Effect of temperature and sorbitol in improving the solubility of carboxylesterases protein CpCE-1 from Cydia pomonella and biochemical characterization
Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella , CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E . coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni 2+ -NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k cat / K m of 100 s −1 μM −1 for α-NA, and the value is 29.78 s −1 μM −1 for β-naphthyl acetate. The V max and K m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu 2+ and Mg 2+ significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C . pomonella in vitro. This is the first report of the characterization of CEs protein from C . pomonella .
危险品化学品经营许可证（带存储）