Cloning and expression of the carbaryl hydrolase gene mcbA and the identification of a key amino acid necessary for carbaryl hydrolysis

摘要

Highlights•A carbaryl-degrading strainPseudomonassp. XWY-1was isolated.•A carbaryl hydrolase genemcbA was expressed inE. coliand purified.•Characteristics and substrate range of McbA were investigated.•His504 was identified as a key residue for McbA function.AbstractCarbamate hydrolase is the initial and key enzyme for degradation of carbamate pesticides. In the present study, we report the isolation of a carbaryl-degrading strainPseudomonassp. XWY-1, the cloning of its carbaryl hydrolase gene (mcbA) and the characterization of McbA. Strain XWY-1 was able to utilize carbaryl as a sole carbon source and degrade it using 1-naphthol as an intermediate. Transposon mutagenesis identified a mutant of XWY-1M that was unable to hydrolyze carbaryl. The transposon-disrupted genemcbA was cloned by self-formed adaptor PCR, then expressed inEscherichia coliBL21(DE3) and purified. McbA was able to hydrolyze carbamate pesticides including carbaryl, isoprocarb, fenobucarb, carbofuran efficiently, while it hydrolyzed aldicarb, and propoxur poorly. The optimal pH of McbA was 7.0 and the optimal temperature was 40°C. The apparentKmandkcatvalues of McbA for carbaryl were 77.67±12.31μM and 2.12±0.10s−1, respectively. Three amino acid residues (His467, His477 and His504) in the predicted polymerase/histidinol phosphatase-like domain were shown to be closely related to the activity of McbA, with His504 being the most important, as a replacement of His504 led to the complete loss of activity. This is the first study to identify key amino acids in McbA.