Alpha-amylase variants and polynucleotides encoding same

• US 10,619,141 B2
• Filed: 05/29/2019
• Issued: 04/14/2020
• 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 E., Friis, Esben Peter, Micheelsen, Pernille Ollendorff, Viksoee-Nielsen, Anders, Deinhammer, Randy, Shen, Xinyu
• Primary Examiner(s): Monshipouri, Maryam
• Application Number:
  US16/425,184
• Publication Number:
  US 20190316105A1
• Time in Patent Office: 321 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 a position corresponding to position 176 of SEQ ID NO:

1 and an alteration at a position corresponding to position 239 of SEQ ID NO;

1, wherein the variant has at least 90% and less than 100% sequence identity to SEQ ID NO;

14; and

the variant has alpha-amylase activity.

2. The alpha-amylase variant of claim 1, wherein the substitution at a position corresponding to position 176 is with Leu.

3. The alpha-amylase variant of claim 1, wherein the alteration at a position corresponding to position 239 is a substitution with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.

4. The alpha-amylase variant of claim 2, wherein the alteration at a position corresponding to position 239 is a substitution with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.

5. The alpha-amylase variant of claim 1, wherein the alteration at a position corresponding to position 239 is a substitution with Ala or Gln.

6. The alpha-amylase variant of claim 2, wherein the alteration at a position corresponding to position 239 is a substitution with Ala or Gln.

7. The alpha-amylase variant of claim 1, wherein the variant has at least 95% sequence identity to SEQ ID NO:

14.

8. The alpha-amylase variant of claim 1, further comprising an alteration at one or more positions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 185, 201, 205, 213, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO:

1.

9. The alpha-amylase variant of claim 8, wherein the alteration at one or more positions is selected from the group consisting of:

a substitution at a position corresponding to position 1 with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 2 with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 68 with Ala, Arg, Asn, Asp, Cys, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 71 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 126 with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 133 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 142 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 144 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Va;

a substitution at a position corresponding to position 156 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val;

a substitution at a position corresponding to position 158 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val;

a substitution at a position corresponding to position 185 with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 201 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 205 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 213 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 279 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 316 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 318 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 360 with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 416 with Ala, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

a substitution at a position corresponding to position 437 with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and

a substitution at a position corresponding to position 450 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

10. The alpha-amylase variant of claim 8, wherein the alteration at one or more positions is selected from the group consisting of:

a substitution at a position corresponding to position 1 with His or Trp;

a substitution at a position corresponding to position 2 with His or Trp;

a substitution at a position corresponding to position 68 with Phe, Tyr or Trp;

a substitution at a position corresponding to position 71 with Phe, His, Tyr or Trp;

a substitution at a position corresponding to position 126 with Phe, His, Tyr or Trp;

a substitution at a position corresponding to position 133 with Phe, Tyr or Trp;

a substitution at a position corresponding to position 142 with Phe, Tyr or Trp;

a substitution at a position corresponding to position 144 with Phe, His, Tyr or Trp;

a substitution at a position corresponding to position 156 with Phe, or Trp;

a substitution at a position corresponding to position 158 with Phe, His or Trp;

a substitution at a position corresponding to position 185 with Pro;

a substitution at a position corresponding to position 201 with Tyr;

a substitution at a position corresponding to position 205 with Tyr;

a substitution at a position corresponding to position 213 with Thr;

a substitution at a position corresponding to position 279 with His, Tyr or Trp;

a substitution at a position corresponding to position 316 with Phe, Tyr or Trp;

a substitution at a position corresponding to position 318 with Phe, His, Tyr or Trp;

a substitution at a position corresponding to position 360 with Ser;

a substitution at a position corresponding to position 416 with Val;

a substitution at a position corresponding to position 437 with Phe, His, Tyr or Trp; and

a substitution at a position corresponding to position 450 with Phe, Tyr or Trp.

11. The alpha-amylase variant of claim 1, which is an isolated alpha-amylase variant.

12. The alpha-amylase variant of claim 1, further comprising a substitution at one or more positions corresponding to positions 201, 205, 213, 360, 416, and 437.

13. The alpha-amylase variant of claim 12, wherein the substitution is 201Y, 205Y, 213T, 360S, 416V, or 437W.

14. The alpha-amylase variant of claim 3, further comprising a substitution at one or more positions corresponding to positions 201, 205, 213, 360, 416, and 437.

15. The alpha-amylase variant of claim 14, wherein the substitution is 201Y, 205Y, 213T, 360S, 416V, or 437W.

16. The alpha-amylase variants of claim 1, which comprises substitutions at positions corresponding to position 176, 201, 205Y, 213, S239, 360, 416, and 437.

17. The alpha-amylase variants of claim 16, wherein the substitutions are K176L, F201Y, H205Y, K213T, S239Q, Q360S, D416V, and R437W.

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

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

20. 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.