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Cloning and Biochemical Characterization of a Novel Carbendazim (Methyl-1 H -Benzimidazol-2-ylcarbamate)-Hydrolyzing Esterase from the Newly Isolated Nocardioides sp. Strain SG-4G and Its Potential for Use in Enzymatic Bioremediation

Pandey, Gunjan, Dorrian, Susan J., Russell, Robyn J., Brearley, Clint, Kotsonis, Steven, Oakeshott, John G.·2010·Applied and Environmental Microbiology
DOI 10.1128/aem.02990-09
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Abstract

A highly efficient carbendazim (methyl-1 H -benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converted to the previously unknown metabolite 2-hydroxybenzimidazole. The initial steps of this novel metabolic pathway were confirmed by growth and enzyme assays and liquid chromatography-mass spectrometry (LC-MS) studies. The enzyme responsible for carrying out the first step was purified and subjected to N-terminal and internal peptide sequencing. The cognate gene, named mheI (for MBC-hydrolyzing enzyme), was cloned using a reverse genetics approach. The MheI enzyme was found to be a serine hydrolase of 242 amino acid residues. Its nearest known relative is an uncharacterized hypothetical protein with only 40% amino acid identity to it. Codon optimized mheI was heterologously expressed in Escherichia coli , and the His-tagged enzyme was purified and biochemically characterized. The enzyme has a K m and k cat of 6.1 μM and 170 min −1 , respectively, for MBC. Radiation-killed, freeze-dried SG-4G cells showed strong and stable MBC detoxification activity suitable for use in enzymatic bioremediation applications.

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Metadata

Authors
Pandey, G., Dorrian, S. J., Russell, R. J., Brearley, C., Kotsonis, S., & Oakeshott, J. G.
Affiliations
  • CSIRO Entomology, Clunies Ross St., Acton, Australian Capital Territory 2601, Australia
  • Orica Australia Limited, 1 Nicholson St., Melbourne, Victoria 3000, Australia
Venue
Applied and Environmental Microbiology
Publisher
American Society for Microbiology
Published
2010-05
Volume
76
Issue
9
Pages
2940-2945
ISSN
  • 0099-2240
  • 1098-5336
DOI
10.1128/aem.02990-09
Cited by
60
References
0
Language
en
Licence
journals.asm.org/non-commercial-tdm-license (tdm)

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