# US 9,434,932 B2 · Alpha-amylase variants > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,434,932 B2 - Document ID: US9434932B2 - Status: Granted - Kind: B2 - Application number: 14/129,565 - Filed: 2012-06-29 - Published: 2014-05-22 - Granted: 2016-09-06 - Earliest claimed: 2011-07-01 - Assignees: Novozymes A/S - Applicants: Svend Kaasgaard; Jens Oebro; Signe Eskildsen Larsen; Allan Svendsen; Annette Helle Johansen; Michael Skjoet; Carsten Andersen; Lars Beier; Esben Peter Friis; Miguel Duarte Guilherme Pereira Toscano; Mads Bjoernvad; Frank Winther Rasmussen; Liv Spaanger Christiansen - Inventors: Svend Kaasgaard; Jens Oebro; Signe Eskildsen Larsen; Allan Svendsen; Annette Helle Johansen; Michael Skjoet; Carsten Andersen; Lars Beier; Esben Peter Friis; Miguel Duarte Guilherme Pereira Toscano; Mads Bjoernvad; Frank Winther Rasmussen; Liv Spaanger Christiansen - Web page: https://patentgrep.com/patent/US9434932B2 ## CPC - C12N 9/2417: {from microbiological source} - C12N 9/2414: {Alpha-amylase (3.2.1.1.)} - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - C12Y 302/01001: Alpha-amylase (3.2.1.1) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract The present invention relates to variants of a parent alpha-amylase. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants. ## Claims ### Claim 1 An isolated variant of a parent alpha-amylase, comprising at least two substitutions at positions corresponding to 140 and 206 of the polypeptide of SEQ ID NO: 1, wherein the variant has at least 90% sequence identity with the polypeptide of SEQ ID NO: 1, and has alpha-amylase activity, and wherein the substitution at position 206 is V206Y. ### Claim 2 (depends on 1) The variant according to claim 1, which comprises a substitution at three positions selected from the group consisting of G304, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. ### Claim 3 (depends on 1) The variant according to claim 1, which comprises a substitution at four positions selected from the group consisting of G304, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. ### Claim 4 (depends on 1) The variant according to claim 1, which further comprises a substitution at two, three or four positions selected from the group consisting of G304, E260 and G476. ### Claim 5 (depends on 1) The variant of claim 1, which further comprises two or more substitutions selected from the group consisting of G304R, W140YF, W189EGT, D134E, E260GHIKNRTY, W284DFR, W439RG, G476EK, G477EKMR. ### Claim 6 (depends on 1) The variant according to claim 1, which comprises substitutions at two, three or four positions selected from the group consisting of G304R, W140YF, E260GHIKNRTY and G476EK. ### Claim 7 (depends on 6) The variant according to claim 6, wherein the substitutions at the two, three or four positions are selected from the group consisting of G304R, W140Y, E260G and G476K. ### Claim 8 (depends on 1) The variant of claim 1, further comprising one or more substitutions selected from the group consisting of N195F, Y243F, G109A, G273DV, G337N, K72R, R181H, S303G and Y100I. ### Claim 9 (depends on 1) The variant according to claim 1, wherein the number of alterations is 2-20. ### Claim 10 (depends on 1) The variant according to claim 1, which has at least 95% sequence identity with the polypeptide of SEQ ID NO: 1. ### Claim 11 (depends on 1) The variant according to claim 1, which comprises substitutions in the positions, corresponding to the positions of the polypeptide of SEQ ID NO: 1, selected from the group consisting of: W140Y+N195F+V206Y+Y243F+E260G+G477E, W140Y+N195F+V206Y+Y243F+E260T+W284D, W140Y+N195F+V206Y+Y243F+W284D, G109A+W140Y+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G+G476E, W140Y+W189G+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G+S303G, W140Y+W189T+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G+W284D, Y100I+W140Y+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G+G337N, W140Y+N195F+V206Y+Y243F+E260G+W439R, G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G, G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E, T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G, T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R, T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E, W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K, W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K, and W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R. ### Claim 12 (depends on 1) The variant according to claim 1, which consists of substitutions in the positions, corresponding to the positions of the polypeptide of SEQ ID NO: 1, selected from the group consisting of: W140Y+N195F+V206Y+Y243F+E260G+G477E, W140Y+N195F+V206Y+Y243F+E260T+W284D, W140Y+N195F+V206Y+Y243F+W284D, G109A+W140Y+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G+G476E, W140Y+W189G+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G+S303G, W140Y+W189T+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G+W284D, Y100I+W140Y+N195F+V206Y+Y243F+E260G, W140Y+N195F+V206Y+Y243F+E260G+G337N, W140Y+N195F+V206Y+Y243F+E260G+W439R, G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G, G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E, T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G, T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R, T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E, W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K, W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K, W140Y+N195F+V206Y+Y243F+E260G+W284F+G477K. ### Claim 13 (depends on 1) The variant according to claim 1, which further comprises deletions at positions corresponding to positions G182*+D183* or D183*+G184* of SEQ ID NO:1. ### Claim 14 (depends on 1) The variant according to claim 1, which comprises alterations in the positions, corresponding to the positions of the polypeptide of SEQ ID NO: 1, selected from the group consisting of: D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G477E, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260T+W284D, D183*+G184*+W140Y+N195F+V206Y+Y243F+W284D, D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G476E, D183*+G184*+W140Y+W189G+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+S303G, D183*+G184*+W140Y+W189T+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284D, D183*+G184*+Y100I+W140Y+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G337N, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W439R, D183*+G184*+G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G, D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E, D183*+G184*+T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G, D183*+G184*+T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R, D183*+G184*+T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K, and D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R. ### Claim 15 (depends on 1) The variant according to claim 1, which consists of alterations in the positions, corresponding to the positions of the polypeptide of SEQ ID NO: 1, selected from the group consisting of: D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G477E, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260T+W284D, D183*+G184*+W140Y+N195F+V206Y+Y243F+W284D, D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G476E, D183*+G184*+W140Y+W189G+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+S303G, D183*+G184*+W140Y+W189T+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284D, D183*+G184*+Y100I+W140Y+N195F+V206Y+Y243F+E260G, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G337N, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W439R, D183*+G184*+G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G, D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E, D183*+G184*+T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G, D183*+G184*+T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R, D183*+G184*+T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K, D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K, and D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R. ### Claim 16 (depends on 1) An isolated polynucleotide encoding the variant of claim 1. ### Claim 17 (depends on 16) A nucleic acid construct comprising the polynucleotide of claim 16. ### Claim 18 (depends on 16) An expression vector comprising the polynucleotide of claim 16. ### Claim 19 (depends on 16) A host cell comprising the polynucleotide of claim 16. ### Claim 20 (depends on 19) A method of producing a variant of a parent alpha-amylase, comprising cultivating the host cell of claim 19 under conditions suitable for the expression of the variant; and recovering the variant. ### Claim 21 An isolated variant of a parent alpha-amylase, comprising substitutions at positions corresponding to 140 and 206 of the polypeptide of SEQ ID NO: 1, wherein the variant has at least 99% sequence identity with the polypeptide of SEQ ID NO: 1, wherein the substitution at position 206 is characterized as V206Y, and wherein the variant has alpha-amylase activity.