Activity improvement of a regioselective nitrilase from Acidovorax facilis and its application in the production of 1-(cyanocyclohexyl) acetic acid

作者：Zhang, Xin-Hong; Liu, Zhi-Qiang; Xue, Ya-Ping; Zheng, Yu-Guo^*

来源：Process Biochemistry, 2014, 49(12): 2141-2148.

DOI：10.1016/j.procbio.2014.08.018

• Acidovorax facilis
• Nitrilase
• Regioselectivity
• 1-Cyanocyclohexylacetonitrile
• 1-(Cyanocyclohexyl) acetic acid

A nitrilase gene from Acidovorax facilis ZJB09122 was cloned and expressed in Escherichia coli BL21 (DE3). To improve the activity of this nitrilase, a key amino acid Phe168 was selected and mutated by site-directed mutagenesis, based on the homology modeling and previously described "hot spot" mutation. After mutation and screening, a mutant (Mut-F168V) with higher activity and stability was obtained. The nitrilase activity of Mut-F168V to hydrolyze 1-cyanocyclohexylacetonitrile was 39.52-fold compared with wild type A. facilis nitrilase (Wt-Acf-Nit). The values of K-m and V-max of Mut-F168V were markedly decreased to 1.89-fold and increased to 50.34-fold as compared to Wt-Acf-Nit, respectively. The biotransformation study showed that 1.0 M of 1-cyanocyclohexylacetonitrile could be regioselectively hydrolyzed to 1-(cyanocyclohexyl) acetic acid with 90% yield. The yield of 1-(cyanocyclohexyl) acetic acid by Mut-F168V was 66.19-fold compared to Wt-Acf-Nit after 1 h at the concentration of 1.0 M 1-cyanocyclohexylacetonitrile as substrate. The 1-(cyanocyclohexyl) acetic acid was subsequently isolated and characterized. The mutant (Mut-F168V) appears promising for potential applications for the industrial production of 1-(cyanocyclohexyl) acetic acid.

• 出版日期2014-12
• 单位浙江工业大学