糖化酶基因的克隆和在酿酒酵母中表达

CLONING AND EXPRESSION OF GLUCOAMYLASE GENE IN SACCHAROMYCES CEREVISIAE

以穿梭质粒pLCl为载体,大肠杆菌C600为受体构建了扣囊拟内孢霉(Endomycopsis fibuligera)Sau34基因文库。从基因文库中提取重组质粒DNA,转化酿酒酵母BJ1991,选出具有淀粉水解酶活性的转化子,它含有编码扣囊拟内孢霉胞外糖化酶的基因片段,能发酵淀粉。琼脂糖凝胶电泳证明所插入DNA片段为5.3kb,SDS-聚丙烯酰胺凝胶电泳测得糖化酶的分子量为54000。酶活最适温度为50℃,最适pH为5.5。

Endomycopsis fibuligera genomic library was constucted in E. coli using E. coli-yeast shuttle vector pLCl. E. coli clones with glucoamylase activity were screened from the library by the specificity of utilizing starch. The recombinant plasmid was isolated from E. coli transformant with enzyme activity, and then, this plasmid was introduced into S. cerevisiae. The result indicated that the plasmid carrying extracellular glucoamylase gene could express in yeast, and this gene product could hydrolyze starch. Gel electrophoresis results showed that the size of the inserted DNA fragment was 5.3 kb. The molecular weight of the glucoamylase determined by SDS-polyacrylamide gel eiectrophoresis was 54 000, the optimal temperature and pH of the enzyme activity was 50℃ and 5.5, respectively.