Nitrilase mutant and use thereof in catalytic synthesis of 2-chloronicotinic acid

Patent metadata

Field	Value
Publication number	US-12516359-B2
Application number	US-202118018561-A
Country	US
Kind code	B2
Filing date	Apr 19, 2021
Priority date	Oct 9, 2020
Publication date	Jan 6, 2026
Grant date	Jan 6, 2026

Abstract

The present invention discloses a nitrilase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid, and belongs to the technical field of enzyme engineering. The nitrilase mutant has an amino acid sequence shown in SEQ ID NO. 4, that is, 167th tryptophan W of a parent nitrilase is mutated into glycine G. According to the nitrilase mutant provided by the present invention, the hydration activity of the parent nitrilase to 2-chloronicotinonitrile is eliminated, a byproduct of 2-chloronicotinamide is not generated in the catalytic process, the nitrile hydrolysis activity is greatly improved, and 2-chloronicotinonitrile can be specifically subjected to catalytic hydrolysis to synthesize 2-chloronicotinic acid. Therefore, the nitrilase mutant has an important potential in enzymatic industrial synthesis of 2-chloronicotinic acid.

First claim

What is claimed is:

1 . A nitrilase mutant having the amino acid sequence of SEQ ID NO: 4.

2 . A gene having the nucleotide sequence of SEQ ID NO: 3.

3 . A recombinant plasmid comprising the gene according to claim 2 .

4 . The recombinant plasmid according to claim 3 , wherein the recombinant plasmid is a pET-28b (+) vector that comprises the gene.

5 . A recombinant engineered bacterium comprising the recombinant plasmid according to claim 3 .

6 . The recombinant engineered bacterium according to claim 5 , wherein the recombinant engineered bacterium is a Escherichia coli BL21 cell that comprises the recombinant plasmid.

7 . A method of catalytic hydrolysis of 2-chloronicotinonitrile to synthesize 2-chloronicotinic acid, comprising:
culturing a cell transformed with a nucleic acid encoding the protein of SEQ ID NO: 4 to express the protein of SEQ ID NO: 4; sonicating the cell to obtain the protein of SEQ ID NO: 4; reacting the protein of SEQ ID NO: 4 with 2-chloronicotinonitrile in a reaction medium that is a NaH 2 PO 4 —Na 2 HPO 4 buffer solution at a pH between 6 and 8 at a temperature of 25-45° C. to produce 2-chloronicotinic acid, and purifying the 2-chloronicotinic acid.

8 . The method according to claim 7 , wherein the concentration of 2-chloronicotinonitrile in the reaction medium is 50-500 mM.

9 . The method according to claim 8 , wherein the NaH 2 PO 4 —Na 2 HPO 4 buffer solution used as the reaction medium has a pH value of 7, the concentration of 2-chloronicotinonitrile in the reaction medium is 300 mM, and the reaction is performed at 30° C. for 30-40 hours.

10 . A recombinant engineered bacterium comprising the recombinant plasmid according to claim 4 .

Assignees

• Univ Zhejiang Technology

Inventors

• Zheng Renchao
• Dai Andi
• Wu Zheming
• Zheng Yuguo

Classifications

• C12Y305/05001 Primary

  Nitrilase (3.5.5.1) · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12N9/78 Primary

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

• C12P17/12 Primary

  containing a six-membered hetero ring · CPC title