Method for screening alpha-amylases

Patent metadata

Field	Value
Publication number	US-11718840-B2
Application number	US-201916531247-A
Country	US
Kind code	B2
Filing date	Aug 5, 2019
Priority date	Jun 30, 2011
Publication date	Aug 8, 2023
Grant date	Aug 8, 2023

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.

First claim

The invention claimed is:

1. A method for screening variant alpha-amylases of a parent alpha-amylase of SEQ ID NO: 2, comprising the steps of
a) determining the binding of the alpha-amylases to a solid or an immobilized substrate for the alpha amylase; and
b) selecting variant alpha-amylases having a lower binding to solid starch and higher wash performance at low temperature compared with the parent alpha-amylase, wherein the variant alpha-amylases have at least 80% sequence identity but less than 100% sequence identity to SEQ ID NO: 2 and comprise a deletion of positions 181+182, 182+183, or 183+184, and further comprise one or two or more substitutions in any of positions corresponding to W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, W347, W469, G476 and G477 in SEQ ID NO: 2.

2. The method of claim 1 , wherein the variant alpha-amylases have high wash performance at low temperature.

3. The method of claim 1 , wherein the alpha-amylases have less than 90% of the binding to starch than the alpha-amylase of SEQ ID NO: 2.

4. A method for selecting variants of a parent alpha-amylase of SEQ ID NO: 2, comprising the steps of
a) generating variants having at least 80% sequence identity but less than 100% sequence identity to SEQ ID NO: 2 and comprising a deletion of positions 181+182, 182+183, or 183+184, and further comprising one or two or more substitutions in any of positions corresponding to W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, W347, W469, G476 and G477 in SEQ ID NO: 2;
b) testing the variants for binding to a solid or an immobilized substrate for the alpha-amylase; and
c) selecting variants having a lower binding to the solid or the immobilized substrate than the parent alpha-amylase.

5. The method of claim 4 , wherein the variants have less than 90% of the binding to starch than the parent alpha-amylase.

6. A variant polypeptide having alpha-amylase activity and having at least 80% sequence identity but less than 100% sequence identity to SEQ ID NO: 2, wherein the variant comprises a deletion of positions 181+182, or 182+183, or 183+184 and further comprises one or two or more substitutions in any of positions corresponding to W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, W347, W469, G476 and G477 in SEQ ID NO: 2, and wherein the variant alpha-amylase has lower binding to solid starch and higher wash performance at low temperature compared with the parent alpha-amylase.

7. The variant alpha-amylase of claim 6 , wherein an amino acid residue in one or more substrate binding sites at the surface of the molecule has been modified.

8. The variant alpha-amylase of claim 6 , having at least 90% sequence identity to SEQ ID NO: 2 and comprising substitutions in positions corresponding to the following positions in SEQ ID NO: 2:
H183*+G184*+Q169N;
H183*+G184*+Q169A;
H183*+G184*+W189Y+E190P;
H183*+G184*+N260D;
H183*+G184*+G477E;
H183*+G184*+G477Q;
H183*+G184*+G477K;
H183*+G184*+W189E+E190P;
H183*+G184*+E194D;
H183*+G184*+Y160S;
H183*+G184*+W189F+E190P;
H183*+G184*+F262A;
H183*+G184*+Y363H;
H183*+G184*+G476E;
H183*+G184*+N260P+G477E;
H183*+G184*+G109A+M202L+Y203G;
H183*+G184*+E194S;
H183*+G184*+E345D+G477R;
H183*+G184*+W167F+N260D+F262P+P380Q+G477K;
H183*+G184*+W159Y+W167Y+N260D+G476E+G477Q;
H183*+G184*+N260D+F262P+W469Y+G476R+G477Q;
H183*+G184*+W167Y+L2301+N260P+W469Y;
H183*+G184*+W159Y+W167Y+N260P+E439Y+G476Q+G477Q; and
H183*+G184*+W167Y+F262P+W469Y+G476R+G477Q.

9. The variant alpha-amylase of claim 8 , which consists of SEQ ID NO: 2 with a substitution selected among:
H183*+G184*+Q169N;
H183*+G184*+Q169A;
H183*+G184*+W189Y+E190P;
H183*+G184*+N260D;
H183*+G184*+G477E;
H183*+G184*+G477Q;
H183*+G184*+G477K;
H183*+G184*+W189E+E190P;
H183*+G184*+E194D;
H183*+G184*+Y160S;
H183*+G184*+W189F+E190P;
H183*+G184*+F262A;
H183*+G184*+Y363H;
H183*+G184*+G476E;
H183*+G184*+N260P+G477E;
H183*+G184*+G109A+M202L+Y203G;
H183*+G184*+E194S;
H183*+G184*+E345D+G477R;
H183*+G184*+W159Y+W167F+F262P+W469Y+G476R;
H183*+G184*+W167F+N260D+F262P+P380Q+G477K;
H183*+G184*+W159Y+W167Y+N260D+G476E+G477Q;
H183*+G184*+N260D+F262P+W469Y+G476R+G477Q;
H183*+G184*+W167Y+L2301+N260P+W469Y;
H183*+G184*+W159Y+W167Y+N260P+E439Y+G476Q+G477Q; and
H183*+G184*+W167Y+F262P+W469Y+G476R+G477Q.

10. A detergent composition comprising the variant alpha-amylase of claim 6 .

11. The variant polypeptide of claim 6 , wherein the polypeptide has at least 90% sequence identity but less than 100% sequence identity to SEQ ID NO: 2.

12. The variant polypeptide of claim 6 , wherein the polypeptide has at least 95% sequence identity but less than 100% sequence identity to SEQ ID NO: 2.

13. The variant polypeptide of claim 6 , wherein the variant alpha-amylase has lower binding to solid starch and higher wash performance at low temperature compared with the parent alpha-amylase, wherein low temperature is a temperature of 5-35° C.

14. The variant polypeptide of claim 6 , wherein the variant alpha-amylase has lower binding to solid starch and higher wash performance at low temperature compared with the parent alpha-amylase, wherein low temperature is a temperature of 5-20° C.

Assignees

• Novozymes As

Inventors

• Kaasgaard Svend
• Larsen Signe Eskildsen
• Oebro Jens
• Beier Lars
• Pontoppidan Connie
• Damager Iben
• Andersen Carsten
• Svendsen Allan

Classifications

• C12N9/2417 Primary

  from microbiological source · CPC title

• C12N9/2414 Primary

  Alpha-amylase (3.2.1.1.) · CPC title

• C12Q1/40

  involving amylase · CPC title

• C11D3/386

  Preparations containing enzymes {, e.g. protease or amylase} · CPC title

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title