BioChem - DOS

Enhanced activity of Thermomyces lanuginosus lipase by site-saturation mutagenesis for efficient biosynthesis of chiral intermediate of pregabalin

Biochemical engineering journal

Zheng, R.C.; Ruan, L.T.; Ma, H.Y.; Tang, X.L.; Zheng, Y.G.

Thermomyces lanuginosus lipase (TLL) variants with enhanced activity for kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) were constructed by site-saturation mutagenesis. Single mutant S83T and double mutant S58L/S83T exhibited 2.69 and 5.46-fold improvement in their specific activity for CNDE over the wild type TLL. The catalytic efficiency of S83T and S58L/S83T mutants were significantly increased, with k<SUB>cat</SUB>/K<SUB>m</SUB> values of 11.3 and 27.3mM<SUP>-1</SUP>min<SUP>-1</SUP>, which was 2.97 and 7.18 times higher than that of the wild type. The whole cell catalysis of 3M CNDE by Escherichia coli harboring mutant S58L/S83T (5% w/v) resulted in 44.8% yield and >96% ee<SUB>P</SUB> within 24h. These encouraging results demonstrated the great potential of the modified TLL for efficient production of (S)-2-carboxyethyl-3-cyano-5-methylhexaoic acid used as chiral intermediate for pregabalin.

2016

Elsevier

1369-703x

113

pp.12-18

10.1016/j.bej.2016.05.007

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART75931347

Pregabalin; Site-saturation mutagenesis; 2-Carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester; Kinetic resolution; Thermomyces lanuginosus lipase