黑曲霉糖化酶在酿酒酵母中的表达和分泌

从黑曲霉糖化酶高产株T2l合成的糖化酶cDNA，经5’端和3’端改造后克隆到酵母质粒YFDl8上，转化酿酒酵母。转化子的淀粉培养基平板检测，培养滤液蛋白电泳和糖化酶活力分析都表明，含有糖化酶基因表达质粒的酵母转化子能有效地分泌有功能的糖化酶到细胞外。实验证明酵母a园子启动子和分泌信号序列能促使黑曲霉糖化酶cDNA在酵母中表达和分泌．实验还表明．黑曲霉糖化酶原的翻译后加工序列很可能亦能被酵母识别，加工生成有功能的成熟的糖化酶。以上成功为构建有实用意义的淀粉水解酵母工程菌迈出了重要的一步。

Expression and Secretion of Glucoamylase of Aspergillus niger in Saccharomyces cerevisiae

Glucoamylase cDNA synthesized from A. niger mutant T21 was modified at 5'and 3'ends in order to clone it into yeast shuttle plasmid YFD18 and to make fusion between cDNA and leader region of yeast mating pheromone a-factor. The modified cDNA was then inserted into YFD18 at Hind I site. Saccharomyces cerevisiae Y33 was transformed with the resultant recombinant plasmid YFD18HH6. Analysis of transfor-mants including halo formation on starch medium plate, SDS-PAGE of culture filtration and determination of glucoamylase activity showed that the yeast transformed with plasmid containing glucoamylase cDNA efficiently secreted glucoamylase into the medium. This fact indicated that yeast a-factor was able to direct the synthesis and secretion of functional glucoamylase of Aspergillus. In addition the proteolytic cleavage site involved in the maturation of glucoamylase in.A. niger also worked in 5. cerevisiae.