Alpha-amylase variants and polynucleotides encoding same

• US 10,351,836 B2
• Filed: 03/07/2016
• Issued: 07/16/2019
• Est. Priority Date: 10/17/2011
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Andersen, Carsten, Larsen, Signe, Friis, Esben Peter, Micheelsen, Pernille Ollendorff, Viksoee-Nielsen, Anders, Deinhammer, Randy, Shen, Xinyu
• Primary Examiner(s): Monshipouri, Maryam
• Application Number:
  US15/062,644
• Publication Number:
  US 20160177280A1
• Time in Patent Office: 1,226 Days
• CPC Class Codes: C11D 3/386 Preparations containing enzymes , e.g. protease or amylase
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2417 from microbiological source
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12Y 302/01001 Alpha-amylase (3.2.1.1)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention relates to alpha-amylase variants. 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

1. An alpha-amylase variant, comprising a substitution at position 176 with L and a substitution at position 201 with Y, wherein

(a) each position corresponds to the positions of SEQ ID NO:

1;

(b) the variant has at least 90% and less than 100% sequence identity to SEQ ID NO;

14; and

(c) the variant has alpha-amylase activity.

2. The alpha-amylase variant of claim 1, which has at least 95% and less than 100% sequence identity to SEQ ID NO:

14.

3. The alpha-amylase variant of claim 1, further comprising a substitution at one or more positions corresponding to positions 68, 71, 126, 133, 142, 144, 156, 158, 185, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO:

1.

4. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 68 of SEQ ID NO:

1.

5. The alpha-amylase variant of claim 4, wherein the substitution at a position corresponding to position 68 is 68F, 68Y, or 68W.

6. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 185 of SEQ ID NO:

1.

7. The alpha-amylase variant of claim 6, wherein the substitution at a position corresponding to position 185 is 185P.

8. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 205 of SEQ ID NO:

1.

9. The alpha-amylase variant of claim 8, wherein the substitution at a position corresponding to position 205 is 205Y.

10. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 213 of SEQ ID NO:

1.

11. The alpha-amylase variant of claim 10, wherein the substitution at a position corresponding to position 213 is 213T.

12. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 360 of SEQ ID NO:

1.

13. The alpha-amylase variant of claim 12, wherein the substitution at position 360 is 360S.

14. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 416 of SEQ ID NO:

1.

15. The alpha-amylase variant of claim 14, wherein the substitution at a position corresponding to position 416 is 416V.

16. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 437 of SEQ ID NO:

1.

17. The alpha-amylase variant of claim 16, wherein the substitution at a position corresponding to position 437 is 437W.

18. The alpha-amylase variant of claim 1, selected from the group consisting of:

68W+176L+185P+201Y+205Y+213T+360S+416V+437W,

68W+176L+201Y+205Y+213T+360S+416V+437W,

126W+176L+201Y+205Y+213T+360S+416V+437W,

133Y+158W+176L+185P+201Y+205Y+213T+360S+416V+437W,

133Y+176L+201Y+205Y+213T+360S+416V+437W,

142W+158W+176L+185P+201Y+205Y+213T+360S+416V+437W,

142W+176L+185P+201Y+205Y+213T+360S+416V+437W,

142W+176L+201Y+205Y+213T+360S+416V+437W,

156W+158W+176L+185P+201Y+205Y+213T+360S+416V+437W,

156W+176L+201Y+205Y+213T+360S+416V+437W,

158W+176L+185P+201Y+205Y+207V+209D+316W,

158W+176L+185P+201Y+205Y+213T+316L+318W+360S+416V+437W,

158W+176L+185P+201Y+205Y+213T+316W+360S+416V+437W,

158W+176L+185P+201Y+205Y+213T+360S+416V+437W,

158W+176L+201Y+205Y+213T+360S+416V+437W,

176L+185P+201Y+205Y+213T+360S+416V+437W,

176L+185P+201Y+205Y+437W,

176L+201Y+205Y+213T+239Q+360S+416V+437W,

176L+201Y+205Y+213T+316W+360S+416V+437W,

176L+201Y+205Y+213T+318W+360S+416V+437W,

176L+201Y+205Y+213T+360S+416V+437W,

176L+201Y+205Y+213T+360S+437W,

176L+201Y+205Y+213T+416V+437W, and

176L+201Y+205Y+360S+416V+437W.

19. A detergent composition comprising the alpha-amylase variant of claim 1 and a surfactant.

20. A method of cleaning a hard surface or a textile, comprising contacting the hard surface or the textile with the alpha-amylase variant of claim 1.

21. A method of producing liquefied starch, comprising liquefying a starch-containing material with the alpha-amylase variant of claim 1.

22. A method of producing a fermentation product, comprising

(a) liquefying a starch-containing material with the alpha-amylase variant of claim 1 to produce a liquefied mash;

(b) saccharifying the liquefied mash to produce fermentable sugars; and

(c) fermenting the fermentable sugars in the presence of a fermenting organism.

23. An isolated polynucleotide encoding the alpha-amylase variant of claim 1.

24. A nucleic acid construct or expression vector comprising the polynucleotide of claim 23, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in a host cell.

25. A host cell comprising the nucleic acid construct or expression vector of claim 24.

26. A method of producing an alpha-amylase variant, comprising:

(a) cultivating the host cell of claim 25 under conditions suitable for expression of the variant; and

(b) recovering the alpha-amylase variant.