α-淀粉酶基因在毕赤酵母中的表达及酶学性质研究

Studies on Expression of Xanthomonas campestris α-Amylase Gene in Pichia pastoris and Enzymatic Properties

以克隆自野油菜黄单胞菌的α-淀粉酶基因质粒(pHN8004)为模板,通过PCR方法将α-淀粉酶基因克隆到毕赤酵母表达载体pHBM905A中,转化毕赤酵母GS115.利用甲醇对重组毕赤酵母GS115(pHBM905AM1)进行诱导,实现了表达.培养温度为28℃,用体积分数为1.0%的甲醇诱导,第7天分泌表达的α-淀粉酶酶活性最高,为1 081 U.mL-1,该酶的最适反应温度和最适反应pH值分别为50℃和5.9.

The active α-amylase gene from Xanthomonas campestris was cloned into the expression vector pHBM905A, and the recombinant plasmid was named pHBM905AM 1. The recombinant plasmid pHBM905AM 1 was linearized and transformed into P. pastori GS115. Activity of α-amylase secreted from the recombinant GS115 （pHBM905AM1） reached 1 081 U·mL^-1 on the 7th d when the recombinant GS115 was fermented with high density liquid media and induced by 1.0% methanol at culture temperature 28 ℃. The optimal temperature and pH value of a-amylase was 50 ℃. and 5.9, respectively.