【摘要】 用PCR方法从酵母(Saccharomycescerevisiae)AH109中扩增出α-半乳糖苷酶的Mel1基因,将其克隆至整合型载体pGAPZαA中构建成组成型分泌表达酶产物的重组质粒pGAPZα-Mel1。将线性化的重组质粒pGAPZα-Mel1电击转化至毕赤酵母(Pichiapastoris)KM71,在含有100mg/mLzeocin和预先涂布有X-α-gal的YPDS平板上选择蓝色阳性菌落。发酵培养酵母的上清经SDS-PAGE分析,在53kD处有特异带;经非变性PAGE凝胶电泳,与显色底物的反应,检测到α-半乳糖苷酶活性带。重组菌pGAPZα-Mel1/KM71摇瓶发酵6d后,培养液α-半乳糖苷酶粗酶活性为12U/mL。更多还原

【Abstract】 Mel1 gene was amplified from yeast(Saccharomyces cerevisiae ) AH109 with PCR, and cloned into integrated vector pGAPZαA, constitutive and secreted expression α-galactosidase plasmid pGAPZα-Mel1 was constructed. The linearized recombinant plasmid pGAPZ α-Mel1 was transformed into Pichia pastoris KM71 by electropotation, and the blue positive colonies were screened out on YPDS plate with X-α-gal and 100 mg/mL zeocin. There was a special 53 kD band by SDS-PAGE from supernatant of yeast culture and a color band was observed by soak the native PAGE gel in X-α-gal to illustrate the α-galactosidase activity; The α-galactosidase enzyme activity was 12 U/mL after fermenting pGAPZα-Mel1 /KM71 for 6 days.更多还原