【目的】原核表达茶尺蠖EoCarE592羧酸酯酶(carboxlesterase, CarE)基因EoCarE592，并探讨重组蛋白对农药的降解能力。【方法】构建原核表达载体 pET-32a-EoCarE592，转化大肠杆菌Escherichia coli BL21异源表达，SDS-PAGE和Western blot鉴定重组蛋白，并对包涵体蛋白变复性；通过固蓝B盐比色法的含量标准曲线测定重组EoCarE592酶活性以及温度、pH和金属离子对酶活性的影响；利用气相色谱检测EoCarE592在30 ℃pH 7.0和0, 4, 8, 12, 16, 20及24 h时对200 mg/L的高效氯氟氰菊酯(lambda-cyhalothrin)、甲基对硫磷(methyl-parathion)和异丙威(isoprocarb)降解能力。【结果】通过大肠杆菌异源表达得到重组EoCarE592包涵体蛋白，经尿素变复性后得到酶活性29.8 U的重组EoCarE592，其最适温度 30 ℃左右、最适pH 7.0~8.0左右、Mg²⁺和K⁺对酶活有促进作用；重组EoCarE592能在30 ℃, pH 7.0, 24 h内对初始浓度为200 mg/L的高效氯氟氰菊酯、甲基对硫磷和异丙威降解率分别为81.30%, 83.94%和79.83%。【结论】羧酸酯酶EoCarE592能降解高效氯氟氰菊酯、甲基对硫磷和异丙威，可能参与茶尺蠖的解毒过程。本研究为环境和果蔬中农药残留的降解奠定了基础。

关键词: 茶尺蠖, 羧酸酯酶, 原核表达, 酶活性, 农药降解

Abstract:

【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 the 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 under pH 7.0 at30 ℃and 0, 4, 8, 12, 16, 20 and 24 h to 200 mg/L lambda-cyhalothrin, methyl-parathion and isoprocarb. 【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 around30 ℃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 at30 ℃, 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.

Key words: Ectropis obliqua')">

Ectropis obliqua, carboxylesterase, prokaryotic expression, enzyme activity, pesticide degradation