Cloning and biochemical characterization of a novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation.
A highly efficient carbendazim (methyl-1H-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 muM 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.
CSIRO Entomology, Clunies Ross St., Australian Capital Territory, Australia. gunjan.pandey@csiro.au
Gunjan
G
Pandey
Susan J
SJ
Dorrian
Robyn J
RJ
Russell
Clint
C
Brearley
Steven
S
Kotsonis
John G
JG
Oakeshott
D001483:Base Sequence; D001562:Benzimidazoles / Q000378:metabolism*; D001673:Biodegradation, Environmental; D002219:Carbamates / Q000378:metabolism*; D003001:Cloning, Molecular; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000302:isolation & purification; D006868:Hydrolysis; D008969:Molecular Sequence Data
D001562:Benzimidazoles; D002219:Carbamates; D004950:Esterases; C006698:carbendazim