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

Paper ID Volume ID Publish Year Pages File Format Full-Text
2736 129 2016 7 PDF Available
Title
Enhanced activity of Thermomyces lanuginosus lipase by site-saturation mutagenesis for efficient biosynthesis of chiral intermediate of pregabalin
Abstract

•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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Keywords
Thermomyces lanuginosus lipase; Site-saturation mutagenesis; 2-Carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester; Kinetic resolution; Pregabalin
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Enhanced activity of Thermomyces lanuginosus lipase by site-saturation mutagenesis for efficient biosynthesis of chiral intermediate of pregabalin
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 113, 15 September 2016, Pages 12–18
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us