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

• Site-saturation mutagenesis was used for engineering of TLL.
• TLL mutants with enhanced activity were obtained and characterized.
• The key residues responsible for higher catalytic efficiency of TLL were revealed.
• Kinetic resolution of CNDE at high substrate loading was achieved by whole-cell biocatalysts.

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 kcat/Km values of 11.3 and 27.3 mM−1 min−1, which was 2.97 and 7.18 times higher than that of the wild type. The whole cell catalysis of 3 M CNDE by Escherichia coli harboring mutant S58L/S83T (5% w/v) resulted in 44.8% yield and >96% eeP within 24 h. 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.

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