Cloning and Expression of the Isoprocarb Hydrolase Gene cehA from the Newly Isolated Isoprocarb-Degrading Strain Sphingobium sp. R-7
摘要
To remove the isoprocarb residues from the environment, a bacterial strain that was capable of degrading isoprocarb was isolated from agricultural soils where isoprocarb has been applied for a long period, and named R-7. On the basis of its cellular morphology and phenotypic features and 16S rRNA phylogenetic analysis, strain R-7 was preliminarily identified as Sphingobium sp. This strain could grow on isoprocarb as a sole carbon source and degrade 98.3% of 0.5 mM of isoprocarb within 16 h in batch liquid culture. The metabolite produced was identified as 2-isopropylphenol by high-performance liquid chromatography and tandem mass spectrometry (HPLC–MS/MS) analysis. Strain R-7 hydrolysed the ester bond of isoprocarb to generate 2-isopropylphenol, but this metabolite was not further degraded. Upon the genome comparison, the isoprocarb hydrolase gene cehA was cloned from strain R-7 and expressed in Escherichia coli BL21. The purified CehAR-7 displayed maximal enzymatic activity at 40 °C and pH 7.0. The apparent Km and kcat values of CehAR-7 for isoprocarb were 169.12 ± 7.74 µmol L−1 and 347 ± 17.73 min−1, respectively. CehAR-7 could hydrolyse carbaryl and isoprocarb efficiently, although its ability to hydrolyse carbofuran, oxamyl and methomyl was poor. In conclusion, this study provided an efficient isoprocarb-degrading microorganism, and identified the isoprocarb hydrolase CehA from strain R-7, which has potential applications for microbial remediation of isoprocarb-polluted environments.