Abstract: The bg gene encoding an extracellular glucosidase from Trichoderma viride was cloned into vector pPIC9k-His6 and expressed in Pichia pastoris GS115 strain under the control of AOX 1 promoter. After 120 h of 5 L-scaled fermentation, wet cells weight of the recombinant P. pastoris Mut+ strain reached 360.6 g/L, and the glucosidase concentration and enzyme activity in the supernatant were 2.1 mg/mL and 73.5 U/mL, respectively. The activity of glucosidase was improved 42.6 fold by affinity chromatography with a final yield of 73.6% and the specific activity of the purified enzyme was 71.9 U/mg. The purified glucosidase, analyzed by SDS-PAGE and western blotting, showed only one homogeneous band and the purity was 95.6% by HPLC. The pI of the enzyme was 5.0. Then the factors affecting the glucosidase activity were evaluated. The optimal temperature and pH were 50℃ and pH 6.5, respectively. Metallic ions Ag+, Ca2+, Cu2+, Fe2+, and SDS could inhibit the enzyme activity, whereas 1 mol/L Mg2+ enhanced 120% of the enzyme activity.

Key words: glucosidase, Trichoderma viride, Pichia pastoris, expression, one-step purification

摘要： 从绿色木霉中克隆了葡萄糖苷酶bg基因，构建入表达载体pPIC9k-His6中，然后在AOX1启动子的控制下，在毕赤酵母GS115菌株中表达. 在5 L发酵罐中发酵120 h，重组P. pastoris Mut+菌株湿重达360.6 g/L，葡萄糖苷酶浓度和酶活分别为2.1 mg/mL和73.5 U/mL. 经亲和层析一步纯化后，得到了电泳纯的葡萄糖苷酶. HPLC分析显示其纯度为95.6%，比酶活为71.9 U/mg. 纯化过程酶得率为73.6%，纯化倍数为42.6. 纯酶的等电点为5.0，最适温度为50℃，最适pH为6.5. 金属离子Ag+, Ca2+, Cu2+, Fe2+及SDS对葡萄糖苷酶活性有抑制作用，而Mg2+, Mn2+, K+能增强葡萄糖苷酶活性，其中1 mol/L Mg2+能使酶活提高20%.

关键词: 葡萄糖苷酶, 绿色木霉, 毕赤酵母, 表达, 一步纯化