Lipase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-11268076-B2
Application number	US-201515526834-A
Country	US
Kind code	B2
Filing date	Dec 8, 2015
Priority date	Dec 9, 2014
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

The present invention relates to a variant of a parent lipase, which parent lipase has the amino acid sequence of SEQ ID NO: 2, wherein the variant has lipase activity, comprises a substitution at a position corresponding to position 51 and comprises a substitution or remains unaltered at a position corresponding to position 96 of SEQ ID NO: 2. 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 lipase variant which
(a) comprises a V at a position corresponding to position 51 of SEQ ID NO: 2 and comprises a substitution or is unaltered at a position corresponding to position 96 of SEQ ID NO: 2,
(b) has at least 97% but less than 100% sequence identity to SEQ ID NO: 2,
(c) has improved stability as compared to the polypeptide of SEQ ID NO: 2, and
(d) has lipase activity.

2. The lipase variant of claim 1 , which is unaltered at a position corresponding to position 96 of SEQ ID NO: 2.

3. The lipase variant of claim 1 , which comprises a substitution at a position corresponding to position 96 of SEQ ID NO: 2.

4. The lipase variant of claim 3 , wherein the substitution at a position corresponding to position 96 of SEQ ID NO: 2 is with Asp, Leu, or Trp.

5. The lipase variant of claim 1 , further comprising one or more amino acid substitutions corresponding to the following substitutions of SEQ ID NO: 2, selected from the group consisting of: V2F/G/H/K/R/Y; N33H/K/R/P/Q; S54T; E56H/K/R; D57G/N; T64S; L69H/K/R; K98E/D/I/R/T/V; E87E/F/H/I/K/M/Q/R; N101D/E; K163G/P; V176L; E210H/K/R; Y220F; A243P/S/T; W260F/Y; and L269I/V.

6. The lipase variant of claim 2 , further comprising one or more amino acid substitutions corresponding to the following substitutions of SEQ ID NO: 2, selected from the group consisting of: V2F/G/H/K/R/Y; N33H/K/R/P/Q; S54T; E56H/K/R; D57G/N; T64S; L69H/K/R; K98E/D/I/R/T/V; E87E/F/H/I/K/M/Q/R; N101D/E; K163G/P; V176L; E210H/K/R; Y220F; A243P/S/T; W260F/Y; and L269I/V.

7. The lipase variant of claim 4 , further comprising one or more amino acid substitutions corresponding to the following substitutions of SEQ ID NO: 2, selected from the group consisting of: V2F/G/H/K/R/Y; N33H/K/R/P/Q; S54T; E56H/K/R; D57G/N; T64S; L69H/K/R; K98E/D/I/R/T/V; E87E/F/H/I/K/M/Q/R; N101D/E; K163G/P; V176L; E210H/K/R; Y220F; A243P/S/T; W260F/Y; and L269I/V.

8. The lipase variant of claim 1 , comprising a set of amino acid substitutions corresponding to the following substitutions of SEQ ID NO: 2, selected from the group consisting of:
a. V2K+N33K+F51V+S54T+L69R+K98E+V176L+E210K+W260Y;
b. V2K+N33K+F51V+L69R+K98E+V176L+V187P+E210K+L227G;
c. V2K+N33K+F51V+L69R+K98E+V176L+E210K+S224F+L227G;
d. V2K+N33K+F51V+L69R+K98E+V176L+E210K+L227G;
e. V2K+N33K+F51V+L69R+K98E+V176L+E210K+L227G+V228P;
f. V2K+N33K+F51V+L69R+K98E+E210K+L227G;
g. V2K+N33K+F51V+V187P;
h. V2K+N33K+F51V+S224F;
i. V2K+N33K+F51V+L227D;
j. V2K+N33K+F51V+V228P;
k. V2K+F51V;
I. V2Y+N33K+F51V;
m. V2Y+N33K+F51V+E56K+L69R+E96D+K98E; and
n. V2Y+N33K+F51V+E56K+L69R+K98E.

9. The lipase variant of claim 1 , comprising a set of amino acid substitutions corresponding to the following substitutions of SEQ ID NO: 2, selected from the group consisting of:
a. N33K+F51V;
b. N33K+F51V+S54T+E56K+L69R+K98E+W260Y;
c. N33K+F51V+S54T+E56K+K98E+E210K+W260Y;
d. N33K+F51V+S54T+E56K+K98I;
e. N33K+F51V+S54T+E56K+K98I+T143A;
f. N33K+F51V+S54T+L69R+E87K+K98E+E210K+Y220F;
g. N33K+F51V+S54T+K98E+W260Y;
h. N33K+F51V+S54T+K98I;
i. N33K+F51V+E56K+L69R+E96D+K98E;
j. N33K+F51V+E56K+L69R+E96D+K98E+V187D;
k. N33K+F51V+E56K+L69R+E96D+K98E+G225P;
l. N33K+F51V+E56K+L69R+K98E;
m. N33K+F51V+E56K+L69R+K98E+V176L+E210K+Y220F;
n. N33K+F51V+E56K+L69R+K98E+V176L+E210K+W260Y;
o. N33K+F51V+E56K+L69R+K98E+V187D;
p. N33K+F51V+E56K+L69R+K98E+E210K;
q. N33K+F51V+E56K+L69R+K98E+G225P;
r. N33K+F51V+E56K+L69R+K98E+T256P;
s. N33K+F51V+E56K+K98E+K163P+E210K;
t. N33K+F51V+E56K+K98E+V176L+E210K+Y220F;
u. N33K+F51V+D57G+K98I;
v. N33K+F51V+L69R+K98E;
w. N33K+F51V+L69R+K98E+V176L+E210K+L227G;
x. N33K+F51V+L93I+N94R+F95V
y. N33K+F51V+K98E;
z. N33K+F51V+K98E+E210R+W260Y;
aa. N33K+F51V+K98I;
bb. N33K+F51V+K98I +H198S+Y220F;
cc. N33K+F51V+K98I+Y220F;
dd. N33P+F51V;
ee. N33P+F51V+D57G+E210K; and
ff. N33P+F51V+K98E.

10. The lipase variant of claim 1 , comprising a set of amino acid substitutions corresponding to the following substitutions of SEQ ID NO: 2, selected from the group consisting of:
a. F51V;
b. F51V+E56K+L69R+K98E;
c. F51V+E56K+E87K+K98E;
d. F51V+D57G+N101D+E210K;
e. F51V+L69R+E87K+K98E;
f. F51V+L69R+K98E+E210K;
g. F51V+I86P+E87A+G91A+N92D+F95Y+D96T+L97P+K98Q+T252I+T255I;
h. F51V+E87K+K98E;
i. F51V+K98E+N101D;
j. F51V+K98E+E210K; and
k. F51V+V176L.

11. The lipase variant of claim 1 , further comprising an alteration at any of positions: 4, 27, 33, 38, 57, 58, 60, 83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225, 227, 231, 249, 254, 255, 256, 263, 264, 265, 266, 267, and 269 of SEQ ID NO: 2.

12. The lipase variant of claim 1 , wherein the improved stability is stability in detergent compositions, stability in detergent compositions containing protease, stability in the presence of protease, stability under storage conditions, stability under storage conditions in the presence of protease, or thermostability, as compared to the lipase of SEQ ID NO: 2.

13. A detergent composition comprising the lipase variant of claim 1 and a surfactant.

14. A detergent composition comprising the lipase variant of claim 2 and a surfactant.

15. A detergent composition comprising the lipase variant of claim 3 and a surfactant.

16. A detergent composition comprising the lipase variant of claim 4 and a surfactant.

Assignees

• Novozymes As

Inventors

• Gylstorff Christian Lundager
• Jackel Christian
• Vind Jesper
• Svendsen Allan
• Malten Marco
• Poulsen Thomas Agersten

Classifications

• C12Y301/01

  Carboxylic ester hydrolases (3.1.1) · CPC title

• C11D3/38627

  containing lipase · CPC title

• C12N9/20 Primary

  Triglyceride splitting, e.g. by means of lipase · CPC title