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›› 2009, Vol. 9 ›› Issue (5): 981-986.

• 生化工程专栏 • 上一篇    下一篇

葡萄糖苷酶在毕赤酵母中的重组表达及一步纯化

周进 银鹏   

  1. 清华大学深圳研究生院生命学部 南京林业大学理学院
  • 收稿日期:2009-04-07 修回日期:2009-06-22 出版日期:2009-10-20 发布日期:2009-10-20
  • 通讯作者: 周进

Expression and One-step Purification of Recombinant Glucosidase in Pichia pastoris

ZHOU Jin YIN Peng   

  1. Life Science Division of Graduate School at Shenzhen, Tsinghua University College of Science, Nanjing Forest University
  • Received:2009-04-07 Revised:2009-06-22 Online:2009-10-20 Published:2009-10-20
  • Contact: ZHOU Jin

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

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

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