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Expression, characterization, and improvement of a newly cloned halohydrin dehalogenase from Agrobacterium tumefaciens and its application in production of epichlorohydrin

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A gene encoding halohydrin dehalogenase (HHDH) from Agrobacterium tumefaciens CCTCC M 87071 was cloned and expressed in Escherichia coli. To increase activity and stability of HHDH, 14 amino acid residues around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected as targets for site-directed mutagenesis. The studies showed that the mutant HHDH (Mut-HHDH) enzyme had a more accessible substrate-binding pocket than the wild-type HHDH (Wt-HHDH). Molecular docking revealed that the distance between the substrate and active site was closer in mutant which improved the catalytic activity. The expressed Wt-HHDH and Mut-HHDH were purified and characterized using 1,3-dichloro-2-propanol (1,3-DCP) as substrates. The specific activity of the mutant was enhanced 26-fold and the value of k cat was 18.4-fold as compared to the Wt-HHDH, respectively. The Mut-HHDH showed threefold extension of half-life at 45 °C than that of Wt-HHDH. Therefore it is possible to add 1,3-DCP concentration up to 100 mM and epichlorohydrin (ECH) was produced at a relatively high conversion and yield (59.6 %) using Mut-HHDH as catalyst. This Mut-HHDH could be a potential candidate for the upscale production of ECH.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 21176224), National Basic Research Program of China (973 Program) (No. 2011CB710806), National Major Project of Scientific Instruments Development of China (No. 2012YQ150087) and the Natural Science Foundation of Zhejiang Province of China (No. Z4080032 and R3110155).

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  1. Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, Zhejiang, People’s Republic of China

    Zhi-Qiang Liu, Ai-Cun Gao, Ya-Jun Wang, Yu-Guo Zheng & Yin-Chu Shen

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  1. Zhi-Qiang Liu
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Correspondence to Yu-Guo Zheng.

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Liu, ZQ., Gao, AC., Wang, YJ. et al. Expression, characterization, and improvement of a newly cloned halohydrin dehalogenase from Agrobacterium tumefaciens and its application in production of epichlorohydrin. J Ind Microbiol Biotechnol 41, 1145–1158 (2014). https://doi.org/10.1007/s10295-014-1443-2

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  • DOI: https://doi.org/10.1007/s10295-014-1443-2

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