Altered insecticide hydrolase activity through multiple site mutagenesis of carboxylesterase 001C from the cotton bollworm Helicoverpa armigera

CarE001C variants with multiple mutations were generated and expressed in E. coli . • The mutants showed significantly reduced esterase activity toward α -naphthyl acetate. • Several mutants acquired enhanced catalytic efficiency toward specific insecticides. Carboxylesterases (CarEs) are key phase I detoxification enzymes that either directly metabolize insecticides or sequester insecticide molecules, thereby reducing their toxicity. Previous studies demonstrated that the A127D mutation in carboxylesterase 001C (CarE001C) of Helicoverpa armigera significantly enhanced hydrolase activity toward the model organophosphate substrate diethyl 4-methylumbelliferyl phosphate (dEUP). To further explore the functional implications of this mutation, six CarE001C variants containing multiple site mutations were generated using the CarE001C A127D mutant as a template through a multiple site-directed mutagenesis approach. The recombinant proteins were successfully expressed in Escherichia coli , purified, and confirmed by SDS-PAGE and Western blot analyses. Enzyme kinetics assays revealed that all multiple-site mutants exhibited significantly reduced esterase activity toward α -naphthyl acetate compared with the wild-type CarE001C. In vitro metabolism assays indicated that several of these mutants exhibited improved hydrolase activity toward specific insecticides. Among them, three mutants (OP4, OP5, and OP6) showed elevated hydrolase activities toward chlorpyrifos, with specific activities ranging from 26.3 to 38.8 nmol·min −1 ·mg protein −1 . Additionally, two mutants demonstrated increased detoxification capacities for beta -cypermethrin and methomyl, exhibiting specific activities of 13.2 and 1.7 nmol·min −1 ·mg protein −1 , respectively. Collectively, these results suggest that multiple site mutations can significantly alter the catalytic properties of H. armigera CarE001C, leading to qualitative changes in insecticide hydrolase activity. This work provides new insights into the adaptive potential of insect esterases in insecticide detoxification, thereby deepening our understanding of their roles in resistance evolution and potential applications in bioremediation. Download: Download high-res image (149KB) Download: Download full-size image