Alpha amylase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-9267124-B2
Application number	US-201214131148-A
Country	US
Kind code	B2
Filing date	Jul 6, 2012
Priority date	Jul 6, 2011
Publication date	Feb 23, 2016
Grant date	Feb 23, 2016

Abstract

The present invention relates to variants of 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.

First claim

The invention claimed is:

1. An alpha amylase variant, comprising a substitution at one or more positions corresponding to positions 128, 143, 141, 192, 20, 76, 123, 136, 142, 165, 219, 224, 265, 383, and 410 of the mature polypeptide of SEQ ID NO: 2, wherein the variant has alpha amylase activity, and wherein the variant alpha amylase has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 2.

2. The variant of claim 1 , selected from the group consisting of:
a) a polypeptide having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2;
b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complementary strand of (i);
c) a polypeptide encoded by a polynucleotide with at least 95% identity with the mature polypeptide coding sequence of SEQ ID NO: 1; or
d) a fragment of the mature polypeptide of SEQ ID NO: 2, which has alpha amylase activity.

3. The variant of claim 2 , wherein the variant alpha amylase consists of the mature polypeptide of SEQ ID NO: 2 having a substitution, at one or more positions corresponding to positions 128, 143, 141, 192, 20, 76, 123, 136, 142, 165, 219, 224, 265, 383, and 410 of the mature polypeptide of SEQ ID NO: 2, and wherein the variant has alpha amylase activity.

4. The variant of claim 1 , wherein the mature polypeptide of SEQ ID NO: 2 is the polypeptide of SEQ ID NO: 3.

5. The variant of claim 1 , wherein the number of alterations is 1-20.

6. The variant according to claim 1 , wherein the variant further comprises a linker and a carbohydrate binding module.

7. The variant of claim 1 , which comprises one or more (several) substitutions selected from the group consisting of G20S, A76G, S123H, G128D, K136F, Y141W, Y141R, N142D, D143N, D165M, K192R, P219C, P224A, P224R, A265C, N383R, and V410A.

8. The variant of claim 7 , wherein the variant comprises at least one of the following substitutions or combinations of substitutions:
D165M; or
Y141W; or
Y141R; or
K136F; or
K192R; or
P224A; or
P224R; or
S123H+Y141W; or
G20S+Y141W; or
A76G+Y141W; or
G128D+Y141W; or
G128D+D143N; or
P219C+Y141W; or
N142D+D143N; or
Y141W+K192R; or
Y141W+D143N; or
Y141W+N383R; or
Y141W+P219C+A265C; or
Y141W+N142D+D143N; or
Y141W+K192R V410A; or
G128D+Y141W+D143N; or
Y141W+D143N+P219C; or
Y141W+D143N+K192R; or
G128D+D143N+K192R; or
Y141W+D143N+K192R+P219C; or
G128D+Y141W+D143N+K192R; or
G128D+Y141W+D143N+K192R+P219C.

9. The variant according to claim 6 , wherein the carbohydrate-binding module is a polypeptide comprising an amino acid sequence which has at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

10. The variant according to claim 9 , wherein the linker has at least 90% sequence identity to SEQ ID NO:19 and carbohydrate-binding module has at least 90% sequence identity to SEQ ID NO: 36.

11. The variant according to claim 10 , wherein the linker has at least 90% sequence identity to SEQ ID NO:21 and carbohydrate-binding module has at least 90% sequence identity to SEQ ID NO: 38.

12. A nucleic acid construct comprising the polynucleotide encoding the variant of claim 1 .

13. An expression vector comprising the polynucleotide of claim 12 .

14. A host cell comprising the polynucleotide of claim 13 .

15. A method of producing a variant alpha amylase, comprising:
a) cultivating the host cell of claim 14 under conditions suitable for the expression of the variant; and
b) recovering the variant.

16. A method for producing a fermentation product from starch-containing material comprising the steps of:
(a) liquefying starch-containing material using a variant alpha-amylase according to claim 1 ;
(b) saccharifying the liquefied material obtained in step (a) using a glucoamylase; and
(c) fermenting the saccharified material using a fermenting organism.

17. A method for producing a fermentation product from starch-containing material comprising:
(a) saccharifying starch-containing material with a variant alpha amylase according to claim 1 , and a glycoamylase at a temperature below the initial gelatinization temperature of said starch-containing material,
(b) fermenting using a fermenting organism.

18. The method according to claim 16 , wherein the fermentation product is ethanol.

19. A method for producing an enzymatically modified starch derivative, comprising using a variant having alpha-amylase activity according to claim 1 for liquefying and/or saccharifying starch.

Assignees

• Matsui Tomoko
• Tomiki Aki
• Coward-Kelly Guillermo
• Novozymes As

Inventors

• Matsui Tomoko
• Tomiki Aki
• Coward-Kelly Guillermo

Classifications

• C12N9/2417 Primary

  from microbiological source · CPC title

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title

• C12P19/20

  produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose · CPC title

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title