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中国农学通报

中国农学通报 ›› 2010, Vol. 26 ›› Issue (21): 54-58.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

拟南芥硫代葡糖苷酶基因TGG1的克隆、表达和酶学特性

张盛敏 孙雪飘 张家明   

  • 收稿日期:2010-04-12 修回日期:2010-07-19 出版日期:2010-11-05 发布日期:2010-11-05
  • 基金资助:

    国家科技支撑计划项目

Cloning and Overexpression of Myrosinase Gene TGG1 from Arabidopsis thaliana and Characterization of Recombinant Protein

  • Received:2010-04-12 Revised:2010-07-19 Online:2010-11-05 Published:2010-11-05

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摘要：

本研究克隆拟南芥硫代葡糖苷酶基因TGG1，利用毕氏酵母表达。重组蛋白用镍亲和柱纯化，获得高纯度芥子酶。重组蛋白分子量78 kD，与天然TGG1分子量接近，为糖基化蛋白。TGG1信号肽在酵母中具有分泌功能，约25%芥子酶分泌到培养基中。TGG1重组蛋白具有广泛的pH适应性，最适反应温度40℃左右。其活性被低浓度维生素C激活，被高浓度维生素C抑制。用Hanes plot方法计算TGG1重组蛋白以Sinigrin为底物时，Km为65μmol/L，最大反应速率Vmax=3.28μmol.min-1.mg-1。

关键词: 甘蔗, 甘蔗, 品种, 蔗茎产量, 含糖量

Abstract:

Myrosinase gene TGG1 of Arabidopsis thaliana was cloned and overexpressed in Pichia pastoris. The recombinant protein of TGG1 was purified with Ni-NTA.The recombinant protein has a molecular weight of 78 kD, larger than the deduced naked protein (58 kD), but similar to the molecular weight of the natural TGG1 protein. The TGG1 signal peptide has some secretion function in yeast, resulted in approximately 25% myrosases in the culture medium. The TGG1 recombinant protein was active in a wide pH and temperature range. The optimal reaction temperature was around 40℃, and the pH between 6-9. Myrosinase activity was activated by low concentrations of ascorbic acid, and suppressed by high concentrations. The apparent Km and Vmax were 65 μmol/L and 3.28 μmol ? min-1 ? mg-1, respectively when sinigrin was the substrate.