Recombinant expression of carboxylesterase EoCarE592 of Ectropis obliqua(Lepidoptera:Geometridae)and determination of its ability to degrade pesticides
[Aim]Prokaryotic expression of EoCarE592,a carboxylesterase(CarE)gene from Ectropis obliqua,and exploration of the ability of the recombinant protein to degrade pesticides.[Methods]The prokaryotic expression vector pET-32a-EoCarE592 was constructed to be transformed to the Escherichia coli BL21 for heterologous expression.The recombinant protein was identified by SDS-PAGE and Western blot,and the inclusion body protein was denatured and renatured.The enzyme activity of the recombinant EoCarE592,and the effects of temperature,pH,and metal ions on the enzyme activity were determined by using the content standard curve of solid blue B salt colorimetry.Gas chromatography was used to detect the degradation ability of EoCarE592 to 200 mg/L lambda-cyhalothrin,methyl-parathion and isoprocarb under pH 7.0 at 30 ℃ and 0,4,8,12,16,20 and 24 h.[Results]Recombinant EoCarE592 inclusion body protein was obtained through heterologous expression in E.coli.After urea renaturation,the recombinant EoCarE592 with the enzyme activity of 29.8 U was obtained.The optimal temperature and pH were determined to be around 30℃and 7.0-8.0,respectively,and Mg2+and K+had a promoting effect on the enzyme activity of EoCarE592.Recombinant EoCarE592 can degrade lambda-cyhalothrin,methyl-parathion and isoprocarb at an initial concentration of 200 mg/L within 24 h at 30 ℃,pH 7.0,with the degradation rates of 81.30％,83.94％,and 79.83％,respectively.[Conclusion]The carboxylase EoCarE592 can degrade lambda-cypermethrin,methyl-parathion,and isoprocarb,and may be involved in the detoxification process of E.obliqua.This study lays a foundation for the degradation of pesticide residues in the environment and fruits and vegetables.
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