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Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester

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Abstract

The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4′-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (−)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipAL315S and LipAS271F, were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipAL315S, LipAS271F, and their combination LipAL315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipAS271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipAS271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m. Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (−)-MPGM.

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 21276082 & 21536004) and Shanghai Commission of Science and Technology (No. 15JC140040).

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China

    Ke-Cai Chen, Ming-Min Zheng, Jiang Pan, Chun-Xiu Li & Jian-He Xu

  2. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China

    Jian-He Xu

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  1. Ke-Cai Chen
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  2. Ming-Min Zheng
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  3. Jiang Pan
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  4. Chun-Xiu Li
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Correspondence to Chun-Xiu Li or Jian-He Xu.

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Chen, KC., Zheng, MM., Pan, J. et al. Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester. Appl Biochem Biotechnol 183, 543–554 (2017). https://doi.org/10.1007/s12010-017-2543-z

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  • DOI: https://doi.org/10.1007/s12010-017-2543-z

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