黑曲霉葡萄糖氧化酶基因在毕赤酵母SMD1168中的表达

Expression of Aspergillus niger Glucose Oxidase Gene in Pichia pastoris SMD1168

在毕赤酵母SMD1168中利用乙醇氧化酶AOX1强启动子表达黑曲霉葡萄糖氧化酶(Glucose oxidase,GOD)。提取黑曲霉Aspergillus niger PCTC的基因组DNA,以此为模板进行PCR扩增获得葡萄糖氧化酶基因,将目的基因插入到具有AOX1强启动子的表达载体p PICZαA上,经电转化导入毕赤酵母SMD1168中。经zeocin抗性平板初筛、摇床复筛以及SDS-PAGE蛋白质电泳的检测,获得了一株产葡萄糖氧化酶活力的菌株,该株菌在30℃、200 r/min的培养条件下,经体积分数1.0%的甲醇诱导发酵1 d可获得1.12 U/mL的酶活。对该菌株进行了摇瓶产酶条件优化,其最佳发酵条件为:在pH 5、30℃下经体积分数1.5%甲醇诱导7 d,酶活为32 U/mL。

Aspergillus niger glucose oxidase(GOD) was expressed in Pichia pastoris SMD1168 with the alcohol oxidase gene promoter(AOX1).A gene of glucose oxidase from Aspergillus niger PCTC was cloned.The gene was fused to the p PICZαA plasmid which had the alcohol oxidase gene promoter(AOX1) and expressed in Pichia pastoris SMD1168.After screening by zeocin gradient resistance plate,shake culture,and the SDS-PAGE analysis of proteins in the culture,one strain was obtained,which gave the higest enzyme activity(1.12 U/mL) after one day induction by 1%methanol.Through the optimization of the shake flask experiments,the highest enzyme activity of 32 U/mL was achieved in the flask by induction for 7 d under optimal conditions(30 ℃,200 r/min,pH5 and 1.5% methanol).