Subtilase variants

Patent metadata

Field	Value
Publication number	US-12529016-B2
Application number	US-202318401382-A
Country	US
Kind code	B2
Filing date	Dec 30, 2023
Priority date	Dec 15, 2014
Publication date	Jan 20, 2026
Grant date	Jan 20, 2026

Abstract

The present invention relates to novel protease variants exhibiting improved stability and or improved wash performance in liquid detergent. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention.

First claim

The invention claimed is:

1 . A subtilase variant having at least 90% sequence identity to SEQ ID NO: 3 wherein the variant has a glutamic acid residue (E) in position 101, wherein the subtilase variant further comprises one or more substitutions selected from the group consisting of S156D, L262E, S3T, and Y209W, wherein the variant has increased stability in a liquid detergent composition compared to the subtilase having the amino acid sequence of SEQ ID NO: 3 and wherein the positions correspond to the positions of SEQ ID NO: 1.

2 . The subtilase variant of claim 1 , wherein the subtilase further has improved wash performance compared with the subtilase having SEQ ID NO: 3.

3 . The subtilase variant of claim 1 , wherein the subtilase further has improved stability compared with the subtilase having SEQ ID NO: 3.

4 . The subtilase variant of claim 1 , wherein the subtilase variant further comprises one or more substitutions selected from the group consisting of: R45E,D,Q; T58L; G61D; S87E; G97S; A98E; S160A; N117E; H120D,K,V; P129D; E136Q; Q137H; S161T; S163A,G; V199M; M222Q; and N261T.

5 . The subtilase variant of claim 1 , wherein the variant is selected from the group consisting of:
SEQ ID NO: 3+S3T, SEQ ID NO: 3+Y209W, SEQ ID NO: 3+L262N,Q,D,E SEQ ID NO: 3+S156D+L262E SEQ ID NO: 3+N238E+L262E SEQ ID NO: 3+S3T+N76D+S156D+Y209W SEQ ID NO: 3+S163G+N128Q+N238E+L262E SEQ ID NO: 3+V104T+H120D+S156D+L262E SEQ ID NO: 3+S3T+V41+N261D SEQ ID NO: 3+S3T+V41+A228V SEQ ID NO: 3+N76D+A228V+L262E SEQ ID NO: 3+Q137H+S141H+R145H+N238E+L262E SEQ ID NO: 3+S3T+N76D+Q137H+S141H+R145H+S156D+Y209W SEQ ID NO: 3+S3T+Q59D+N76D SEQ ID NO: 3+S3T+N76D+H120D SEQ ID NO: 3+S3T+N76D+A194P+G195E+V199M+V2051 SEQ ID NO: 3+S3T+N76D+S156D SEQ ID NO: 3+S3T+N76D+Y209W+N261D SEQ ID NO: 3+S3T+N76D+H120D+Y209W SEQ ID NO: 3+S3T+N76D+S156D+Y209W SEQ ID NO: 3+S3T+V41+N76D+A228V+N261D SEQ ID NO: 3+S3T+V41+N76D+H120D SEQ ID NO: 3+N76D+A228V+L262E SEQ ID NO: 3+N261D+L262E SEQ ID NO: 3+S87E+L262E SEQ ID NO: 3+Q59D+L262E SEQ ID NO: 3+K27Q+L262E SEQ ID NO: 3+H120D+L262E SEQ ID NO: 3+K27Q+S156D SEQ ID NO: 3+H120D+S163G+L262E SEQ ID NO: 3+S3T+N76D SEQ ID NO: 3+S3T+H120D SEQ ID NO: 3+S3T+S163G SEQ ID NO: 3+S156D+S163G SEQ ID NO: 3+S3T+T58L SEQ ID NO: 3+T58L+S156D SEQ ID NO: 3+S3T+N76D+H120D SEQ ID NO: 3+S3T+N76D+A228V SEQ ID NO: 3+S3T+N76D+S156D SEQ ID NO: 3+S3T+N76D+Y209W SEQ ID NO: 3+S3T+N76D+Y209W+V244T SEQ ID NO: 3+N76D+S156D SEQ ID NO: 3+H120D+S156D SEQ ID NO 3+R45E+L262E SEQ ID NO 3+S87E+L262E SEQ ID NO 3+G61D+L262E SEQ ID NO 3+Q59D+L262E SEQ ID NO 3+Q59D+S156D SEQ ID NO 3+S156D+L262E SEQ ID NO 3+S163G+N238E+L262E SEQ ID NO 3+S3T+V41+S163G+N261D SEQ ID NO 3+S3T+V41+S163G+N261D SEQ ID NO 3+S87E+S163G+L262E SEQ ID NO 3+S156D+S163G+L262E SEQ ID NO 3+T58L+S163G+N261D SEQ ID NO 3+S156D+S163G+L262E and SEQ ID NO 3+S3T+N76D+Y209W+N261D+L262E.

6 . A nucleotide sequence encoding a variant according to claim 1 .

7 . An expression vector comprising the nucleotide sequence of claim 6 .

8 . A recombinant host cell comprising the nucleotide sequence of claim 6 or the expression vector of claim 7 .

9 . A method for producing the variant according to claim 1 , comprising:
Providing a recombinant host cell comprising a nucleotide sequence encoding a subtilase variant having at least 90% sequence identity to SEQ ID NO: 3 wherein the variant has a glutamic acid residue (E) in position 101, wherein the subtilase variant further comprises one or more substitutions selected from the group consisting of S156D, L262E, S3T, and Y209W, wherein the variant has increased stability in a liquid detergent composition compared to the subtilase having the amino acid sequence of SEQ ID NO: 3 and wherein the positions correspond to the positions of SEQ ID NO: 1; Culturing the recombinant host cell under conditions leading to expression of the variant; and Isolating the variant.

10 . The variant of claim 1 , wherein the variant has at least 95% sequence identity to SEQ ID NO: 3.

11 . The variant of claim 1 , wherein the variant has at least 96% sequence identity to SEQ ID NO: 3.

12 . The variant of claim 1 , wherein the variant has at least 97% sequence identity to SEQ ID NO: 3.

13 . The variant of claim 1 , wherein the variant has at least 98% sequence identity to SEQ ID NO: 3.

14 . The variant of claim 1 , wherein the subtilase variant further comprises two or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S124M, P129D, E136Q, S143W, S161T, S163A,G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D.

15 . The variant of claim 1 , wherein the subtilase variant further comprises three or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S124M, P129D, E136Q, S143W, S161T, S163A,G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D.

Assignees

• Novozymes As

Inventors

• Toscano Miguel Duarte Guilherme Pereira
• Friis Esben Peter
• Lenhard Rolf Thomas
• Larsen Signe Eskildsen
• Munch Astrid
• Lue Bena-Marie
• Bauer Mikael

Classifications

• C12Y304/21112

  Site-1 protease (3.4.21.112), i.e. subtilisin kexin isozyme-1 · CPC title

• C12N9/54 Primary

  bacteria being Bacillus · CPC title

• C11D3/38618 Primary

  Protease or amylase in liquid compositions only · CPC title