木薯Linamarase基因的克隆、表达及酶学特性分析

Cloning,Expression of Linamarase in Cassava and Characterization of Its Recombinant Protein Activity

亚麻仁苷酶(Linamarase)是存在于木薯、巴豆等作物中的降解生氰葡萄糖苷Linamarin(亚麻仁苷)的酶,在结构上与芥子酶有很大的相似性,对它的研究有利于揭示生氰葡萄糖苷酶和芥子酶之间的关系以及芥子酶的起源问题。通过真核表达的方法,克隆了木薯Linamarase基因,构建酵母表达载体,转化毕赤酵母GS115,经诱导表达和亲和纯化,获得纯化的Linamarase重组蛋白。SDS-PAGE分析表明,重组蛋白分子量集中在71 kD左右。该酶最适反应温度在37℃左右,最适pH约为5。以pNPG为底物时,Km为1.70 mmol/L,最大反应速率Vmax为8.36μmol/(min.mg)。

Abstract： Linamarase is a kind of enzyme, which can degrade Linamarin in cassava, croton and other crops. The structure of this enzyme is very similar with rnyrosinase. Our study on this enzyme was avail to announce the relationship between cyanogen glycosidase enzyme and myrosinase, at the same time also reveal the origin of myrosinase. Linamarase gene was cloned from cassava, the yeast expression vector was constructed and then transformed into piehia pastorios GSll5. Purified recombinant protein of Linamarase was obtained after induction and affinity purification. SDS-PAGE analysis showed that, the recombinant protein concentrated was in the 71 kD molecular weight around. The optimal temperature of this enzyme was about 37℃, and the optimal pH was about 5. The Km was 1.70 mmol/L, and Vmax was 8.36μmol/（min. mg）, while pNPG was used as the substrate.