Preliminary Study Of Mechanism Of Chymotrypsin For Metabolism Of Deltamethrin

Development of vector resistance to pyrethroid insecticides has been reported all over the world,which is a major obstacle to control of vector-borne diseases.In an effort to elucidate the mechanisms of the resistance,our laboratory has previously identified and reported a number of genes associated with resistance to a widely used pyrethroid deltamethrin,including a Culex chymotrypsin gene.Subsequent experiments with over-expression and RNAi further established that chymotrypsin upregulation was a causal factor of deltamethrin-resistance. To test if chymotrypsin directly acts on deltamethrin,in vitro hydrolytic reactions of deltamethrin catalyzed by chymotrypsin were carried out.In addition,the kinetic parameters were measured.Deltamethrin degradation was assessed by measuring deltamethrin disappearance with GC-MS and UV-vis spectrophotometry following incubation of various concentrations(4.96-24.80μM)of deltamethrin withα-chymotrypsin.The retention time of crude products of deltamethrin metabolic reactions(including undegraded substrate deltamethrin)were 37.968 min,37.415 min,36.490 min,and 35.895 min. Compared with NIST Mass Spectral Database,the peak that appeared in 37.968 min was undegraded deltamethrin,and the other peaks represented degradation products of deltamethrin catalyzed by chymotrypsin.The UV-vis spectrophotometric peak absorbance of deltamethrin was at 264 nm,and the peak absorbance of deltamethrin metabolic products were at 250 nm and 296 nm respectively. Michaelis-Menten metabolic rate constants(Vmax and Km)were calculated by nonlinear regression for chymotrypsin(from bovine)using GraphPad Prism 4.0,the Vmax was 97.97±26.57 nmol/L/min and Km was 7.84±3.83μM.The larvicidal bioassay tests indicated that the mixture from the degradation reaction showed LC₅₀increased significantly(p＜0.05)when applied on both sensitive and resistant strains of Cx.pipiens pallens,indicating the metabolites have reduced or no toxicity.The acute oral toxicity of the metabolic to Wistar rats was much lower than that of deltamethrin.The data provided a mechanistic explanation for the correlation between chymotrypsin upregulation and deltamethrin resistance.It is the first report to our knowledge on the deltamethrin metabolism by chymotrypsin.Our work also showed that chymotrypsin could directly degrade small,non-protein molecules such as deltamethrin. The results also suggest that chymotrypsin could be attractive candidate for the development of biocatalyst for the control of residual insecticides in the environment and on agricultural products.