【摘要】 以α-半乳糖苷酶基因为筛选标记,构建GAL1诱导型启动子介导的酿酒酵母表达载体YGM-α-gal质粒,将枯草芽孢杆菌的β-1,3-1,4-葡聚糖酶基因克隆到此载体中,构建质粒YGMPA-α-gal,转化宿主酵母后,实现β-1,3-1,4-葡聚糖酶在酿酒酵母中的分泌表达.结果表明:在2%半乳糖诱导下,摇瓶发酵24h后分泌表达的β-葡聚糖酶活性达到411.9U爛mL-1,而在培养60h后,发酵液中α-半乳糖苷酶活性可达64.2U爛mL-1.说明α-半乳糖苷酶基因可用作酿酒酵母表达载体转化的有效筛选标记,为食品级酿酒酵母表达系统的构建提供了新选择.更多还原

【Abstract】 YGM-α-gal,an expression vector for Saccharomyces cerevisiae,was constructed containing α-galactosidase gene as selection marker and an inducible GAL1 promoter.The β-1,3-1,4-glucanase gene cloned from Bacillus subtilis was inserted into the multiple cloning sites of the plasmid YGM-α-gal to generate YGMPA-α-gal,then to transform the host yeast,and β-1,3-1,4-glucanase was efficiently expressed as a secretive protein in S.cerevisiae.The results indicated that the β-1,3-1,4-glucanase activity in the ferment liquid in shake flask was about 411.9 U·mL-1 after adding 2% galactose for 24 h,and the α-galactosidase activity reached 64.2 U·mL-1 after adding 2% galactose for 60 h.So the α-galactosidase gene can be used as an effective selection marker for screening the positive transformants of food-grade yeast expression system.更多还原