Lipase Variants and Polynucleotides Encoding Same

• US 20170314001A1
• Filed: 12/01/2015
• Published: 11/02/2017
• 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
• Granted Patent: US 10,457,921 B2
• Time in Patent Office: 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 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.

2. The variant of claim 1 selected from the group consisting of:

a. a polypeptide having at least 80% but less than 100% sequence identity to SEQ ID NO;

2;

b. a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO;

1, or (ii) the full-length complement of (i);

c. a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the polypeptide coding sequence of SEQ ID NO;

1; and

d. a fragment of SEQ ID NO;

2, which has lipase activity.

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

2;

SEQ ID NO;

4;

or SEQ ID NO;

6.

4. The variant of claim 1, wherein the substitution at position 92 is with Asp or Lys.

5. The variant of claim 1, wherein the substitution at position 96 is with Ile, Leu, or Thr.

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

G92D, G92K, E961, E96L, E96T, G92D+E961, G92D+E96L, G92D+E96T, G92K+E96I, G92K+E96L, and G92K+E96T.

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

2, preferably R81Q;

S83T;

R84H;

S85T;

I86UP/W;

E87A/K/T;

N88Q;

I90L/M;

G91A/L;

L93F;

N94D/K;

F95A/L/Y;

L97F/P; and

K98D/Q.

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

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

9. The variant of claim 1 comprising or constituting the substitutions selected from the group consisting of:

a. S83T E87K G91L N92D F95Y D96T L97P K98Q;

b. R81Q S83T R84H S85T I86L E87T N88Q I90M G91A N92K L93F N94K F95A D96L K98Q;

c. S83T E87A G91A N94D D961 L97P K98D;

d. I86P E87A G91A N92D F95Y D96T L97P K98Q I252T I255T;

e. I86W E87A G91A N92D F95Y D96T K98Q I252T I255T;

f. I86W E87A G91A N92D F95Y D96T L97P K98Q I252T I255T;

g. I86P E87A G91A N92D L93F F95Y D96T L97P K98Q I252T I255T;

h. I86P E87T G91L N94D D961 L97F K98D;

i. I90L F95L F211L I252T;

j. S83T G91A N92D E96T;

k. S83T G91A N92D F95Y E96L L97P K98Q;

I. S83T I86P G91A N92D D96T K98Q;

m. F51V I86P E87A G91A N92D F95Y D96T L97P K98Q T2521 T2551;

n. S83T E87T G91L N92D D961 L97F K98D;

o. E87T G91A N92D N94D D96L K98Q;

p. E87K G91A N92D N94D D96L K98Q;

q. S83T E87T G91A N92D F95Y D96T L97P K98Q;

r. S83T I86W G91L N92D F95Y;

s. S83T I86P G91A N92D D96L L97P K98Q;

t. S83T I86P G91A N92D F95Y D96L;

u. S83T I86P G91L N92D D96L;

v. S83T F95Y D96L;

w. S83T I86W G91L N92D F95Y D96T;

x. G91A N92D D96L K98Q;

y. S83T I86P G91L N92D D96L L97P;

z. S83T I86P G91A N92D D96L K98Q;

aa. S83T I86W G91L N92D F95Y;

bb. S83T I86P G91L N92D F95Y D96T;

cc. S83T G91L N92D K98Q P136H; and

dd. I86P G91L N92K L97P

ee. N33K F51V S83T I86P G91T N92D N94D F95A E96L K98Q

ff. S83T I86P E87T G91A N92D F95Y E96T K98Q

gg. S83T I86P E87A G91A N92D F95Y E96T L97P

hh. S83T I86P E87K G91A N92D F95Y E96D

ii. I86P E87T G91A N92D E96L L97P K98Q

jj. E87A G91L N92D L97P K98E E96D

kk. S83T I86P E87T G91A N92D E96T

ll. S83T E87K G91L N92D F95Y K98Q E96D

mm. S83T I86P G91A N92D F95Y E96T

nn. S83T E87T G91A N92D F95Y E96L K98Q

oo. N33K F51V S83T R84H G91A N92D L93F F95A E96L K98E

pp. N33K F51V G91A N92D L93F N94D F95Y E96L K98E

qq. N33K F51V S83T I90M G91A N92D F95Y E96L L97P K98Q

rr. S83T E87R G91A N92D F95Y E96T

ss. S83T E87A G91L N92D F95Y E96L

tt. N33K F51V I86W I90M G91A N92D F95A E96L F211 L

uu. N33K F51V S83T I86P I90F G91L N92D L93F E96L L259F

vv. N33K F51V L93I N94R F95V E96P E99T

ww. N33K F51V L93I N94R F95V E96H

xx. N33K F51V L93W N94R F95V E96P

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

10. The variant of claim 1, which has at least one improved property in comparison with the parent lipase, wherein said 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.

11. A composition comprising the variant of claim 1.

13. Method for cleaning a surface comprising the step of contacting said surface with the variant of claim 1.

14. A polynucleotide encoding the variant of claim 1.

15. A nucleic acid construct comprising the polynucleotide of claim 14.

16. An expression vector comprising the polynucleotide of claim 14.

17. A host cell comprising the polynucleotide of claim 1.

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

(a) cultivating the host cell of claim 17 under conditions suitable for expression of the variant; and

(b) recovering the variant.

19. 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.

12. (canceled)