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Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin

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Abstract

Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.

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Acknowledgements

This work was financially supported by the National High Technology Research and Development Program of China (no. 2012AA022201), the Key Scientific and Technology Programs of Zhejiang Province (no. 2012C03005-2) and the Natural Science Foundation of Zhejiang Province (no. Z4090612).

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  1. Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China

    Xiao-Jun Li, Ren-Chao Zheng, Hong-Ye Ma & Yu-Guo Zheng

  2. Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China

    Xiao-Jun Li, Ren-Chao Zheng, Hong-Ye Ma & Yu-Guo Zheng

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  1. Xiao-Jun Li
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Correspondence to Yu-Guo Zheng.

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Li, XJ., Zheng, RC., Ma, HY. et al. Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin. Appl Microbiol Biotechnol 98, 2473–2483 (2014). https://doi.org/10.1007/s00253-013-5136-y

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