Protease variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-10400230-B2
Application number	US-201515535883-A
Country	US
Kind code	B2
Filing date	Dec 18, 2015
Priority date	Dec 19, 2014
Publication date	Sep 3, 2019
Grant date	Sep 3, 2019

Abstract

The present invention relates to protease variants and methods for obtaining protease 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.

First claim

The invention claimed is:

1. A protease variant comprises one or more substitutions selected from the group consisting of Q70F, Q70A, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein the positions corresponds to the positions of SEQ ID NO: 3, wherein the variant has a sequence identity to SEQ ID NO: 3 of at least 80% and wherein the variant has protease activity.

2. A protease variant of claim 1 , which has reduced Ki compared to the parent or compared to the protease with SEQ ID NO: 3.

3. A protease variant of claim 1 , wherein the variant is selected from the group consisting of:
a) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 2;
b) a fragment of the mature polypeptide of SEQ ID NO: 2, which has protease activity; and
c) a protease variant having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 2, and comprising an alteration selected from the group consisting of:
S114Q,S173P,S175P,F180Y;
K146T,S173P,S175P,F180Y;
K146N,S173P,S175P,F180Y;
K146W,S173P,S175P,F180Y;
K146F,S173P,S175P,F180Y;
K146A,S173P,S175P,F180Y;
I150A,S173P,S175P,F180Y;
I150N,S173P,S175P,F180Y;
S27K,I150N,S171N,S173P,G174R,S175P,F180Y,Q198E,T297P;
S27K,K146P,S148R,A151R,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,T297P;
S173P,S175P,N176Y,F180Y;
S173P,S175P,I178Y,F180Y;
S173P,S175P,I178F,F180Y;
S173P,S175P,I178F,F180Y;
S173P,S175P,I178P,F180Y;
S173P,S175P,F180Y,L184F;
S173P,S175P,F180Y,L184F;
S27K,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297P;
S27K,I121V,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297P;
S27K,Q70N,G107N,I121V,E127Q,S173P,S175P,F180Y,L184F,Q198E,T297P;
S27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199K,T297P;
S27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199R,T297P;
S173P,S175P,F180Y,L184Y;
S27K,S171N,S173P,G174R,S175P,F180Y,L184Y,Q198E,T297P;
S27K,S173P,G174K,S175P,F180Y,L184Y,Q198E,N199K,T297P;
S27K,S173P,G174K,S175P,F180Y,L184Y,Q197K,Q198E,T297P;
S173P,S175P,F180Y,L184W;
S27K,S171N,S173P,G174R,S175P,F180Y,L184W,Q198E,T297P;
S173P,S175P,F180Y,L184D;
S173P,S175P,F180Y,R224D;
S173P,S175P,F180Y,R224G;
S27K,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,R224G,T297P;
S173P,S175P,F180Y,R224S;
S27K,I121V,S171N,S173P,G174R,S175P,F180Y,Q198E,R224S,T297P;
S27K,S171D,S173P,G174R,S175P,G182A,L184F,Q198E,N199K,R224G,T297P;
S27K,S171D,S173P,G174R,S175P,G182A,L184F,Q198E,N199K,T297P;
S27K,S171N,S173P,G174R,S175P,F180Y,G182A,L184F,Q198E,N199K,R224G,T297P;
S27K,S171N,S173P,G174R,S175P,F180Y,G182A,L184F,Q198E,N199K,T297P;
S27K,V162T,S173P,G174K,S175P,F180Y,L184F,Q197K,Q198E,T297P;
S27K,V162T,S173P,G174K,S175P,F180Y,Q197K,Q198E,T297P;
Q70F;
Q70A;
S111R;
S111E;
S111D;
S114A;
S144R;
A145E;
I150N;
I150S;
G182A;
L184F;
L184Y;
R224G; and
Y240R.

4. A protease variant of claim 1 , wherein the variant has at least 85% sequence identity to the mature polypeptide of SEQ ID NO: 3.

5. A protease variant of claim 1 , wherein the total number of alterations compared to SEQ ID NO: 3 is 1-20 alterations.

6. A detergent composition comprising a protease variant according to claim 1 and one or more detergent components.

7. The detergent composition according to claim 6 , further comprising one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanses, xyloglucanses, pectinses, pectin lyases, xanthanses, peroxidases, haloperoxygenses, catalases and manannases, or any mixture thereof.

8. The detergent composition according to claim 6 in form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

9. A method for obtaining a protease variant, comprising introducing into a parent protease one or more of the following substitutions: Q70F, Q70A, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R of SEQ ID NO 3, wherein the variant has an amino acid sequence which is at least 60% identical to SEQ ID NO: 3, and recovering the variant.

10. The method of claim 9 , wherein the variant comprises two, three, four or five substitutions at positions corresponding to positions 114, 146, 150, 151, 176, 178, 184 or 224 of the mature polypeptide of SEQ ID NO: 3.

11. The method according to claim 9 , wherein the protease variant has at least 85% sequence identity to the mature polypeptide of SEQ ID NO: 3.

12. The method according to claim 9 , wherein the parent protease is selected from the group consisting of:
a) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 2; and
b) a fragment of the mature polypeptide of SEQ ID NO: 2, which has protease activity.

13. The method according to claim 9 , wherein the parent protease has at least 85% sequence identity to SEQ ID NO: 3.

14. The variant according to claim 1 , wherein the variant has a sequence identity to SEQ ID NO: 3 of at least 90%.

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

16. The method according to claim 9 , wherein the variant has at least 85% sequence identity to SEQ ID NO: 3.

17. The method according to claim 9 , wherein the variant has at least 90% sequence identity to SEQ ID NO: 3.

18. The method according to claim 9 , wherein the variant has at least 95% sequence identity to SEQ ID NO: 3.

Assignees

• Novozymes As

Inventors

• Rasmussen Frank W
• Toscano Miguel Duarte Guilherme Pereira
• Christensen Lars L H
• Friis Esben Peter

Classifications

• C12N9/54 Primary

  bacteria being Bacillus · CPC title

• C12N9/50

  Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)} · CPC title

• C11D3/38663

  Stabilised liquid enzyme compositions · CPC title

• C12N9/96 Primary

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C11D3/386

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