Arginine deiminase mutant and methods of using the same

Patent metadata

Field	Value
Publication number	US-10557130-B2
Application number	US-201815907340-A
Country	US
Kind code	B2
Filing date	Feb 28, 2018
Priority date	Mar 2, 2017
Publication date	Feb 11, 2020
Grant date	Feb 11, 2020

Abstract

An arginine deiminase mutant with improved enzyme activity and temperature stability and application thereof were provided, belonging to the technical field of genetic engineering and enzyme engineering. The arginine deiminase mutant is proline, namely Gly292 Pro, mutated from glycine near an enzyme active center. A wild-type arginine deiminase arcA coding gene is molecularly modified by a site-directed mutation technique to obtain a mutant enzyme ADIG292P, which has glycine at position 292 of an amino acid sequence of the wild type arginine deiminase mutated to proline. The arginine deiminase, modified by site-directed mutation, of the present invention has 1.5 times of increase in enzyme activity and 5.43 times of increase in half-life period at 40° C. compared with the wild-type enzyme, which solves the problems of low catalytic ability and temperature stability during the catalytic synthesis of citrulline using arginine deiminase, and lays a foundation for industrial production of efficient synthesis of citrulline and medication application.

First claim

What is claimed is:

1. An arginine deiminase mutant, comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein the mutant comprises mutation Gly292Pro compared to a parent arginine deiminase, greater enzyme activity and greater temperature stability than the parent arginine deiminase.

2. The arginine deiminase mutant of claim 1 , wherein the mutant is engineered starting with arginine deiminase sequences obtained from bacteria, archaebacteria, or eukaryotic cells.

3. The arginine deiminase mutant of claim 2 , wherein the bacteria is obtained from Enterococcus faecalis SK32.001.

4. The arginine deiminase mutant of claim 1 , wherein the mutant is encoded by SEQ ID NO:2.

5. The arginine deiminase mutant of claim 1 , wherein temperature stability of the mutant is increased at least 5-fold at 45° C. compared to the parent arginine deiminase.

6. The arginine deiminase mutant of claim 1 , wherein the enzyme activity is increased at least 2.5-fold higher than the parent arginine deiminase.

Assignees

• Univ Jiangnan

Inventors

• Zhang Tao
• Jiang Bo
• Jiang Hangyu
• Mu Wanmeng
• Miao Ming

Classifications

• A61K38/50

  acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• A61P35/02

  specific for leukemia · CPC title

• C12N9/78 Primary

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C12P13/10

  Citrulline; Arginine; Ornithine · CPC title