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

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.

1 . An  Arabis alpina  nitrilase comprising the amino acid sequence shown in SEQ ID No. 1. 
     
     
         2 . A nitrilase encoding gene comprising the nucleoside sequence shown in SEQ ID No. 2. 
     
     
         3 . A recombinant vector comprising the nitrilase encoding gene of  claim 2 . 
     
     
         4 . A recombinant bacterial strain comprising the recombinant vector of  claim 3 . 
     
     
         5 . A method of producing (S)-3-cyano-5-methylhexanoic acid comprising hydrolysis of racemic 2-isobutylsuccinimide by the nitrilase of  claim 1 . 
     
     
         6 . The method of  claim 5 , wherein a nitrilase gene-containing, recombinant bacterial strain is fermented and grown to provide wet cells comprising the nitrilase 
     
     
         7 . The method of  claim 6 , where the amount of nitrilase gene-containing, recombinant bacterial strain used for hydrolysis is 50 g/L based on the weight of the wet cells. 
     
     
         8 . The method of  claim 6 , wherein (a) the nitrilase gene-containing recombinant bacterial strain is inoculated into a liquid LB broth containing 50 μg/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 μg/mL of kanamycin at 2% (v/v); (c) the nitrilase gene-containing, recombinant bacterial strain is grown to cell concentration (OD 600 ) 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 nitrilase-gene containing, 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. 
     
     
         9 . The method of  claim 6 , wherein hydrolysis of racemic 2-isobutylsuccinimide takes place at a pH 5.0-10.0 buffer, at 25-45° C. while stirring at 150 rpm 
     
     
         10 . The method of  claim 6 , wherein (S)-3-cyano-5-methylhexanoic acid is isolated and purified from the reaction mixture following hydrolysis. 
     
     
         11 . The method of  claim 7 , wherein the concentration of racemic 2-isobutylsuccinimide is 0.15-1.5 mol/L.