Alpha-Amylase Variants and Polynucleotides Encoding Same

• US 20190316105A1
• Filed: 05/29/2019
• Published: 10/17/2019
• 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 E., Friis, Esben Peter, Micheelsen, Pernille Ollendorff, Viksoee-Nielsen, Anders, Deinhammer, Randy, Shen, Xinyu
• Granted Patent: US 10,619,141 B2
• Time in Patent Office: 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, in particular the substitution K176L, in combination with 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, 239, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO;

1;

wherein the variant has at least 60% and less than 100% sequence identity to (i) the mature polypeptide of any of SEQ ID NOs;

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO;

1, amino acids 1 to 483 of SEQ ID NO;

2, amino acids 1 to 485 of SEQ ID NO;

3, amino acids 1 to 482 of SEQ ID NO;

4, amino acids 1 to 484 of SEQ ID NO;

5, amino acids 1 to 483 of SEQ ID NO;

6, amino acids 1 to 485 of SEQ ID NO;

7, amino acids 1 to 485 of SEQ ID NO;

8, amino acids 1 to 485 of SEQ ID NO;

9, amino acids 1 to 485 of SEQ ID NO;

10, amino acids 1 to 485 of SEQ ID NO;

11, amino acids 1 to 480 of SEQ ID NO;

12, amino acids 1 to 483 of SEQ ID NO;

13 or amino acids 1 to 481 of SEQ ID NO;

14; and

wherein the variant has alpha-amylase activity.

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

3. The variant of claim 1, which comprises a substitution at one or more positions corresponding to any of positions 68, 185, 201, 205, 213, 360, 416 and 437.

4. The variant of claim 1, which comprises a substitution at one or more positions corresponding to any of positions 2, 68, 133, 142, 156, 158, 279 and 437.

5. The variant of claim 1 or 2, which comprises an alteration at two or more positions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450.

6. The variant of claim 1, which comprises a substitution at two or more positions corresponding to any of positions 68, 185, 201, 205, 213, 360, 416 and 437.

7. The variant of claim 1, which comprises a substitution at two or more positions corresponding to any of positions 2, 68, 133, 142, 156, 158, 279 and 437, in particular two or more substitutions selected from the group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W and R437W.

8. The variant of claim 1, wherein the variant has at least 65%, but less than 100%, sequence identity to (i) the mature polypeptide of any of SEQ ID NOs:

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO;

1, amino acids 1 to 483 of SEQ ID NO;

2, amino acids 1 to 485 of SEQ ID NO;

3, amino acids 1 to 482 of SEQ ID NO;

4, amino acids 1 to 484 of SEQ ID NO;

5, amino acids 1 to 483 of SEQ ID NO;

6, amino acids 1 to 485 of SEQ ID NO;

7, amino acids 1 to 485 of SEQ ID NO;

8, amino acids 1 to 485 of SEQ ID NO;

9, amino acids 1 to 485 of SEQ ID NO;

10, amino acids 1 to 485 of SEQ ID NO;

11, amino acids 1 to 480 of SEQ ID NO;

12, amino acids 1 to 483 of SEQ ID NO;

13 or amino acids 1 to 481 of SEQ ID NO;

14.

9. The variant of claim 1, which is a variant of a parent alpha-amylase selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to (i) the mature polypeptide of any of SEQ ID NOs;

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO;

1, amino acids 1 to 483 of SEQ ID NO;

2, amino acids 1 to 485 of SEQ ID NO;

3, amino acids 1 to 482 of SEQ ID NO;

4, amino acids 1 to 484 of SEQ ID NO;

5, amino acids 1 to 483 of SEQ ID NO;

6, amino acids 1 to 485 of SEQ ID NO;

7, amino acids 1 to 485 of SEQ ID NO;

8, amino acids 1 to 485 of SEQ ID NO;

9, amino acids 1 to 485 of SEQ ID NO;

10, amino acids 1 to 485 of SEQ ID NO;

11, amino acids 1 to 480 of SEQ ID NO;

12, amino acids 1 to 483 of SEQ ID NO;

13 or amino acids 1 to 481 of SEQ ID NO;

14;

or

(b) a fragment of the mature polypeptide of any of SEQ ID NOs;

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, which has alpha-amylase activity.

11. An isolated polynucleotide encoding the variant of claim 1.

12. A nucleic acid construct comprising the polynucleotide of claim 11.

13. An expression vector comprising the polynucleotide of claim 11.

14. A host cell comprising the polynucleotide of claim 11.

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

a. cultivating the host cell of claim 14 under conditions suitable for expression of the variant; and

b. recovering the variant.

10. (canceled)

16. (canceled)