in Table 1 (wherein the amino acid sequence of the polypeptides is indicated relative to the sequence of the parent alpha-amylase of SEQ ID NO: 1).

[0192] Table 1

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] In a specific embodiment, the variant α-amylase of the present invention has enhanced wash performance at both 40°C and 50°C. Such variant α-amylases can be selected from

[0205] (a) SEQ ID NO: 1 having mutations V238A, S334T, and W482Y;

[0206] (b) SEQ ID NO: 1 having mutations Y243F and S334T;

[0207] (c) SEQ ID NO: 1 having mutations W48F, W167Y, H210N, Y299F, S339A, and V366I;

[0208] (d) SEQ ID NO: 1 having mutations W48F, W167F, H210N, and S339A;

[0209] (e) SEQ ID NO: 1 having mutation W48F; and

[0210] (f) SEQ ID NO: 1 having mutations W167Y, H210N, and S339A,

[0211] and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is based on the amino acid sequence listed in SEQ ID NO:2.

[0212] Preparation of the polypeptide of the present invention

[0213] The variant α-amylases of the present invention can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semisynthetic gene construction, random mutagenesis, shuffling, and the like.

[0214] Site-directed mutagenesis is a technique for introducing one or more (eg, several) mutations at one or more defined sites in the polynucleotide encoding the parent.

[0215] Site-directed mutagenesis can be achieved in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis can also be performed by cassette mutagenesis, which involves cutting at a site in a plasmid containing a polynucleotide encoding a parent by a restriction enzyme and then connecting an oligonucleotide containing the mutation to the polynucleotide. Typically, the restriction enzyme that digests the plasmid is the same as the restriction enzyme that digests the oligonucleotide, thereby allowing the sticky ends of the plasmid and the insert to be connected to each other. See, for example, Scherer and Davis, 1979, (Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States]) 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. [Nucleic Acids Research] 18: 7349-4966.

[0216] Site-directed mutagenesis can also be achieved in vivo by methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0217] Any site-directed mutagenesis procedure may be used in the present invention.There are many commercially available kits that can be used to prepare variants.

[0218] Synthetic gene construction requires in vitro synthesis of designed polynucleotide molecules to encode the polypeptide of interest. Gene synthesis can be performed using a variety of techniques, such as multiplexed microchip-based techniques described by Tian et al. (2004, Nature 432: 1050-1054), and similar techniques for synthesizing and assembling oligonucleotides on optically programmable microfluidic chips.

[0219] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by a relevant screening procedure, such as those described by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0220] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the importance of individual amino acid residues in a polypeptide to be rapidly determined.

[0221] The many aspects of realizing semisynthetic gene construction by combining synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or reorganization.The semisynthetic construction typically utilizes the process of synthetic polynucleotide fragments in conjunction with PCR technology.Therefore, the restricted region of gene can be synthesized from scratch, and other regions can use site-specific mutagenic primers to increase, yet other regions can carry out fallibility PCR or non-fallibility PCR amplification.Then the polynucleotide subsequence can be reorganized.

[0222] Polynucleotide

[0223] The present invention also relates to polynucleotides encoding variants of the present invention. Therefore, the present invention relates to polynucleotides encoding variant polypeptides having alpha-amylase activity and exhibiting enhanced washing performance compared to the polypeptide of SEQ ID NO: 1. In particular, the present invention relates to polynucleotides encoding variant polypeptides comprising mutations at one or more positions corresponding to the following 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, 211 4, 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, 339, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474, and 482, wherein the numbering is based on the amino acid sequence set forth in SEQ ID NO:2.

[0224] Nucleic acid construct

[0225] The present invention also relates to a nucleic acid construct comprising a polynucleotide encoding a variant polypeptide of the present invention, operably linked to one or more control sequences, which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Therefore, the present invention relates to a nucleic acid construct comprising a polynucleotide encoding a variant polypeptide having alpha-amylase activity and exhibiting enhanced washing performance compared to the polypeptide of SEQ ID NO: 1, which polynucleotide is operably linked to one or more control sequences, which direct the expression of the coding sequence in a suitable host cell under conditions compatible with these control sequences. In particular, the present invention relates to nucleic acid constructs encoding variant polypeptides comprising mutations at one or more positions corresponding to the following 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, 211 4, 416, 434, 460, 469, 474, and 482, wherein the numbering is based on the amino acid sequence listed in SEQ ID NO:2, and these nucleic acid constructs are operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0226] Polynucleotides can be manipulated in a variety of ways to provide expression of variants. Depending on the expression vector, manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0227] The control sequence can be a promoter, i.e. a polynucleotide recognized by a host cell for expressing the polynucleotide. The promoter comprises a transcriptional control sequence that mediates the expression of the variant. The promoter can be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0228] Examples of suitable promoters for directing transcription of the nucleic acid construct of the invention in a bacterial host cell are promoters obtained from the following genes: Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular coli lac operon, the E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), the Streptomyces coelicolor agar hydrolase gene (dagA) and the prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Other promoters are described in "Useful proteins from recombinant bacteria" by Gilbert et al., 1980, Scientific American, 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0229] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900). 00 / 56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei β-xylosidase, and the NA2-tpi promoter (a modified promoter from an Aspergillus neutral α-amylase gene in which the untranslated leader is replaced by the untranslated leader of an Aspergillus triose phosphate isomerase gene; non-limiting examples include a modified promoter from an Aspergillus niger gene encoding a neutral α-amylase in which the untranslated leader has been replaced with an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae gene encoding a triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof.

[0230] In yeast hosts, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0231] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3'-end of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used.

[0232] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0233] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0234] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described in Romanos et al., 1992, supra.

[0235] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0236] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0237] The control sequence may also be a leader sequence, a non-translated mRNA region that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the variant. Any leader that works in the host cell may be used.

[0238] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0239] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0240] The control sequence may also be a polyadenylation sequence, a sequence that is operably linked to the 3'-terminus of the variant coding sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence that functions in the host cell may be used.

[0241] Preferred polyadenylation for filamentous fungal host cells is obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger-alpha glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0242] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0243] The control sequence can also be a signal peptide coding region, encoding a signal peptide connected to the N-terminus of the variant, and directing the variant to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide can inherently contain a signal peptide coding sequence, which is naturally connected to the segment of the coding sequence encoding the variant in the translation reading frame. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding sequence that is foreign to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an external signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence, so as to enhance the secretion of the variant. However, any signal peptide coding sequence directing the expressed variant to enter the secretory pathway of the host cell can be used.

[0244] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for Bacillus sp. NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prs A. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0245] Effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0246] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (1992, supra).

[0247] The control sequence can also be a propeptide coding sequence encoding a propeptide located at the N-terminal of a variant. The polypeptide generated is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptide is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the gene of bacillus subtilis alkaline protease (aprE), bacillus subtilis neutral protease (nprT), thermophilic myceliophthora laccase (WO 95 / 33836), miehei rhizomucor aspartic protease and saccharomyces cerevisiae α-factor.

[0248] Where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0249] It is also desirable to add a regulatory sequence that regulates the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of a gene to be turned on or off in response to chemical or physical stimulation, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include lac, tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter, and Aspergillus oryzae glucoamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include a dihydrofolate reductase gene amplified in the presence of methotrexate and a metallothionein gene amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operably connected to the regulatory sequence.

[0250] Expression vector

[0251] The present invention also relates to a recombinant expression vector comprising a polynucleotide encoding a variant polypeptide of the present invention, a promoter, and a transcription and translation termination signal. Therefore, the present invention relates to a recombinant expression vector comprising a polynucleotide, a promoter, and a transcription and translation termination signal, the polynucleotide encoding a variant polypeptide having alpha-amylase activity and exhibiting enhanced washing performance compared to the polypeptide of SEQ ID NO: 1. Specifically, the present invention relates to a recombinant expression vector comprising a polynucleotide, a promoter, and a transcription and translation termination codon, the polynucleotide encoding a variant polypeptide comprising a mutation at one or more positions corresponding to the following 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, 2 404, 416, 434, 460, 469, 474, and 482, wherein the numbering is based on the amino acid sequence set forth in SEQ ID NO:2.

[0252] Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more suitable restriction sites to allow insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotides may be expressed by inserting a polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. When producing an expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked to a suitable control sequence for expression.

[0253] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0254] The carrier can be an autonomously replicating carrier, i.e., a carrier existing as an extrachromosomal entity, which replicates independently of chromosome replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The carrier can include any device for ensuring self-replication. Alternatively, the carrier can be such a carrier that is integrated into the genome and replicates with the chromosome into which it has been integrated when it is introduced into the host cell. In addition, a single carrier or plasmid or two or more carriers or plasmids (these carriers or plasmids together comprise the total DNA to be introduced into the genome of the host cell) or a transposon can be used.

[0255] The vector preferably contains one or more selectable markers that permit easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0256] Examples of bacterial selectable markers are the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), together with their equivalents. Preferably, the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and the Streptomyces hygroscopicus bar gene are used in Aspergillus cells.

[0257] The vector preferably contains one or more elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0258] For being integrated into the host cell genome, the vector can rely on the polynucleotide sequence encoding the variant or any other element of the vector for being integrated into the genome by homologous or non-homologous recombination.Alternately, the vector can include other polynucleotides for guiding the precise position of the chromosome integrated into the host cell genome by homologous recombination.In order to increase the possibility of integration at the precise position, the elements of these integrations should include a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to improve the possibility of homologous recombination.Integration element can be any sequence homologous to the target sequence in the host cell genome.In addition, integration element can be non-coding polynucleotides or encoding polynucleotides.On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0259] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator that is functional in the cell and mediates autonomous replication. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0260] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

[0261] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0262] Examples of origins of replication used in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00 / 24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing the gene can be accomplished according to the method disclosed in WO 00 / 24883.

[0263] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the production of variants. The increased copy number of the polynucleotide can be obtained by integrating at least one other copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene together with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene and thus other copies of the polynucleotide can be selected by culturing the cell in the presence of an appropriate selective agent.

[0264] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, supra).

[0265] Host cells

[0266] The present invention also relates to recombinant host cells comprising polynucleotides encoding variant polypeptides of the present invention, operably linked to one or more control sequences, which direct the production of variant polypeptides of the present invention. Therefore, the present invention relates to recombinant host cells comprising polynucleotides encoding variant polypeptides having alpha-amylase activity and exhibiting enhanced washing performance compared to the polypeptide of SEQ ID NO: 1, which polynucleotides are operably linked to one or more control sequences that direct the production of variant polypeptides. In particular, the invention relates to a recombinant host cell comprising a polynucleotide encoding a variant polypeptide comprising a mutation at one or more positions corresponding to: 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, 21 460, 474, and 482, wherein the numbering is based on the amino acid sequence listed in SEQ ID NO:2, the polynucleotide being operably linked to one or more control sequences that direct the production of the variant polypeptide.

[0267] The construct or vector comprising the polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication. The choice of a host cell will depend to a large extent on the gene encoding the variant and its source.

[0268] The host cell may be any cell useful in the recombinant production of the variant, eg, a prokaryotic or eukaryotic cell.

[0269] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include but are not limited to Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0270] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus fluorescens, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0271] The bacterial host cell may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0272] The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0273] Introduction of DNA into Bacillus cells can be achieved by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), transformation of competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). Introduction of DNA into E. coli cells can be achieved by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). Introduction of DNA into Streptomyces cells can be achieved by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). Introduction of DNA into Pseudomonas cells can be achieved by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).Introduction of DNA into Streptococcus cells can be accomplished by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell may be used.

[0274] The host cell may also be a eukaryotic organism, such as a plant, or a fungal cell.

[0275] The host cell may be a fungal cell. "Fungi" as used herein include Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota as well as Oomycota and all mitotic spore fungi (as defined by Hawksworth et al., in: Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0276] The fungal host cell may be a yeast cell. "Yeast" as used herein includes ascospore-producing yeasts (Saccharomyces), basidiospore-producing yeasts, and yeasts belonging to imperfect fungi (Blastomyces). Since the classification of yeasts may change in the future, for purposes of the present invention, yeasts should be defined as described in the Biology and Activity of Yeasts (Skinner, Passmore and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9 [Society for Applied Bacteriology Symposium Series 9], 1980).

[0277] The yeast host cell can be a Candida cell, a Hansenula cell, a Kluyveromyces cell, a Pichia cell, a Saccharomyces cell, a Schizosaccharomyces cell, or a Yarrowia cell, such as a Kluyveromyces lactis cell, a Saccharomyces carlsbergensis cell, a Saccharomyces cerevisiae cell, a Saccharomyces diastaticus cell, a Saccharomyces douglasii cell, a Saccharomyces kluyveri cell, a Saccharomyces norbensis cell, a Saccharomyces oviformis cell, or a Yarrowia lipolytica cell.

[0278] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subphyla of Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitin, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeasts (such as Saccharomyces cerevisiae) is by budding of a unicellular thallus, and carbon catabolism may be fermentative.

[0279] The filamentous fungal host cell can be an Acremonium, Aspergillus, Bjerkandera, Ceroplastes, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neotrichia, Neurospora, Paecilomyces, Penicillium, Pseudocercospora, Phlebia, Chytridiomyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Toxicodendron, Trametes, or Trichoderma cell.

[0280] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis a eirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium feces, or Chrysosporium spp. merdarium), rent spores, Chrysosporium queenslandicum, tropical chrysosporium, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, rod-shaped fusarium, cereal fusarium, Kuwei fusarium, broadsword fusarium, graminearum fusarium, graminearum fusarium, heterospore fusarium, albizia fusarium, sharp fusarium, multi-branch fusarium, pink fusarium, elder fusarium, color fusarium, pseudo-branch spore fusarium, sulfur-colored fusarium, round fusarium, pseudo-spore fusarium, embellished fusarium, insolens humicola, soft hair humicola, rice black mold, thermophilic myceliophthora, rough chain spore, purple Penicillium, Phanerochaete chrysosporium, radiata fusarium, Pleurotus eryngii, eryngii, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0281] Fungal cells can be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 81: 1470-1474 and Christensen et al., 1988, Bio / Technology [Biology / Technology] 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al. 1989, Gene [Gene] 78: 147-156 and WO 96 / 00787. Yeast can be transformed using methods described in Becker and Guarente, in Abelson, JN and Simon, MI, eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0282] Production method

[0283] The present invention also relates to methods for producing variant α-amylases, comprising: (a) culturing a host cell of the present invention under conditions suitable for expression of the variant polypeptide; and (b) recovering the variant polypeptide.

[0284] Host cells can be cultured in a nutrient medium suitable for producing variants using methods known in the art. For example, it is possible to culture by shaking bottles, or in a suitable medium and under conditions allowing variant expression and / or separation, carry out small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) in a laboratory or industrial fermentor tank to cultivate cells. Cultivation is to use procedures known in the art, and occurs in a suitable nutrient medium, which comprises carbon and nitrogen sources and inorganic salts. Suitable substratum can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalog of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be directly recovered from the substratum. If the variant is not secreted, it can be recovered from a cell lysate.

[0285] Variants can be detected using methods known in the art. Suitable detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the alpha-amylase activity of a variant (see Examples).

[0286] The variant may be recovered using methods known in the art. For example, the variant may be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

[0287] Variants may be purified by a variety of procedures known in the art to obtain substantially pure variants, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, eds., VCH Publishers, New York, 1989).

[0288] In alternative aspects, the variant is not recovered, but rather a host cell of the invention expressing the variant is used as a source of the variant.

[0289] Composition

[0290] The alpha-amylase polypeptides of the present invention can be added to detergent compositions and thus become components of detergent compositions. Therefore, the present invention relates to compositions comprising variant polypeptides having alpha-amylase activity and exhibiting enhanced washing performance compared to the polypeptide of SEQ ID NO: 1. In particular, the present invention relates to compositions comprising variant polypeptides comprising mutations at one or more positions corresponding to the following 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, 339, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474, and 482, wherein the numbering is based on the amino acid sequence listed in SEQ ID NO:2.

[0291] Therefore, another aspect of the present invention provides a detergent composition comprising a polypeptide of the present invention and a surfactant (such as an anionic surfactant, a cationic surfactant, a nonionic surfactant and / or a zwitterionic surfactant). Also provided is a concentrate or additive for preparing such a detergent composition, the concentrate or additive comprising a polypeptide of the present invention and optionally a surfactant.

[0292] As discussed in detail below, the detergent composition may further comprise one or more additional components selected from the group consisting of oxidizing agents, bleach activators, fillers, builders, buffers, structurants, chelating agents, optical brighteners, defoamers, enzymes, perfumes, anti-redeposition agents, skin conditioning agents, softeners, emulsifiers, and colorants.

[0293] In one embodiment, the composition is a liquid or powder laundry detergent composition.

[0294] In further embodiments, the composition is a liquid or powder automatic dishwashing (ADW) detergent composition.

[0295] For example, the detergent compositions of the present invention may be formulated as hand or machine cleaning compositions, including laundry additive compositions suitable for pre-treating stained fabrics and rinse-added fabric softener compositions, or as detergent compositions for use in general household hard surface cleaning operations, or as detergent compositions for use in hand or machine dishwashing operations.

[0296] In a specific aspect, the invention provides a detergent concentrate / additive comprising an α-amylase polypeptide of the invention. The detergent concentrate / 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, such as another α-amylase, a glucoamylase, a maltogenic amylase, a CGTase and / or a cellulase, a mannanase (such as MANNAWAY TM , from Novozymes (Denmark), pectinase, pectin lyase, cutinase and / or laccase.

[0297] In general, the enzyme(s) will be selected to have properties that are compatible with the chosen detergent (ie, pH optimum, compatibility with other enzymes and non-enzymatic ingredients, etc.), and will be present in an effective amount.

[0298] Proteases: Suitable proteases include those of animal, plant or microbial origin. Preferred are microbial origin. Chemically modified mutants 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. Examples of alkaline proteases are subtilisins, particularly those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89 / 06279). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin) and the Fusarium proteases described in WO 89 / 06270 and WO 94 / 25583.

[0299] Examples of useful proteases are variants as described in WO 92 / 19729, WO 98 / 20115, WO 98 / 20116 and WO 98 / 34946, in particular variants substituted at one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274. Preferred commercially available proteases include (SEQ ID NO:3), and (From (Novozymes A / S)), MAXACAL, PURAFECT (Genencor International Inc.).

[0300] Lipases: Suitable lipases include those of bacterial or fungal origin, including chemically modified mutants or protein engineered mutants. Examples of useful lipases include lipases from the genus Humicola (synonymous with Thermomyces), for example 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; Pseudomonas lipases, for example from Pseudomonas alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012); Bacillus lipase, for example from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B. stearothermophilus (JP 64 / 744992) or B. pumilus (WO 91 / 16422). Other examples of lipase variants are 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.

[0301] Preferred commercially available lipases include LIPOLASE< TM > and LIPOLASE ULTRA< TM >(Novozymes A / S, SEQ ID NO:4 herein).

[0302] Amylase: Suitable amylases (α- and / or β-amylases) include amylases derived from bacteria or fungi. Chemically modified mutants or protein engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, for example, a specific strain of Bacillus licheniformis (described in more detail in GB 1,296,839). Examples of useful α-amylases are variants described in WO 94 / 02597, WO 94 / 18314, WO 96 / 23873, and WO 97 / 43424, especially variants with substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444. A commercially available α-amylase is DURAMYL< TM > LIQUEZYME TM TERMAMYL TM >、NATALASE< TM >、FUNGAMYL< TM >and BAN< TM >(Novozymes A / S), Preferenz S100, Preferenz S110, Preferenz S1000, ExcellenzS110, Excellenz S1000, Excellenz S2000, RAPIDASE< TM > and PURASTAR< TM > (from Genencor International Inc.). Thus, a suitable amylase may be any of those listed herein as SEQ ID NOs: 5, 6, 7, 8, 9, 10, and 11, and variants thereof.

[0303] Cellulase:Suitable cellulases include those of bacterial or fungal origin. Include chemically modified mutants or protein engineered mutants. Suitable cellulases include cellulases from Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example, from Humicola insolens, thermophilic Myceliophthora and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89 / 09259. Particularly suitable cellulases are alkaline or neutral cellulases with color care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are cellulase variants, such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307 and PCT / DK 98 / 00299.

[0304] Commercially available cellulases include and (Novozymes A / S), and PURADAX (Genencor International Inc.) and (Kao Corporation).

[0305] Peroxidase / oxidase: Suitable peroxidase / oxidases include those of plant, bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus (e.g., from Coprinus cinereus), and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include (Novozymes A / S).

[0306] Lechinase: Suitable lechinase include those of bacterial or fungal origin. They may be chemically modified or protein engineered. Examples of useful lechinase include those set forth in SEQ ID NO: 3, 4, and 5 (Novozymes A / S) and WO 99 / 06516 (Henkel KGAA). Examples of other suitable lichinase that may be chemically modified or protein engineered are those shown in SEQ ID NO: 12, 13, 14, and 15 herein.

[0307] Detergent enzymes may be included in a detergent composition by adding a single additive comprising one or more enzymes, or by adding a combined additive comprising all of these enzymes. The detergent additive of the present invention (i.e., a single additive or a combined additive) may be formulated, for example, as a granule, a liquid, a slurry, etc. Preferred detergent additive formulations are granules, especially non-dusting granules; liquids, especially stabilized liquids; or slurries.

[0308] Non-dusting particles can be produced, for example, as disclosed in US 4,106,991 and 4,661,452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1000 to 20000; ethoxylated nonylphenols with from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols with from 12 to 20 carbon atoms and 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for use by fluidized bed technology are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (e.g., propylene glycol), sugars or sugar alcohols, lactic acid or boric acid according to established methods. Protected enzymes can be prepared according to the method disclosed in EP238 216.

[0309] The detergent compositions of the invention may be in any convenient form such as a bar, tablet, powder, granulate, paste or liquid.Liquid detergents may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or may be non-aqueous.

[0310] The detergent composition comprises one or more surfactants which may be nonionic (including semi-polar) and / or anionic and / or cationic and / or zwitterionic. Surfactants are typically present at a level of from 0.1% to 60% by weight.

[0311] When included therein, the detergent will typically contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzene sulfonates, α-olefin sulfonates, alkyl sulfates (fatty alcohol sulfates), alcohol ethoxysulfates, secondary alkyl sulfonates, α-sulfo fatty acid methyl esters, alkyl or alkenyl succinic acid or soap.

[0312] When included therein, the detergent typically contains from about 0.2% to about 40% of a nonionic surfactant, such as alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethylamine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides, polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine ("glucamides").

[0313] The detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenediaminetriaminopentaacetic acid, alkyl or alkenyl succinic acid, soluble silicate, or layered silicate (e.g., SKS-6 from Hoechst). Other complexing agents may be methylglycine diacetic acid (MGDA) and glutamic acid diacetic acid (GLDA), which are particularly useful in phosphate-free automatic dishwashing detergents.

[0314] The detergent may comprise one or more polymers. Examples are sulfonated polymers, carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates (such as polyacrylates), maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers.

[0315] The detergent may comprise a bleaching system which may comprise a H2O2 source, such as perboric acid or percarbonic acid, which may be combined with a peracid-forming bleach activator, such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. In addition, the bleaching catalyst may be a component based on Mn and / or Co. Alternatively, the bleaching system may comprise a peracid, for example of the amide, imide or sulfone type.

[0316] The one or more enzymes of the detergent composition of the present invention may be stabilized using conventional stabilizers, for example polyols, such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, such as aromatic borate esters, or phenylboronic acid derivatives, such as 4-formylphenylboronic acid, and the composition may be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708.

[0317] The detergent may also contain other conventional detergent ingredients such as, for example, fabric conditioners including clays, foam boosters, foam suppressors, corrosion inhibitors, soil suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

[0318] It is currently expected that any enzyme, especially the α-amylase polypeptide of the present invention, is added to the detergent composition in an amount equivalent to adding 0.01 mg-100 mg enzyme protein per liter of washing liquid, preferably adding 0.05 mg-5 mg enzyme protein per liter of washing liquid, and particularly preferably adding 0.1 mg-1 mg enzyme protein per liter of washing liquid.

[0319] Additionally, the alpha amylase polypeptides of the present invention may be incorporated into detergent formulations as disclosed in WO 2006 / 002643, which is incorporated herein by reference.

[0320] Examples of dishwashing detergent compositions of the present invention

[0321] The alpha-amylase polypeptides of the present invention can be used in a dishwashing detergent composition comprising:

[0322] (a) Powdered Automatic Dishwashing Compositions

[0323]

[0324] (b) Powdered automatic dishwashing compositions

[0325]

[0326] (c) Powdered automatic dishwashing compositions

[0327]

[0328]

[0329] (d) Powdered automatic dishwashing compositions

[0330] (e) Powdered automatic dishwashing compositions

[0331]

[0332] (f) Powder and liquid dishwashing compositions having a cleaning surfactant system

[0333] (p) Non-aqueous liquid automatic dishwashing compositions

[0334]

[0335]

[0336] (q) Non-aqueous liquid dishwashing compositions

[0337] (r) Thixotropic liquid automatic dishwashing compositions

[0338]

[0339] (s) Liquid automatic dishwashing compositions

[0340]

[0341] (t) Liquid automatic dishwashing detergent compositions comprising protected bleach particles

[0342] (u) An automatic dishwashing composition as described in (a), (b), (c), (d), (f) and (j) wherein the perborate is replaced by percarbonate.

[0343] (v) An automatic dishwashing composition as described in (a) to (f), which additionally comprises a manganese catalyst. The manganese catalyst may, for example, be one of the compounds described in: "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639.

[0344] use

[0345] The present invention is also directed to methods of using the alpha-amylase polypeptides of the present invention in detergents, particularly laundry detergent compositions and dishwashing detergent compositions.

[0346] Thus, the invention provides the use of an alpha-amylase polypeptide or composition of the invention in a domestic or industrial cleaning process.

[0347] In one embodiment, the use is the cleaning of fabrics, such as laundry.

[0348] In another embodiment, the use is the cleaning of ceramic, plastic or glass materials, such as dishwashing.

[0349] Thus, the alpha-amylase polypeptides of the present invention are useful as components of washing, dishwashing and hard surface cleaning detergent compositions, either in domestic or industrial settings.

[0350] The alpha-amylase variants of the present invention have valuable properties that allow various other industrial applications. For example, the alpha-amylase polypeptides of the present invention can be used in starch processes, particularly starch conversion, especially the liquefaction of starch (see, for example, US 3,912,590, European Patent Application Nos. 252 730 and 63 909, WO 99 / 19467 and WO 96 / 28567, all references are incorporated herein by reference). Compositions for starch conversion purposes are also contemplated, which, in addition to the variants of the present invention, may also include glucoamylase, pullulanase and other alpha-amylases.

[0351] In addition, the α-amylase variants of the present invention are also particularly suitable for producing sweeteners and ethanol from starch or whole grains (see, e.g., U.S. Pat. No. 5,231,017, incorporated herein by reference), such as fuel ethanol, drinking ethanol, and industrial ethanol.

[0352] The alpha-amylase variants of the invention are also used in desizing of textiles, fabrics and garments (see, e.g., WO 95 / 21247, U.S. Patent 4,643,736, EP 119,920, incorporated herein by reference), beer making or brewing, pulp and paper production.

[0353] Starch conversion

[0354] Conventional starch conversion processes (eg, liquefaction and saccharification processes) are described, for example, in US Pat. No. 3,912,590 and European Patent Publication Nos. 252,730 and 63,909, which are incorporated herein by reference.

[0355] In one embodiment, the starch conversion process degrades starch into lower molecular weight carbohydrate components, such as sugars or fat substitutes, and the process includes a debranching step.

[0356] When converting starch to sugars, the starch is depolymerized. Such a depolymerization process may consist of, for example, a pretreatment step and two or three consecutive process steps, namely, a liquefaction process, a saccharification process and, depending on the desired end product, an optional isomerization process.

[0357] (i) Pretreatment of native starch

[0358] Natural starch consists of microscopic granules that are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually rupture, dispersing the starch molecules into the solution. During this "gelatinization" process, there is a significant increase in viscosity. Since the solids level in a typical industrial process is 30%-40%, the starch has to be diluted or "liquefied" to enable it to be handled. Today, the reduction in viscosity is mainly achieved through enzymatic degradation.

[0359] (ii) Liquefaction

[0360] In the liquefaction step, alpha-amylase degrades long-chain starch into shorter units of branches and linear chains (maltodextrins). The liquefaction process is carried out at 105°C-110°C for 5 to 10 minutes, followed by 1-2 hours at 95°C. The pH value is between 5.5 and 6.2. To ensure optimal enzyme stability under these conditions, 1 mM calcium (40 ppm free calcium ions) is added. After this treatment, the liquefied starch will have a "dextrose equivalent" (DE) of 10-15.

[0361] (iii) Saccharification

[0362] After the liquefaction process, the liquefaction is carried out by adding a glucoamylase (e.g., AMG) and a debranching enzyme (e.g., isoamylase (U.S. Pat. No. 4,335,208) or a pullulanase (e.g., Promozyme TM ) U.S. Pat. No. 4,560,651) converts maltodextrin into dextrose. Prior to this step, the pH is lowered to a value below 4.5 and a high temperature (above 95°C) is maintained to inactivate the liquefying α-amylase to reduce the formation of short oligosaccharides called "panose precursors" that cannot be properly hydrolyzed by debranching enzymes.

[0363] The temperature was lowered to 60°C and glucoamylase and debranching enzyme were added. The saccharification process was carried out for 24-72 hours.

[0364] Typically, when α-amylase is denatured after the liquefaction step, about 0.2% to 0.5% of the saccharification products are branched trisaccharides that cannot be degraded by pullulanase. <2> - α-Glucosylmaltose (panose). If active amylase from the liquefaction step is present during saccharification (ie not denatured), the level can be as high as 1%-2%, which is highly undesirable as it significantly reduces the saccharification yield.

[0365] (iv) Isomerization

[0366] When the desired final sugar product is, for example, high fructose syrup, dextrose syrup can be converted to fructose. After the saccharification process, the pH is increased to a value in the range of 6-8, preferably pH 7.5, and calcium is removed by ion exchange. Then, the saccharification reaction is carried out using, for example, an immobilized glucose isomerase (such as Sweetzyme< TM >IT) converts dextrose syrup into high fructose syrup.

[0367] Ethanol production

[0368] In general, the production of alcohol (ethanol) from whole grains can be divided into 4 main steps:

[0369] - Grinding

[0370] -liquefaction

[0371] -Saccharification

[0372] - Fermentation

[0373] (i) Grinding

[0374] The grains are ground to open up the structure and allow further processing. The two processes used are wet or dry grinding. In dry grinding, the whole grain is ground and used in the rest of the process. Wet grinding is very good at separating the germ and meal (starch granules and protein) and is used in several locations in parallel production of syrups except in a few cases.

[0375] (ii) Liquefaction

[0376] During liquefaction, starch granules are solubilized by hydrolysis into maltodextrins with a DP mostly above 4. Hydrolysis can be performed by acid treatment or enzymatically by α-amylase. Acid hydrolysis is used to a limited extent. The raw material can be ground whole grain or a side stream from starch processing.

[0377] Enzymatic liquefaction is typically carried out in a three-step hot slurry process. The slurry is heated to between 60°C-95°C (preferably 80°C-85°C) and one or more enzymes are added. The slurry is then jet cooked between 95°C-140°C (preferably 105°C-125°C), cooled to 60°C-95°C and more enzymes are added to obtain final hydrolysis. The liquefaction process is carried out at a pH of 4.5-6.5, typically at a pH between 5 and 6. The ground and liquefied grain is also called mash.

[0378] (iii) Saccharification

[0379] In order to produce low molecular sugars DP1-3 that can be metabolized by yeast, the maltodextrin in the liquefaction process must be further hydrolyzed. Typically, enzymatic hydrolysis is performed by glucoamylase, alternatively, alpha-glucosidase or acid alpha-amylase can be used. The full saccharification step can last for 72 hours, however, typically only a typical 40-90 minutes of pre-saccharification is performed, and then saccharification (SSF) is completed during fermentation. Saccharification is typically performed at a temperature of from 30°C-65°C, typically about 60°C and at pH 4.5.

[0380] (iv) Fermentation

[0381] Yeast, typically from the genus Saccharomyces, is added to the mash and fermentation is continued for 24-96 hours, such as typically 35-60 hours. The temperature is between 26°C-34°C, typically around 32°C, and the pH is from pH 3-6, preferably around pH 4-5.

[0382] Note that the most widely used process is the simultaneous saccharification and fermentation (SSF) process, in which there is no continuous phase of saccharification, meaning that yeast and enzymes are added together. When performing SSF, a pre-saccharification step is usually introduced before fermentation at a temperature above 50°C.

[0383] (v) Distillation

[0384] After fermentation, the mash is distilled to extract the ethanol.

[0385] The ethanol obtained according to the process of the present invention can be used as, for example, fuel ethanol, drinking ethanol, ie, drinkable neutral alcoholic beverage, or industrial ethanol.

[0386] (vi) By-products

[0387] The remainder from the fermentation is the spent grains, which are typically used in animal feed in liquid or dry form.

[0388] Further details on how to accomplish liquefaction, saccharification, fermentation, distillation, and recovery of ethanol are well known to those skilled in the art.

[0389] According to the method of the present invention, saccharification and fermentation can be performed simultaneously or separately.

[0390] Pulp and paper production

[0391] The alkaline alpha-amylase polypeptides of the invention can also be used to produce lignocellulosic materials such as pulp, paper and paperboard from starch-enhanced waste paper and paperboard, especially when repulping occurs at a pH above 7 and when the amylase promotes the decomposition of the waste material by enhancing the degradation of starch. The alpha-amylase of the invention is particularly suitable for use in a process for producing papermaking pulp from starch-coated printing paper. The process can be performed as described in WO 95 / 14807 and comprises the following steps:

[0392] a) disintegrating paper to produce pulp,

[0393] b) treating with a starch degrading enzyme before, during or after step a), and

[0394] c) After steps a) and b), separating the ink particles from the paper pulp.

[0395] The alpha-amylases of the invention are also very suitable for modifying starch, where the enzyme-modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clay. With the alkaline alpha-amylases of the invention it is possible to modify starch in the presence of fillers, thereby allowing a simpler integration process.

[0396] Desizing of textiles, fabrics and apparel

[0397] The alpha-amylase of the present invention is also very useful in textiles, fabrics or clothes desizing. In the textile processing industry, alpha-amylase is traditionally used as an auxiliary agent in the desizing process to promote the removal of starch-containing sizing as a protective coating on the weft yarn during the weaving process. Thorough removal of the sizing coating after weaving is crucial to ensure the best effect in the subsequent process (wherein the fabric is scrubbed, bleached and dyed). Enzymatic starch decomposition is preferred because it does not produce any harmful effects on the fiber material. In order to reduce processing costs and increase the processing capacity of the grinder, the desizing process is sometimes combined with the scrubbing and bleaching steps. In such cases, non-enzymatic auxiliaries such as alkalis or oxidants are typically used to decompose starch because traditional alpha-amylases are not very compatible with high pH levels and bleaching agents. Due to the use of chemicals with considerable corrosiveness, the non-enzymatic decomposition of starch sizing causes some fiber damage. Therefore, it is desirable to use the alpha-amylase of the present invention because they have improved performance in alkaline solutions. When desizing cellulose-containing fabrics or textiles, alpha-amylases can be used alone or in combination with cellulases.

[0398] Desizing and bleaching processes are well known in the art. For example, such processes are described in, for example, WO 95 / 21247, US Pat. No. 4,643,736, EP 119,920 (incorporated herein by reference).

[0399] Commercially available products for desizing include and ULTRA (from Novozymes A / S).

[0400] Beer manufacturing

[0401] The alpha-amylases of the invention may also be very useful in the beer making process; alpha-amylases are typically added during the mashing process.

[0402] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0403] The invention described and claimed in this application is not limited to the scope of the specific aspects disclosed herein, because these aspects are intended to illustrate several aspects of the present invention. Any equivalent aspects are intended to be within the scope of the present invention. In fact, various modifications of the present invention will become apparent to those skilled in the art from the aforementioned description, except for those shown and described herein. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure including the definitions shall prevail.

[0404] Example

[0405] Example 1: Determination of α-amylase activity

[0406] 1. Phadebas Assay

[0407] By adopting The α-amylase activity can be determined by using the tablet as substrate method. Phadebas tablets ( Amylase test substance ( The Amylase Test, supplied by Pharmacia Diagnostic, contains a cross-linked insoluble blue starch polymer that has been mixed with bovine serum albumin and a buffer substance and compressed into a tablet.

[0408] For each single measurement, a piece was suspended in a tube containing 5 ml of 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl2, pH adjusted to the value of interest with NaOH). The test was performed in a water bath at the temperature of interest. The alpha-amylase to be tested was diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylase solution was added to 5 ml of 50 mM Britton-Robinson buffer. The starch was hydrolyzed by alpha-amylase, giving soluble blue fragments. The absorbance of the resulting blue solution was measured at 620 nm with a spectrophotometer, which is a function of the activity of the alpha-starch.

[0409] It is important that the absorbance at 620 nm measured after 10 or 15 minutes of incubation (test time) is within the range of 0.2-2.0 absorbance units at 620 nm. Within this absorbance range, there is a linear relationship between activity and absorbance (Lambert-Beer's law). Therefore, the dilution of the enzyme must be adjusted to fit this standard. Under a specified set of conditions (temperature, pH, reaction time, buffer conditions), 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and will produce a blue color. The color intensity is measured at 620 nm. Under a given set of conditions, the absorbance measured is proportional to the specific activity of the alpha-amylase (activity per milligram of pure alpha-amylase protein).

[0410] 2. Alternative Methods

[0411] The α-amylase activity is determined by a method using a PNP-G7 substrate. PNP-G7 is an abbreviation for p-nitrophenyl-α, D-maltoheptaglycoside, a block oligosaccharide that can be cleaved by an endoamylase. After cleavage, the α-glucosidase included in the kit digests the substrate to release free PNP molecules, which have a yellow color and can thus be measured by visible spectrophotometry at λ=405nm (400nm-420nm). The kit containing the PNP-G7 substrate and α-glucosidase is manufactured by Bohringer-Mannheim (Cat. No. 1054635).

[0412] To prepare the reagent solution, 10 ml of substrate / buffer solution was added to 50 ml of enzyme / buffer solution recommended by the manufacturer. The assay was performed by transferring 20 μL of sample to a 96-well microtiter plate and incubating at 25° C. 200 μL of reagent solution pre-equilibrated to 25° C. was added. The solution was mixed and pre-incubated for 1 minute and the absorbance was measured at OD 405 nm every 30 seconds for 4 minutes in an ELISA reader.

[0413] Under a given set of conditions, the slope of the time-dependent absorption curve is directly proportional to the activity of the α-amylase in question.

[0414] Determination of LAS sensitivity

[0415] The variants were incubated with different concentrations of LAS (linear alkylbenzene sulfonate; Nansa 1169 / P) at 40°C for 10 minutes.

[0416] use The residual activity can be determined by the assay or an alternative method using the PNP-G7 substrate.

[0417] LAS was diluted into 0.1 M phosphate buffer (pH 7.5).

[0418] The following concentrations were used: 500ppm, 250ppm, 100ppm, 50ppm, 25ppm, and 10ppm without LAS. The variants were diluted to a concentration of 0.01mg / l-5mg / l in different LAS buffers, with a total volume of 10ml, and incubated for 10 minutes in a temperature-controlled water bath. Incubation was stopped by transferring a small aliquot to a cold assay buffer. During the activity measurement, it was very important that the LAS concentration was less than 1ppm to avoid affecting the activity measurement.

[0419] Then use the above The residual activity is determined in duplicate by the assay or alternative method. The activity is measured after subtracting the blank. The activity without LAS is 100%.

[0420] Example 2: Evaluation of the washing performance of α-amylase polypeptides of the invention using full-scale automatic dishwashing (ADW)

[0421] In order to evaluate the washing performance of the polypeptides of the present invention in a detergent base composition, washing experiments can be performed using a full-scale automatic dishwashing (ADW). A full-scale ADW apparatus is used to test the washing performance of polypeptides under test conditions that simulate conventional consumer apparatus.

[0422] In this study, the test conditions were a regular 50°C wash program and a short 40°C program using a Miele Dishwasher Miele G4300 SCU machine.

[0423] General description of washing performance.

[0424] Melamine tiles stained with, for example, starch (DM-77 / DM-177 / DM-277 / DM-377 from Test Material Center BV (PO Box 120, 3133 KT, Vlaardingen, The Netherlands)) were used as test material and washed with tap water at 19° dH-20° dH at 40° C. and 50° C. under a set program as follows (see Tables 2, 3 and 4). Detergent and α-amylase were added at a concentration of 1.5 mg polypeptide / wash or 3 mg polypeptide / wash for 40° C. washing and 0.5 mg polypeptide / wash or 1 mg polypeptide / wash for 50° C. washing when the detergent dispenser lid was open in the dishwasher. The test with 0 mg polypeptide / L was used as blank and corresponded to the contribution from the detergent. The full-scale washing performance experiment was performed under the experimental conditions specified below:

[0425] Table 2 Experimental conditions

[0426]

[0427]

[0428] Table 3 ADW standard detergent with bleach

[0429]

[0430] Table 4 ADW standard detergent without bleach

[0431]

[0432] After washing, dry the melamine tiles.

[0433] Washing performance was measured as the difference in reflectance between washed and unwashed tiles. Reflectance measurements were performed using a Color-Eye 7000 (CE7000) for acquiring spectra and performing reflectance difference calculations. Reflectance was measured at 460 nm (no UV light in the illuminant).

[0434] Wash performance is considered to be improved if the improvement factor (IF) is at least 1.0, preferably at least 1.2 under one or more of the conditions listed above, i.e. at 40°C or 50°C, wherein the variant concentration in the 40°C wash is 1.5 mg polypeptide / wash or 3 mg polypeptide / wash, and 0.5 mg polypeptide / wash or 1 mg polypeptide / wash.

[0435] The wash performance of exemplary polypeptides of the invention obtained from full scale washes are shown in Table 5a (40°C) and Table 5b (50°C) (where the wash performance scores are relative to the wash performance of the parent alpha-amylase of SEQ ID NO: 1).

[0436] Table 5a ADW wash performance of exemplary polypeptides of the invention

[0437]

[0438]

[0439] Table 5b ADW wash performance of exemplary polypeptides of the invention

[0440]

[0441]

[0442] Example 3: Evaluation of the wash performance of α-amylase polypeptides of the invention using a full-scale automated mechanical stress assay (AMSA)

[0443] In order to evaluate the washing performance of polypeptide of the present invention in detergent base composition, automatic mechanical stress determination (AMSA) can also be used to carry out washing experiment. Using AMSA test, the washing performance of a large number of small-volume enzyme detergent solutions can be checked. AMSA plate has many slits and lids for test solution, and the lid strongly squeezes the small pieces of textile to be washed to all slit openings. During the wash time, the flat plate, test solution, textile and lid are vigorously vibrated so that the test solution is contacted with the textile and mechanical stress is applied in a regular, periodic oscillation mode. For further description, referring to WO 02 / 42740 (especially the "determining method embodiment" paragraph of the 23rd-24th page, which is disclosed and incorporated herein by reference).

[0444] Overall description of washing performance

[0445] Test solutions were prepared comprising water (21° dH), 3.94 g / L of ADW standard detergent with bleach or 3.45 g / L of ADW standard detergent without bleach (as specified below) (see Tables 6, 7 and 8), and a polypeptide of the invention at a concentration of 0.03 mg, 0.06 mg, 0.12 mg and 0.24 mg polypeptide protein / L (40° C.) or 0.01 mg, 0.03 mg, 0.06 mg and 0.12 mg enzyme protein / L (50° C.). Fabrics stained with starch (CS-28 from the Test Materials Center BV, PO Box 120, 3133 KT, Vlaardingen, the Netherlands) were added and washed at 40° C. and 50° C. for 10 or 20 minutes, as described in detail below. After thorough rinsing under running tap water and drying in the dark, the light intensity values ​​of the stained fabrics were then measured as a measure of the washing performance. The test with 0 mg enzyme protein / L was used as blank and corresponds to the contribution from the detergent. Preferably, a mechanical action is applied during the washing step, for example in the form of shaking, rotating or stirring the washing solution together with the fabrics and tiles. The AMSA washing performance experiment was carried out under the experimental conditions detailed below:

[0446] Table 6 Experimental conditions

[0447]

[0448]

[0449] Table 7 ADW Standard Detergent with Bleach

[0450]

[0451] Table 8 ADW standard detergent without bleach

[0452]

[0453] By adding CaCl2, MgCl2 and NaHCO3 (Ca 2+ Mg 2+ :HCO3 - =4:1:10), the water hardness was adjusted to 21° dH. After washing, the textiles were rinsed with tap water and dried.

[0454] Washing performance can be measured as brightness, expressed as the intensity of light reflected from a sample when illuminated with white light. When a sample is stained, the intensity of its reflected light is lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure washing performance.

[0455] Color measurements were performed using a professional flatbed scanner (EPSON Expression 10000XL, EPSON) which was used to capture images of the washed textiles.

[0456] In order to extract the value of light intensity from the scanned image, the The color pixel value is converted into red, green, and blue (RGB) values. The intensity value (Int) is calculated by adding the RGB values ​​as a vector and then taking the length of the resulting vector:

[0457]

[0458] The wash performance of the variants according to the invention obtained by AMSA is as follows;

[0459] Table 9 AMSA washing performance of exemplary polypeptides of the invention

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

Claims

1. A polypeptide comprising a variant amino acid sequence of SEQ ID NO: 1, wherein the polypeptide has alpha-amylase activity and exhibits enhanced wash performance compared to the polypeptide of SEQ ID NO: 1, wherein the variant amino acid sequence is based on SEQ ID NO: 1 and wherein the substitution at position 118 is K118Q, K118H, K118N, K118R, wherein the numbering is according to the amino acid sequence listed in SEQ ID NO: 2, wherein the enhanced wash performance is evaluated using an automated mechanical stress assay (AMSA) method, wherein the polypeptide exhibits enhanced wash performance under one or more conditions selected from the group consisting of: (a) Standard ADW detergent, with bleach, at 40°C, and a wash cycle of 10 min; (b) standard ADW detergent, no bleach, at 40 °C, and a 10 min wash cycle; (c) standard ADW detergent, with bleach, at 50°C, and a wash cycle of 20 min; as well as (d) Standard ADW detergent, no bleach, at 50°C, and a 20 min wash cycle.

2. The polypeptide of claim 1, wherein the substitution is made at a position corresponding to: A51T+K118N+L202M+N270G, A51T+K118H+L202M+N270G, A51T+K118Q+L202M+N270G, A51T+K118R+L202M+N270G, W48F+K118H+V238A+S334T, A51T+K118H+L202M+N270G, or W48F+K118H+V238A+S334T.

3. A polynucleotide encoding the polypeptide according to any one of claims 1 to 2.

4. A nucleic acid construct comprising the polynucleotide according to claim 3.

5. An expression vector comprising the polynucleotide according to claim 3.

6. A host cell comprising the polynucleotide of claim 3, wherein the host cell is not a plant variety.

7. A method for producing an alpha-amylase polypeptide, the method comprising: (a) culturing the host cell according to claim 6 under conditions suitable for expressing the polypeptide; as well as (b) recovering the polypeptide.

8. A detergent composition comprising the polypeptide according to any one of claims 1 to 2 and a surfactant, a concentrate or an additive for preparing the detergent composition.

9. The composition according to claim 8, wherein the surfactant is selected from the group consisting of: Anionic surfactants, cationic surfactants, nonionic surfactants and amphoteric surfactants.

10. The composition according to claim 8 or 9, further comprising one or more additional components selected from the group consisting of: Oxidants, bleach activators, chelating agents, fillers, builders, buffers, structurants, sequestrants, optical brighteners, defoamers, enzymes, perfumes, anti-redeposition agents, skin conditioning agents, softeners, emulsifiers and colorants.

11. A composition according to any one of claims 8 to 10, wherein the composition is a liquid or powder laundry detergent composition.

12. A composition according to any one of claims 8 to 10, wherein the composition is a liquid or powder automatic dishwashing (ADW) detergent composition.

13. A composition according to any one of claims 8 to 10, wherein the composition is a liquid manual dishwashing detergent composition.

14. Use of a variant according to any one of claims 1 to 2, or a composition according to any one of claims 8 to 13, in a domestic or industrial cleaning process.

15. Use according to claim 14 for cleaning fabrics.

16. The use according to claim 14 for laundry washing.

17. The use according to claim 14, for cleaning ceramic, plastic or glass materials.

18. Use according to claim 17 for dishwashing.

19. A method of removing stains from a surface comprising contacting the surface with a composition according to any one of claims 8 to 13.

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