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Cloning, Overexpression, Purification, and Characterization of the Maleylacetate Reductase from Sphingobium chlorophenolicum Strain ATCC 53874

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Abstract

The final enzyme in the pentachlorophenol (PCP) biodegradation pathway in Sphingobium chlorophenolicum is maleylacetate reductase (PcpE), which catalyzes the reductive dehalogenation of 2-chloromaleylacetate to maleylacetate and the subsequent reduction of malyelacetate to 3-oxoadipate. In this study, the pcpE gene was cloned from S. chlorophenolicum strain ATCC 53874 and overexpressed in Escherichia coli BL21-AI cells. The recombinant PcpE, purified to higher than 95% purity using affinity chromatography, exhibited optimal activity at pH 7.0. The kinetic parameters k cat and K m were 1.2 ± 0.3 s−1 and 0.09 ± 0.04 mM, respectively, against maleylacetate under the optimal pH. In addition, the purified PcpE was able to restore PCP-degrading capability to S. chlorophenolicum strain ATCC 39723, implicating that there was no functional PcpE in the ATCC 39723 strain.

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Acknowledgments

This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada, a New Investigator Establishment Grant from the Saskatchewan Health Research Foundation, and a New Opportunities Fund from the Canada Foundation for Innovation.

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Authors and Affiliations

  1. College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada

    Lifeng Chen, Katharine Maloney, Ed Krol & Jian Yang

  2. Environment Canada, Water Science and Technology Directorate, Saskatoon, Canada

    Bin Zhu

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  1. Lifeng Chen
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  2. Katharine Maloney
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  3. Ed Krol
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  4. Bin Zhu
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  5. Jian Yang
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Correspondence to Jian Yang.

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Chen, L., Maloney, K., Krol, E. et al. Cloning, Overexpression, Purification, and Characterization of the Maleylacetate Reductase from Sphingobium chlorophenolicum Strain ATCC 53874. Curr Microbiol 58, 599–603 (2009). https://doi.org/10.1007/s00284-009-9377-z

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  • DOI: https://doi.org/10.1007/s00284-009-9377-z

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