Amphipathic peptide-lipase conjugate having advanced lipase activity and use thereof

Patent metadata

Field	Value
Publication number	US-9527886-B2
Application number	US-201214123513-A
Country	US
Kind code	B2
Filing date	Aug 10, 2012
Priority date	Aug 10, 2011
Publication date	Dec 27, 2016
Grant date	Dec 27, 2016

Abstract

Disclosed are an amphipathic peptide-lipase conjugate with enhanced lipase activity, a polynucleotide coding for the conjugate, an expression vector carrying the polynucleotide, a transformant anchoring the expression vector therein, a method for preparing the conjugate, a lipolysis method using the conjugate, and a method for producing biodiesel using the lipase.

First claim

The invention claimed is:

1. A method for increasing a lipase activity, comprising fusing an amphipathic peptide at the N-terminus of a lipase, wherein the amphipathic peptide is NKC comprising amino acid sequence of SEQ ID NO:4.

2. The method of claim 1 , wherein the lipase is Photobacterium lipolyticum -derived lipase M37 comprising amino acid sequence of SEQ ID NO:7.

3. The method of claim 1 , further comprising amphipathic peptide linked to the C-terminus of the lipase.

4. The method of claim 1 , wherein the amphipathic peptide is fused to the lipase via a peptide linker.

5. The method of claim 1 , wherein the amphipathic peptide is fused to the lipase via a non-peptidyl linker.

6. The method of claim 5 , wherein the non-peptidyl linker is selected from the group consisting of polyethylene glycol homopolymers, polypropylene glycol homopolymers, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyol, polyvinyl alcohols, polysaccharides, dextran, polyvinyl ethyl ether, lipid polymers and any combination thereof.

7. The method of claim 1 , wherein the peptide fusion comprises amino acid sequence of SEQ ID NO:16.

8. The method of claim 1 , wherein the amphipathic peptide fused to a lipase has at least a 2.2 fold increase in lipase activity for an triglyceride than the lipase without an amphipathic peptide.

9. A method for preparing an amphipathic peptide-lipase conjugate having an increased affinity between the lipase and substrate, comprising:
(a) culturing a transformant comprising a polynucleotide encoding an amphipathic peptide-lipase fusion in which an amphipathic peptide is fused to the N-terminus of a lipase; and
(b) recovering the amphipathic peptide-lipase conjugate from the cell culture or culture medium,
wherein the amphipathic peptide is NKC comprising an amino acid sequence of SEQ ID NO:4.

10. The method of claim 9 , wherein the fusion comprises amino acid sequence of SEQ ID NO:16.

11. A method of lipolysis, comprising contacting an amphipathic peptide-lipase fusion in which an amphipathic peptide is fused to the N-terminus of a lipase with a lipid substrate, wherein the amphipathic peptide is NKC comprising amino acid sequence of SEQ ID NO:4.

12. A method for producing biodiesel, comprising contacting an amphipathic peptide-lipase conjugate in which an amphipathic peptide is fused to the N-terminus of a lipase with fat and/or oil together with alcohol,
wherein the amphipathic peptide is NKC comprising amino acid sequence of SEQ ID NO:4.

Assignees

• Kim Sun-Chang
• Sung Bong Hyun
• Yang Kyung Seok
• Lee Jun Hyoung
• Lim Ki Jung
• Park Myung Keun
• Korea Advanced Inst Sci & Tech
• Intelligent Synthetic Biology Ct

Inventors

• Kim Sun-Chang
• Sung Bong Hyun
• Yang Kyung Seok
• Lee Jun Hyoung
• Lim Ki Jung
• Park Myung Keun

Classifications

• C07K14/47

  from mammals · CPC title

• C07K14/463

  from amphibians · CPC title

• C07K14/001

  by chemical synthesis · CPC title

• C07K7/06 Primary

  having 5 to 11 amino acids · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title