Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, College of Plant Protection, Northwest A & F University, Yangling, 712100, Shaanxi, Peoples's Republic of China.
Appl Microbiol Biotechnol. 2013 Dec;97(24):10423-33. doi: 10.1007/s00253-013-5236-8. Epub 2013 Sep 27.
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.
羧酸酯酶（CEs）是昆虫体内负责解毒杀虫剂的酶。在苹果蠹蛾（Cydia pomonella）中，CEs 参与了合成拟除虫菊酯、新烟碱类、氨基甲酸酯和有机磷杀虫剂的解毒过程。然而，在大肠杆菌（Escherichia coli）系统中，CEs 蛋白的功能过表达常常导致不溶性蛋白的产生。在本研究中，我们在大肠杆菌 BL21（DE3）中表达了融合蛋白 CpCE-1。该重组蛋白在 37°C 下以包涵体形式过量表达，而在较低的生长温度下，它产生了更高比例的可溶性蛋白。在生长培养基中添加山梨醇可以增加可溶性蛋白的产量和酶活性。融合蛋白从裂解物上清液中通过 Ni(2+)-NTA 琼脂糖凝胶柱进行纯化。该酶对α-萘基乙酸酯（α-NA）表现出更高的亲和力和底物特异性，α-NA 的 k cat/K m 值为 100 s(-1) μM(-1)，β-萘基乙酸酯的 k cat/K m 值为 29.78 s(-1) μM(-1)。使用底物α-NA 还确定了 V max 和 K m 分别为 12.9 μmol/min/mg 蛋白和 13.4 μM。最适 pH 值为 7.0，最适温度为 25°C。酶抑制试验表明，PMSF 和 DEPC 强烈抑制酶活性，而金属离子 Cu(2+)和 Mg(2+)则显著激活酶活性。更重要的是，氯氰菊酯、灭多威和乙酰甲胺磷被发现抑制了酶的活性。这里提供的数据为 C. pomonella 中可溶性蛋白的异源表达以及进一步研究昆虫体内杀虫剂代谢提供了信息。这是首次报道苹果蠹蛾 CEs 蛋白的特性。
