Nitrilase from arabis alpina, its encoding gene, vector, recombinant bacterial strain and uses thereof

Patent metadata

Field	Value
Publication number	US-10260059-B2
Application number	US-201715687800-A
Country	US
Kind code	B2
Filing date	Aug 28, 2017
Priority date	Aug 27, 2015
Publication date	Apr 16, 2019
Grant date	Apr 16, 2019

Abstract

The disclosure provides a nitrilase from Arabis alpina , which belongs to genus Arabis , family brassicaceae. The disclosure further provides the encoding gene, vector, recombinant bacterial strain, and the application in the manufacturing of (S)-3-cyano-5-methylhexanoic acid. The wet resting cells containing nitrilase Aa-Nit can kinetically resolve racemic IBSN at 1.2 M with a 42% conversion rate in 15 hr and >99% ee value. The disclosure provides a regio- and stereoselective method for the preparation of (S)-3-cyano-5-methylhexanoic acid. This method provides an atom economical, mild, environmental friendly industrial method to manufacture (S)-3-cyano-5-methylhexanoic acid.

First claim

What we claim:

1. A method of producing (S)-3-cyano-5-methyl hexanoic acid comprising performing hydrolysis of racemic 2-isobutylsuccinimide using an Arabis alpina nitrilase comprising the amino acid sequence of SEQ ID NO: 1.

2. The method of claim 1 , wherein the nitrilase gene comprising, recombinant bacterial strain is fermented and grown to provide wet cells comprising the nitrilase.

3. The method of claim 2 , where the amount of nitrilase gene comprising, recombinant bacterial strain used for hydrolysis is 50 g/L based on the weight of the wet cells.

4. The method of claim 2 , wherein (a) the nitrilase gene comprising, recombinant bacterial strain is inoculated into a liquid LB broth containing 50 pg/mL of kanamycin and is grown for 12 hr at 37° C.; (b) the LB broth of step (a) is inoculated into fresh liquid LB broth containing 50 pg/mL of kanamycin at 2% (v/v); (c) the nitrilase gene comprising, recombinant bacterial strain is grown to cell concentration (OD600) 0.4-0.8 at 37° C.; IPTG is added to the LB broth of step (b) until the concentration reaches 0.2 mM, (d) the gene comprising, recombinant bacterial strain is induced to grow at 28° C. for 12 hr; the LB broth of step (c) is centrifuged at 4° C. at 12000 rpm for 5 min; and (e) the wet cells are collected.

5. The method of claim 2 , wherein hydrolysis of racemic 2-isobutylsuccinimide takes place at a pH 5.0-10.0 in a buffer, at 25-45° C. while stirring at 150 rpm.

6. The method of claim 2 , wherein (S)-3-cyano-5-methylhexanoic acid is isolated and purified from the reaction mixture following hydrolysis.

7. The method of claim 3 , wherein the concentration of racemic 2-isobutylsuccinimide is 0.15-1.5 mol/L.

Assignees

• Univ Zhejiang Technology
• Zhejiang Chiral Medicine Chemicals Co Ltd

Inventors

• Zheng Renchao
• Zheng Yuguo
• Zhang Qin
• Huang Youming
• Weng Jianfeng
• Liu Tianchun
• Fan Weirong

Classifications

• C12Y305/05001

  Nitrilase (3.5.5.1) · CPC title

• C12P13/002

  Nitriles (-CN) · CPC title

• C12N9/78 Primary

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