糖化酵母糖化酶基因在酿酒酵母中的克隆与表达  

Cloning and Expressing Glucoamylase Gene of Saccharomyces diastaticus in Saccharomyces cerevisiae

作　　者：郭彦言[1] 王红蕾[1] 左小明[1] 

机构地区：[1]长春工业大学化学与生命科学学院,吉林长春130012

出　　处：《安徽农业科学》2015年第22期8-10,30,共4页Journal of Anhui Agricultural Sciences

摘　　要：[目的]使酿酒酵母高效表达糖化酵母糖化酶基因。[方法]利用PCR技术从糖化酵母中扩增出大小为2 700 bp左右的带有启动子的糖化酶基因STA1。通过限制性内切酶Bsp DI与Acc65I双酶切,将目的基因STA1连接到穿梭质粒pRS416中构建重组质粒pRS416-sta1,转化酿酒酵母通过筛选。[结果]糖化酶活性最高为120 U/ml,酶的最适温度为60℃,最适p H为5.0,在酶催化反应2 h后,酶剩余活力能达到约90%。[结论]通过基因工程手段得到高效表达糖化酶基因的酿酒酵母工程菌株。[Objective] Highly expressing glucoamylase gene of Saccharomyces diastaticus in Saccharomyces cerevisiae. [Method] The glucoamylase STA1 gene of 2700 bp with promoter from Saccharomyces diastaticus was amplified by PCR. STA1 was digested by BspDI and Acc65 I with Saccharomyces cerevisiae integrative expression vectors pRS416,named pRS416-sta1. This plasmid was introduced into S. cerevisiae. [Result] The glucoamylase levels of Saccharomyces cerevisiae activity was 120 U / ml. The result of determination of enzymatic properties of glucoamylase showed that the optimum temperature of glucoamylase was 60 ℃,the optimum pH of glucoamylase was 5. 0. After the enzyme reacted for 2 hours,the remaining enzyme activity achieved 90%. [Conclusion] The result indicated that the plasmid carrying extracellular glucoamylase gene could express in yeast,and this gene product could hydrolyze starch.

关 键 词：酿酒酵母 糖化酶 糖化酵母 基因表达 

分 类 号：S188.4[农业科学—农业基础科学] Q786[生物学—分子生物学]