CRCU

Chemical Research in Chinese Universities ›› 2012, Vol. 28 ›› Issue (4): 666-671.

• Articles • Previous Articles     Next Articles

Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

REN He-jun1, LU Yang2, ZHOU Rui1, DAI Chun-yan3, WANG Yan1, ZHANG Lan-ying 1   

  1. 1. Key Laboratory of Groudwater Resources and Environment, Ministry of Education, College of Environment and Resources, Jilin University, Changchun 130021, P. R. China;
    2. Jilin Academy of Agricultural Sciences, Changchun 130033, P. R. China;
    3. Institute of Virology and AIDS Research, the First Hospital of Jilin University, Changchun 130061, P. R. China
  • Received:2012-01-19 Revised:2012-03-31 Online:2012-07-25 Published:2012-07-25
  • Supported by:

    Supported by the National Natural Science Foundation of China(Nos.41101226, 50879029) and the Technology Development Project of Jilin Province, China(Nos.201101020, 20090415).

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Abstract: To find new extradiol dioxygenases(EDOs, EC 1.13.11.2), a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity. A novel EDO, designated as Bph­C_A, was identified and heterologously expressed in Escherichia coli. The deduced amino acid sequence of BphC­_A exhibited a homology of less than 60% with other known EDOs. Phylogenetic analysis of BphC­_A suggests that the protein is a novel member of the EDO family. The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol, the preferred substrate of other known EDOs. The optimum activity of purified Bph­C_A occurred at pH=8.5 and 35℃, and Bph­C_A showed more than 40% of its initial activity at 5℃. The activity of purified Bph­C_A was significantly induced by Mn2+ and slightly reduced by Al3+, Cu2+ and Zn2+.

Key words: Extradiol dioxygenase, Metagenome, Cold-active enzyme, Gene cloning, Functional characterization