Polypeptides having trehalase activity

Patent metadata

Field	Value
Publication number	US-11220679-B2
Application number	US-201816635195-A
Country	US
Kind code	B2
Filing date	Aug 6, 2018
Priority date	Aug 8, 2017
Publication date	Jan 11, 2022
Grant date	Jan 11, 2022

Abstract

The present invention relates to trehalase variants of the Myceliophthora sepedonium GH37 trehalase. 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 trehalase variant polypeptide having increased stability against protease degradation and/or increased thermo-stability compared to SEQ ID NO: 1, comprising a substitution at one or more positions corresponding to position 152, 182, 185, or 583 of SEQ ID NO: 1, wherein the variant has at least 80%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

2. The variant of claim 1 , which comprises one or more substitutions selected from the group consisting of P152G, P152E, P152A, F182V, F185R, or G583T.

3. The variant of claim 1 having an increase in stability against protease degradation compared to SEQ ID NO: 1, comprising a substitution or combination of substitutions selected from:
G583T;
P152G;
P152E;
P152A;
F182V;
F185R;
P152G+G583T;
F185R+G583T;
P152A+F182V+G583T; and
and wherein the variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity and wherein the residual activity after 3 days incubation with A. niger protease mixture at 30° C., pH 4.0 is at least 50%, at least 60%, at least 70%, or at least 80%.

4. The variant of claim 1 having an increase in thermo-stability compared to SEQ ID NO: 1, comprising a substitution or combination of substitutions selected from:
P152G;
P152E;
P152A;
F182V;
F185R;
P152G+G583T;

F185R+G583T;
P152A+F182V+G583T; and
F182V+F185R+G583T;
and wherein the variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity and wherein the thermal denaturing temperature measure by TSA assay is at least 65.7° C.

5. The variant of claim 4 , wherein the thermal denaturing temperature is at least 65.8° C., at least 65.9° C., at least 66.0° C., at least 66.1° C., at least 66.2° C., at least 66.3° C., at least 66.4° C., at least 66.5° C., at least 66.6° C., at least 66.6° C., at least 66.8° C., at least 66.9° C., at least 67.0° C., at least 67.1° C., at least 67.2° C., at least 67.3° C., at least 67.4° C., at least 67.5° C.

6. A composition comprising a trehalase variant polypeptide having increased stability against protease degradation and/or increased thermo-stability compared to SEQ ID NO: 1, the trehalase variant polypeptide comprising a substitution at one or more positions corresponding to position 152, 182, 185, or 583 of SEQ ID NO: 1, wherein the variant has at least 80%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

7. A whole broth formulation or cell culture composition comprising the polypeptide of claim 1 .

8. The variant of claim 1 , wherein the variant has at least 85%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

9. The variant of claim 1 , wherein the variant has at least 90%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

10. The variant of claim 1 , wherein the variant has at least 95%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

11. The variant of claim 1 , wherein the variant has at least 97%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

12. The variant of claim 1 , wherein the variant has at least 99%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

13. The composition of claim 6 , wherein the variant has at least 85%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

14. The composition of claim 6 , wherein the variant has at least 90%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

15. The composition of claim 6 , wherein the variant has at least 95%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

16. The composition of claim 6 , wherein the variant has at least 97%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

17. The composition of claim 6 , wherein the variant has at least 99%, but less than 100% sequence identity to amino acids 1 to 674 of SEQ ID NO: 1 and wherein the variant has trehalase activity.

Assignees

• Novozymes As

Inventors

• Tsutsumi Noriko
• Matsui Tomoko
• Kurakata Yuma

Classifications

• C12P2203/00

  Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source · CPC title

• C12N9/2402 Primary

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C12Y302/01028

  Alpha,alpha-trehalase (3.2.1.28) · 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