棉铃虫葡萄糖氧化酶HaGOX在毕赤酵母中的重组表达和性质研究

Expression and Characterization of Glucose Oxidase HaGOX From Helicoverpa armigera in Pichia pastoris

毕赤酵母是使用最广泛、最具有发展前景的异源蛋白表达系统之一。在毕赤酵母GS115中实现了来源于棉铃虫(Helicoverpa armigera)葡萄糖氧化酶基因hagox的异源表达。以棉铃虫hagox基因连接pPICZαA质粒构建重组表达载体,电击转化毕赤酵母GS115H菌株后得到含有hagox基因的重组酵母菌株。通过平板显色、酶活力测定和SDS-PAGE分析,重组酵母中HaGOX成功表达。HaGOX含606个氨基酸,理论分子量66.9 kD,预测等电点pH 4.78。经测试重组HaGOX的最适pH为6.0~7.0,最适温度为40℃,对6-脱氧-葡萄糖和D-葡萄糖具有高催化活力,以D-葡萄糖为底物的比活力为13.63 U·mg^-1,对其他吡喃糖和二糖没有催化活力。

Pichia pastoris is one of the most widely used and most promising heterologous protein expression systems.In the study,Helicoverpa armigera glucose oxidase(hagox)gene was expressed in Pichia pastoris.The hagox gene was ligated into the pPICZαA plasmid to construct the Pichia pastoris recombinant expression vector.The Pichia pastoris recombinant strain containing hagox gene was constructed by electroporating the recombinant plasmid into Pichia pastoris GS115 H.Then,the expression of recombinant protein HaGOX was detected by plate coloring,enzyme activity determination and SDS-PAGE.HaGOX encoded 606 amino acids,theoretical molecular weight was 66.9 kD and isoelectric point was 4.78.The optimal pH and temperature were pH 6.0~7.0 and 40℃,respectively,and optimal substrate were 6-deoxy-glucose and D-glucose.The specific activity of the recombinant enzyme was determined to be 13.63 U·mg^-1 by using D-glucose as substrate,and there was no catalytic activity for other pyranose and disaccharide.