Expression of Nitrilase Gene from Acidovorax facilis in Escherichia coli

doi:10.13560/j.cnki.biotech.bull.1985.2015.01.027

Abstract

Abstract: Plasmid containing Acidovorax facilis gene, was used as a template, and length of 1 080 bp nitrilase（Nitrilase）gene fragment was obtained by PCR. Expressed genes were sequenced and compared to gene sequences found in GenBank. There are 2 bp mutation was found, which can not affect the expression and characterization of the enzyme. The recombinant plasmid was transformed into the Escherichia coli Rosetta（DE3）competent cell, which was induced IPTG for strain- expression. SDS-PAGE analysis was used and showed that the protein molecular weight was about 41.28 kD. Enzyme activity analysis showed that the specific activity of the supernatant was 3 U/mg. Induction conditions were optimized, including induction temperature, IPTG concentration, induction time and a series of conditions. Under optimal conditions and with the expansion of the culture volume, the specific activity can up to 15 U/mg, and activity increased by about five times.

Key words: nitrilase, Acidovorax facilis, Escherichia coli, clone

Guan Qiwang, Ma Jiangfeng, He Aiyong, Jiang Min, Gong Changbin. Expression of Nitrilase Gene from Acidovorax facilis in Escherichia coli[J]. Biotechnology Bulletin, 2015, 31(1): 181-185.

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