Mutant-type lipases and applications thereof

• US 7,049,122 B2
• Filed: 09/04/2002
• Issued: 05/23/2006
• Est. Priority Date: 09/21/2001
• Status: Expired due to Fees

• Current Assignee: Academia Sinica (Government of The Republic of China)
• Original Assignee: Academia Sinica (Government of The Republic of China)
• Inventors: Shaw, Jei-Fu, Chang, Rey-Chang, Chou, Shu-Jen
• Primary Examiner(s): Nashed, Nashaat T.
• Application Number:
  US10/233,443
• Publication Number:
  US 20030109019A1
• Time in Patent Office: 1,357 Days
• Field of Search: 435/195, 435/196, 435/197, 536/23.2
• US Class Current: 435/198
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12Y 301/01003 Triacylglycerol lipase (3.1.1.3)

Abstract

The present invention provides mutant-type lipases which demonstrate superior lipolytic and esterific activities. The mutant-type lipases are characterized by an amino acid alteration at the residue immediately followed either the serine residue or the histidine residue or both residues of the Ser-His-Asp catalytic triad. The Ser-His-Asp catalytic triad is known to be the three residues, although occur far apart in the amino acid sequence of a lipase, that contribute to the hydrolytic activity in the active site of the lipase. The amino acid residue that follows the serine residue of the Ser-His-Asp catalytic triad is alanine. The amino acid residue that follows the histidine residue of the Ser-His-Asp catalytic triad is isoleucine. The wild-type lipase is preferably originated from Staphylococcus, particularly Staphylococcus epidermindis. The present invention also relates to a method for preparing the mutant-type lipases by site-directed mutagenesis using PCR and a method for utilizing the mutant-type lipase to catalyze synthesis of flavor esters to be used in food industry.

Claims

1. A purified mutant lipase comprising a serine-histidine-aspartic acid catalytic triad;

wherein the amino acid residue following said serine residue of said triad is replaced with an alanine;

triad wherein said mutant lipase hydrolyzes fatty acid esters or esterifies fatty acids; and

wherein the wild-type lipase of said mutant lipase is isolated from a gram positive or gram negative bacterium or fungus.

2. The purified mutant lipase according to claim 1, wherein the last amino acid residue of said mutant lipase is further replaced with a glutamic acid residue and 6 histidine residues are fused to the C-terminus of said glutamic acid residue.

3. The purified mutant lipase according to claim 1, wherein said lipase is a mutant from staphylococcus.

4. The purified mutant lipase according to claim 3, wherein said lipase is a mutant lipase from Staphylococcus epidermidis.

5. The purified mutant lipase according to claim 1, wherein said lipase further comprises an isoleucine residue following said histidine residue of said triad.

6. The purified mutant lipase according to claim 4, wherein said lipase has the amino acid sequence of SEQ ID NO:

15.

7. The purified mutant lipase according to claim 1, wherein said lipase comprises the isoleucine residue following said histidine residue of said triad.

8. The purified mutant lipase according to claim 1, wherein said lipase hydrolyzes fatty acid esters.

9. The purified mutant lipase according to claim 1, wherein said lipase esterifies fatty acids with alcohols.

10. The purified mutant lipase according to claim 1, wherein said lipase esterifies fatty acids without using organic solvents.

11. A method for producing the mutant lipase according to claim 1 or 2, comprising:

(a) Synthesizing a mutant gene encoding said mutant lipase by a site-directed mutagenesis using polymerase chain reaction (PCR);

(b) Ligating said mutant gene to a plasmid;

(c) transfecting said ligated plasmid into a host cell; and

(d) Culturing said host cell to overexpress said lipase.

12. The method according to claim 11, wherein said site-directed mutagenesis uses an oligonucleotide primer of SEQ ID NO:

1 corresponding to the N-terminal, an oligonucleotide primer of SEQ ID NO;

2 corresponding to the C-terminal and a mutagenic primer of SEQ ID NO;

6 to produce said mutant gene encoding said mutant lipase which contains said alanine residue following said serine residue of said triad.

13. The method according to claim 11, wherein said mutant gene is digested with restriction endonucleases before ligated to said plasmid.

14. The method according to claim 11, wherein said plasmid is pET-20(b)⁺.

15. The method according to claim 11, wherein said host cell is a prokaryotic cell.

16. The method according to claim 15, wherein said prokaryotic cell is Escherichia coil.

17. The method according to claim 11, wherein said over expressed lipase is further purified by a NiSO₄−

charged His-bind resin column.

18. A method for enhancing flavor in food comprising:

(a) Enzymatically synthesizing a flavor ester from alcohol and fatty acid by the lipase according to claim 5; and

(b) Adding said flavor ester to food.

19. The method according to claim 18, wherein said alcohol is a primary alcohol having carbon chain length of C₂–

C₁₆.

20. The method according to claim 18, wherein said flavor ester is decyl myristate or decyl oleate.

21. A method for enhancing flavor in food comprising:

(a) Enzymatically synthesizing a flavor ester from alcohol and fatty acid by the lipase according to claim 7; and

(b) Adding said flavor ester to said food.

22. The method according to claim 21, wherein said alcohol is a primary alcohol having carbon-chain length of C₂ to C₁₂.

23. The method according to claim 20, wherein said flavor ester is decyl laurate.

24. The purified mutant lipase according to claim 1, wherein said gram positive bacterium is one selected from the group consisting of Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus aureus, Bacillus thermocatenularus, Staphylococcus xylosus, and Staphylococcus warneri.

25. The purified mutant lipase according to claim 1, wherein said gram negative bacterium is one selected from the group consisting of Acinetobacter calcoaceticus, Arthrobacter sp., Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas vulgaris, and Serratia marcescens.

26. The purified mutant lipase according to claim 1, wherein said fungus is one selected from the group consisting of Humicola lanuginose, Candida cylindracea, and Rhizopus oryzae.

27. A purified mutant lipase comprising a serine-histidine-aspartic acid catalytic triad;

wherein the amino acid residue following said histindine residue of said triad is replaced with an isoleucine, wherein said mutant lipase hydrolyzes fatty acid esters or esterifies fatty acids; and

wherein the wild-type lipase of said mutant lipase is isolated from staphylococcus.

28. A purified mutant lipase having the amino acid sequence of SEQ ID NO:

17.