Yang Xue-Qing, Wang Wei, Tan Xiao-Ling, Wang Xiao-Qi, Dong Hui
Key Laboratory of Economical and Applied Entomology of Liaoning Province, College of Plant Protection, Shenyang Agricultural University , Shenyang 110866, China.
Institute of Plant Protection, Chinese Academy of Agricultural Sciences , Beijing 100193, China.
J Agric Food Chem. 2017 Mar 22;65(11):2337-2344. doi: 10.1021/acs.jafc.7b00372. Epub 2017 Mar 14.
On the basis of prior work, cytochrome P450 CYP9A61 was found to be enriched in fat bodies and during feeding stages, and transcription was induced by λ-cyhalothrin in Cydia pomonella. In this study, recombinant CYP9A61 was expressed in Escherichia coli and Pichia pastoris, and its biochemical properties were investigated. Substrate saturation curves and biochemical properties revealed that, in the presence of glycosylation, the yeast-secreted CYP9A61 exhibited a higher affinity for the substrate p-nitroanisole and was found to be more stable at certain pHs and temperatures than bacterially produced CYP9A61. Half-inhibitory concentrations (IC) of three synthetic pyrethroids on both the bacterium- and yeast-expressed CYP9A61 suggested that recombinant CYP9A61 expressed in different hosts exhibits different inhibition properties. Taken together, our findings show that yeast-expressed CYP9A61 exhibits enzyme activity that is better than that expressed in bacteria and might be used for further metabolism assays to reveal the insecticide-detoxifying role of CYP9A61 in C. pomonella.
基于先前的研究工作，发现细胞色素P450 CYP9A61在苹果蠹蛾的脂肪体和取食阶段富集，并且其转录受高效氯氟氰菊酯诱导。在本研究中，重组CYP9A61在大肠杆菌和毕赤酵母中表达，并对其生化特性进行了研究。底物饱和曲线和生化特性表明，在存在糖基化的情况下，酵母分泌的CYP9A61对底物对硝基苯甲醚表现出更高的亲和力，并且发现在特定的pH值和温度下比细菌产生的CYP9A61更稳定。三种合成拟除虫菊酯对细菌和酵母表达的CYP9A61的半数抑制浓度（IC）表明，在不同宿主中表达的重组CYP9A61表现出不同的抑制特性。综上所述，我们的研究结果表明，酵母表达的CYP9A61表现出比细菌表达的更好的酶活性，可用于进一步的代谢分析，以揭示CYP9A61在苹果蠹蛾中的解毒作用。
