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Purification, Gene Cloning, and Characterization of a Novel Halohydrin Dehalogenase from Agromyces mediolanus ZJB120203

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Abstract

A novel halohydrin dehalogenase (HHDH), catalyzing the transformation of 1,3-dichloro-2-propanol (1,3-DCP) to epichlorohydrin (ECH), was purified from Agromyces mediolanus ZJB120203. The molecular mass of the enzyme was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 735-bp nucleotide fragment was obtained based on the N-terminal and internal amino acid sequences of the purified HHDH. The gene codes a protein sequence with 244 amino acid residues, and the protein sequence shows high similarity to Hhe AAD2 (HHDH from Arthrobacter sp. AD2), defined as Hhe AAm, which is the seventh reported HHDH. Expression of Hhe AAm was carried out in Escherichia coli and purification was performed by nickel-affinity chromatography. The recombinant HheAAm possessed an optimal pH of 8.5 and an optimal temperature of 50 °C and manifested a K m of 4.58 mM and a V max of 3.84 μmol/min/mg. The activity of Hhe AAm was not significantly affected by metal ions such as Zn2+, Ca2+, Cu2+, and EDTA, but was strongly inhibited by Hg2+ and Ag+. In particular, the Hhe AAm exhibits an enantioselectivity for the conversion of prochiral 1,3-DCP to (S)-ECH. The applications of the Hhe AAm as a catalyst for asymmetric synthesis are promising.

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Acknowledgments

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

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Authors and Affiliations

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

    Feng Xue, Zhi-Qiang Liu, Nan-Wei Wan & Yu-Guo Zheng

  2. Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Feng Xue, Zhi-Qiang Liu, Nan-Wei Wan & Yu-Guo Zheng

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  1. Feng Xue
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  2. Zhi-Qiang Liu
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Correspondence to Yu-Guo Zheng.

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Xue, F., Liu, ZQ., Wan, NW. et al. Purification, Gene Cloning, and Characterization of a Novel Halohydrin Dehalogenase from Agromyces mediolanus ZJB120203. Appl Biochem Biotechnol 174, 352–364 (2014). https://doi.org/10.1007/s12010-014-1111-z

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  • DOI: https://doi.org/10.1007/s12010-014-1111-z

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