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Enhanced enantioselectivity of a carboxyl esterase from Rhodobacter sphaeroides by directed evolution

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Abstract

The present work created an esterase variant from Rhodobacter sphaeroides (RspE) with enhanced selectivity in hydrolytic kinetic resolutions by directed evolution. A “model” substrate, methyl mandelate, was introduced in the high-throughput screening procedure. E values of a variant CH (Asn62Cys/Leu145His) for six different esters were 10–83, which were a relative improvement compared to 2–20 for the wild type. Our subsequent crystal structure interpretation and molecular dynamics simulations helped shed light on the source of enantioselectivity modified by directed evolution. Though mutations displayed no “direct” interaction with the substrate, they were hypothesized to strengthen the intramolecular interaction in the catalytic cavity of variant. Conformation analysis revealed that the enhanced enantioselectivity of variant CH for the seven substrates applied in this study was derived from the decrease in size of the substrate binding pocket.

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Acknowledgment

This work was financially supported by the Natural Science Foundation of China (grant no. 21176215/21176102), Outstanding Young Scholar of Zhejiang Province (grant no. R4110092) and the Program for Zhejiang Leading Team of S&T Innovation (2011R50007). We are grateful for all editor and reviewers for their helpful advice. We also thank all the other members of Prof. Yu’s group.

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Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Institute of Bioengineering, Zhejiang University, Hangzhou, 310027, People’s Republic of China

    Jingbo Ma, Fei Guo, Jiali Gu, Xiaoling Tang, Ling Jiang, Ji Liu & Hongwei Yu

  2. State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, China

    Lian Wu & Jiahai Zhou

Authors
  1. Jingbo Ma
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  2. Lian Wu
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  3. Fei Guo
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  4. Jiali Gu
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  5. Xiaoling Tang
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  6. Ling Jiang
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  7. Ji Liu
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  8. Jiahai Zhou
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  9. Hongwei Yu
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Correspondence to Jiahai Zhou or Hongwei Yu.

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Ma Jingbo and Wu Lian contributed equally.

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Ma, J., Wu, L., Guo, F. et al. Enhanced enantioselectivity of a carboxyl esterase from Rhodobacter sphaeroides by directed evolution. Appl Microbiol Biotechnol 97, 4897–4906 (2013). https://doi.org/10.1007/s00253-012-4396-2

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