关键词: 葡萄糖氧化酶, 棉铃虫, 毕赤酵母, 异源表达

Abstract: 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 GS115H. 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.

Key words: Glucose oxidase, Helicoverpa armigera, Pichia pastoris, Heterologous expression