Mutant lipase and use thereof

Patent metadata

Field	Value
Publication number	US-10829748-B2
Application number	US-201716305348-A
Country	US
Kind code	B2
Filing date	Jun 8, 2017
Priority date	Jun 10, 2016
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

The present invention further relates to a process for preparing a food product wherein a polypeptide according to the present invention is used.

First claim

The invention claimed is:

1. A polypeptide having lipase activity wherein the polypeptide is
a polypeptide, which, when aligned with the polypeptide according to SEQ ID NO: 1, comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 1 and comprises at least one amino acid substitution resulting in Ser (S), Ala (A) or Leu (L) at position 246, Trp (W) at position 307, Leu (L) at position 345, Ile (I) at position 365, and/or Phe (F) at position 534, wherein the position is defined with reference to SEQ ID NO: 1, wherein Ala(A) at position 1 in SEQ ID NO: 1 is counted as number 1.

2. The polypeptide according to claim 1 , wherein the polypeptide at least comprises one amino acid substitution resulting in Trp (W) at position 307, wherein Ala (A) at position 1 in SEQ ID NO: 1 is counted as number 1, wherein said polypeptide has at least 80% identity to amino acid sequence of SEQ ID NO: 1 and wherein said polypeptide has a higher specificity towards butyrate as a side chain than the specificity towards octanoate and/or palmitate as a side chain.

3. The polypeptide according to claim 1 , wherein the polypeptide least comprises one amino acid substitution resulting in Leu (L) at position 345, wherein Ala (A) at position 1 in SEQ ID NO: 1 is counted as number 1, wherein said polypeptide has a higher specificity towards butyrate as a side chain than the specificity towards octanoate and/or palmitate as a side chain.

4. The polypeptide according to claim 1 , wherein the polypeptide is a polypeptide of SEQ ID NO: 1 and at least comprises one amino acid substitution resulting in Phe (F) at position 534, wherein Ala (A) at position 1 in SEQ ID NO: 1 is counted as number 1, wherein said polypeptide has a higher specificity towards butyrate as a side chain than the specificity towards octanoate and/or palmitate as a side chain.

5. The polypeptide according to claim 1 , wherein the polypeptide at least comprises one amino acid substitution resulting in Ile (I) at position 365, wherein Ala (A) at position 1 in SEQ ID NO: 1 is counted as number 1, wherein said polypeptide has a higher specificity towards butyrate as a side chain than the specificity towards octanoate and/or palmitate as a side chain.

6. The polypeptide according to claim 1 that is an isolated, pure, recombinant, or synthetic polypeptide.

7. The polypeptide according to claim 1 , wherein the polypeptide having lipase activity has a higher specificity towards butyrate as a side chain than the specificity towards octanoate and/or palmitate as a side chain.

8. A composition comprising the polypeptide according to claim 1 .

9. A method for generating a variant polypeptide having lipase activity wherein the method comprises
a. selecting a parent polypeptide comprising at least 80% identity to the amino acid sequence according to SEQ ID NO: 1; and,
b. substituting at least one amino acid into Ser (S), Ala (A) or Leu (L) at position 246, Trp (W) at position 307, Leu (L) at position 345, Ile (I) at position 365, and/or Phe (F) at position 534, wherein said position is defined with reference to SEQ ID NO: 1, wherein alanine at position 1 in SEQ ID NO: 1 is counted as number 1; and
c. generating the variant polypeptide, wherein the polypeptide having lipase activity has a higher specificity towards butyrate than the specificity towards octanoate and/or palmitate.

10. A nucleic acid encoding a lipase, which has at least 80% sequence identity to SEQ ID NO: 2, wherein SEQ ID NO: 2 comprises at least one mutation resulting in Ser (S), Ala (A) or Leu (L) at position 246, Trp (W) at position 307, Leu (L) at position 345, Ile (I) at position 365, and/or Phe (F) at position 534 of the amino acid sequence of SEQ ID NO: 1, wherein the position is defined with reference to SEQ ID NO: 1, wherein alanine at position 1 in SEQ ID NO: 1 is counted as number 1.

11. An expression vector comprising a nucleic acid according to claim 10 operably linked to at least one control sequence that directs expression of the polypeptide in a host cell.

12. A recombinant host cell comprising a nucleic acid according to claim 10 , or an expression vector comprising said nucleic acid.

13. A method for preparing a polypeptide according to claim 1 , comprising cultivating a host cell in a suitable fermentation medium, under conditions that allow expression of the polypeptide, and optionally recovering the polypeptide.

14. A process for preparing a product comprising a lipid comprising bringing the lipid into contact with a polypeptide according to claim 1 .

15. The polypeptide according to claim 1 , for improving flavour in a product comprising a lipid.

16. The polypeptide according to claim 15 , wherein improving flavour comprises reducing the formation of fatty acids having at least 8 carbon atoms, as compared to the use of a polypeptide comprising an amino acid sequence according to SEQ ID NO: 1.

17. The process according to claim 14 , wherein the product comprising a lipid is a food product, optionally a dairy product, optionally butter, cream or cheese.

18. The polypeptide of claim 15 , wherein the product comprising a lipid is a food product, optionally a dairy product, optionally butter, cream or cheese.

19. The process for preparing a product comprising a lipid comprising bringing the lipid into contact with a composition according to claim 8 .

20. The polypeptide of claim 1 , wherein the polypeptide is encoded by a nucleic acid which has at least 80% identity to the nucleotide sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 comprises at least one mutation resulting in an amino acid Ser (S), Ala (A) or Leu (L) at position 246, Trp (W) at position 307, Leu (L) at position 345, Ile (I) at position 365, and/or Phe (F) at position 534 of a polypeptide according to SEQ ID NO: 1, wherein Ala (A) at position 1 in SEQ ID NO: 1 is counted as number 1.

21. The polypeptide of claim 1 , which, when aligned with the polypeptide according to SEQ ID NO: 1, consists of a polypeptide at least 80% identity to the amino acid sequence of SEQ ID NO: 1 and comprises at least one amino acid substitution resulting in Ser (S), Ala (A) or Leu (L) at position 246, Trp (W) at position 307, Leu (L) at-position 345, Ile (I) at position 365, and/or Phe (F) at position 534, wherein the position is defined with reference to SEQ ID NO: 1, wherein Ala(A) at position 1 in SEQ ID NO: 1 is counted as number 1.

Assignees

• Dsm Ip Assets Bv

Inventors

• De Jong Rene Marcel
• Bijleveld Willem
• Van Rij Evert Tjeerd

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12N1/14

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title

• C12N9/20 Primary

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

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• A23C19/082

  Adding substances to the curd before or during melting; Melting salts · CPC title