Lipase variants and polynucleotides encoding same

• US 10,457,921 B2
• Filed: 12/01/2015
• Issued: 10/29/2019
• Est. Priority Date: 12/05/2014
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Svendsen, Allan, Vind, Jesper, Baunsgaard, Lone, Skjold-Joergensen, Jakob, Bhatia, Vikram Kjoeller, Oestergaard, Lars Henrik
• Primary Examiner(s): Raghu, Ganapathirama
• Application Number:
  US15/531,983
• Publication Number:
  US 20170314001A1
• Time in Patent Office: 1,428 Days
• CPC Class Codes: C11D 3/38627 containing lipase
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12Y 301/01 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12Y 301/01003 Triacylglycerol lipase (3.1.1.3)

Abstract

The present invention relates to a variant of a parent lipase, wherein said variant has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, has lipase activity, comprises a substitution at a position corresponding to position 92 and/or 96; and remains unaltered at the positions corresponding to positions 231, 233 and 254 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.

Claims

1. A variant of a parent lipase, wherein the variant has at least 90% but less than 100% sequence identity to SEQ ID NO:

2, has lipase activity, comprises a substitution at position 92 and/or 96; and

remains unaltered at positions 231, 233 and 254 of SEQ ID NO;

2;

wherein the substitution at position 92 is Asp or Lys; and

wherein the substitution at position 96 is IIe, Leu or Thr.

2. The variant of claim 1 wherein the variant has

at least 92% but less than 100% sequence identity to SEQ ID NO:

2.

4. The variant of claim 1, which comprises substitutions selected from the group consisting of:

N92D, N92K, E961, E96L, E96T, N92D+E961, N92D+E96L, N92D+E96T, N92K+E961, N92K+E96L, and N92K+E96T.

5. The variant of claim 1, which further comprises one or more substitutions at positions 81-99 of SEQ ID NO:

2, including R81Q;

S83T;

R84H;

S85T;

I86L/P/W;

E87A/K/T;

N88Q;

190L/M;

G91A/L;

L93F;

N94D/K;

F95A/L/Y;

L97F/P; and

K98D/Q.

6. The variant of claim 1, which further comprises one or more substitutions at positions 51, 136, 211, 252, and 255 of SEQ ID NO:

2, including F51V, P136H, F211L, I252T, and I255T.

7. The variant of claim 1 comprising substitutions selected from the group consisting of:

a. S83T E87K G91L N92D F95Y E96T L97P K98Q;

b. R81Q S83T R84H S85T I86L E87T N88Q I90M G91A N92K L93F N94K F95A E96L K98Q;

c. S83T E87A G91A N94D E961 L97P K98D;

d. I86P E87A G91A N92D F95Y E96T L97P K98Q I252T I255T;

e. I86W E87A G91A N92D F95Y E 96T K98Q I252T I255T;

f. I86W E87A G91A N92D F95Y E 96T L97P K98Q I252T I255T;

g. I86P E87A G91A N92D L93F F95Y E96T L97P K98Q I252T I255T;

h. I86P E87T G91L N94D E961 L97F K98D;

i. S83T G91A N92D E96T;

j. S83T G91A N92D F95Y E96L L97P K98Q;

k. S83T I86P G91A N92D E96T K98Q;

l. F51V I86P E87A G91A N92D F95Y E96T L97P K98Q T252I T255I;

m. S83T E87T G91L N92D E961 L97F K98D;

n. E87T G91A N92D N94D E96L K98Q;

o. E87K G91A N92D N94D E96L K98Q;

p. S83T E87T G91A N92D F95Y E96T L97P K98Q;

g. S83T I86W G91L N92D F95Y;

r. S83T I86P G91A N92D E96L L97P K98Q;

s. S83T I86P G91A N92D F95Y E96L;

t. S83T I86P G91L N92D E96L;

u. S83T F95Y E96L;

v. S83T I86W G91L N92D F95Y E96T;

w. G91A N92D E96L K98Q;

x. S83T I86P G91L N92D E96L L97P;

y. S83T I86P G91A N92D E96L K98Q;

z. S83T I86W G91L N92D F95Y;

aa. S83T I86P G91L N92D F95Y E96T;

bb. S83T G91L N92D K98Q P136H; and

cc. I86P G91L N92K L97P;

dd. N33K F51V S83T I86P G91T N92D N94D F95A E96L K98Q;

ee. S83T I86P E87T G91A N92D F95Y E96T K98Q;

ff. S83T I86P E87A G91A N92D F95Y E96T L97P;

gg. S83T I86P E87K G91A N92D F95Y E96D;

hh. I86P E87T G91A N92D E96L L97P K98Q;

ii. S83T I86P E87T G91A N92D E96T;

jj. S83T I86P G91A N92D F95Y E96T;

kk. S83T E87T G91A N92D F95Y E96L K98Q;

ll. N33K F51V S83T R84H G91A N92D L93F F95A E96L K98E;

mm. N33K F51V G91A N92D L93F N94D F95Y E96L K98E;

nn. N33K F51V S83T 190M G91A N92D F95Y E96L L97P K98Q;

oo. S83T E87R G91A N92D F95Y E96T;

pp. S83T E87A G91L N92D F95Y E96L;

qq. N33K F51V I86W 190M G91A N92D F95A E96L F211L;

rr. N33K F51V S83T I86P 190F G91L N92D L93F E96L L259F; and

ss. E56K S83T G91A N92D N94D E96L K98Q E239P K163G.

8. The variant of claim 1, which has at least one improved property in comparison with the parent lipase, wherein the property is selected from the group consisting of:

Increased lipase activity;

Increased lipase activity at reduced/low levels of Ca;

Increased lipase activity in the presence of EDTA; and

Increased Ca-independency.

9. A composition comprising the variant of claim 1.

10. A method for cleaning a surface, comprising the step of contacting the surface with the variant of claim 1.

11. A polynucleotide encoding the variant of claim 1.

12. A nucleic acid construct comprising the polynucleotide of claim 11.

13. An expression vector comprising the polynucleotide of claim 11.

14. An isolated recombinant host cell comprising the polynucleotide of claim 11.

15. A method of producing a lipase variant, comprising:

(a) cultivating the isolated recombinant host cell of claim 14 under conditions suitable for expression of the variant;

and (b) recovering the variant.

16. A method for obtaining the lipase variant of claim 1, comprising introducing into the parent lipase a substitution at one or more positions corresponding to positions 92 and/or 96 of SEQ ID NO:

2, wherein the variant has lipase activity; and

recovering the variant.

3. The variant of 1, wherein the parent lipase comprises or consists of SEQ ID NO:

2;

SEQ ID NO;

4;

or SEQ ID NO;

6.