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Identification and cloning of a gene encoding dichloromethane dehalogenase from a methylotrophic bacterium, Bacillus circulans WZ-12 CCTCC M 207006

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Abstract

The gene dehalA encoding a novel dichloromethane dehalogenases (DehalA), has been cloned from Bacillus circulans WZ-12 CCTCC M 207006. The open reading frame of dehalA, spanning 864 bp, encoded a 288-amino acid protein that showed 85.76% identity to the dichloromethane dehalogenases of Hyphomicrobium sp. GJ21 with several commonly conserved sequences. These sequences could not be found in putative dichloromethane (DCM) dehalogenases reported from other bacteria and fungi. DehalA was expressed in Escherichia coli BL21 (DE3) from a pET28b(+) expression system and purified. The subunit molecular mass of the recombinant DehalA as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was approximately 33 kDa. Subsequent enzymatic characterization revealed that DehalA was most active in a acidic pH range at 30°, which was quite different from that observed from a facultative bacterium dichloromethane dehalogenases of Methylophilus sp. strain DM11. The Michaelis–Menten constant of DCM dehalogenase was markedly lower than that of standard DCM dehalogenases.

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Abbreviations

DCM:

Dichloromethane

DehalA:

Dichloromethane dehalogenases from Bacillus circulans WZ-12

dehalA :

Gene encoding DehalA

dehA :

Dichloromethane dehalogenases of Hyphomicrobium sp. GJ21

K m :

Michaelis–Menten constant

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

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Acknowledgments

The authors wish to thank the National High Technology Research and Development Program (“863”Program) of China (Grant No. 2006AA06A310) and the Natural Science Foundation of China (Grant No. 20476099) for the support of this study.

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Authors and Affiliations

  1. College of Biological and Environmental Engineering, Zhejiang University of Technology, 310032, Hangzhou, People’s Republic of China

    Shijin Wu, Xiang Yu & Jianmeng Chen

  2. Ningbo Institute of Technology, Zhejiang University, 315100, Ningbo, People’s Republic of China

    Huaxing Zhang

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  1. Shijin Wu
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Correspondence to Jianmeng Chen.

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Wu, S., Zhang, H., Yu, X. et al. Identification and cloning of a gene encoding dichloromethane dehalogenase from a methylotrophic bacterium, Bacillus circulans WZ-12 CCTCC M 207006. Bioprocess Biosyst Eng 32, 845–852 (2009). https://doi.org/10.1007/s00449-009-0311-3

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