有机磷农药降解酶基因Oph2的克隆及甲基对硫磷降解效果研究

Study on cloning of organophosphorus pesticide degradation enzyme gene Oph2 and degradation effect of methyl parathion

目的挖掘可高效表达有机磷农药降解酶的基因,并构建含有该表达基因的工程菌,提供一种高效降解甲基对硫磷的新方法。方法利用基因克隆获得高效表达有机磷农药降解酶基因Oph2,采用基因工程手段将其转化至毕赤酵母(Pichia pastoris)表达系统,对毕赤酵母的发酵产物进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodiumdodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)定性和酶活定量研究,通过与目标底物甲基对硫磷反应,分析工程菌表达降解酶对甲基对硫磷的降解效果。结果本研究成功克隆得到了高效表达有机磷农药降解酶基因Oph2,基因全长1000 bp,编码256个氨基酸,酶蛋白分子量为37 kD,并利用表达载体pPIC9K成功将其转化至毕赤酵母GS115。构建的工程菌GS115-pPIC9K-Oph2表达有机磷农药降解酶活性为11.57 U/mL,对6.8 mg/L的甲基对硫磷降解效率为90%以上。结论本研究得到的工程菌GS115-pPIC9K-Oph2可高效表达有机磷农药降解酶,该酶活性高,对甲基对硫磷降解能力强,可有效应用于甲基对硫磷的降解。

Objective To explore the gene that can efficiently express organophosphorus pesticide degrading enzyme, construct engineering bacteria containing this gene, and provide a new method for efficient degradation of methyl parathion. Methods The gene Oph2 with high expression of organophosphorus pesticide degrading enzyme was cloned and transformed into Pichia pastoris expression system by genetic engineering, the fermentation products of Pichia pastoris were qualitatively studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and quantitatively studied by enzyme activity. Finally, the degradation effect of methyl parathion by engineering bacteria expression degrading enzyme was analyzed. Results In this study, Oph2 gene with high exression of organophosphorus pesticide degrading enzyme was successfully cloned, the full length of the gene was 1000 bp, encoding 256 amino acids, and the molecular weight of the enzyme protein was 37 kD, it was successfully transformed into Pichia pastoris GS115 using expression vector pPIC9K. The constructed engineering strain GS115-pPIC9K-Oph2 expressed organophosphorus pesticide degrading enzyme with the activity of 11.57 U/mL, and the degradation efficiency of 6.8 mg/L methyl parathion was over 90%. Conclusion The engineering strain GS115-pPIC9K-Oph2 obtained in this study can efficiently express organophosphorus pesticide degrading enzyme,which has high activity and strong ability to degrade methyl parathion, and can be effectively used for the degradation of parathion methyl.