为了以酿酒酵母S78为宿主菌异源高效表达糖化酶基因,进一步扩大糖化酶基因在工业生产上的应用.运用RT-PCR法从黑曲霉中克隆得到糖化酶基因(glaA)cDNA,去除其信号肽编码区后的序列重组到酵母表达载体pVT102U/αADH1强启动子下游,并与α因子分泌肽信号序列融合.用PEG/LiAc法将构建的重组表达载体转入酿酒酵母S78菌株,筛选出的转化菌点种到可溶性淀粉平板上培养,用碘染法鉴定重组基因的表达情况.鉴定出了典型水解圈的酵母转化子,转化子接种到YPD培养基中摇瓶培养后,取发酵上清液经SDS-PAGE检测到分子量大小约为80 kDa的目标蛋白带,上清液用DNS法检测其淀粉酶最高酶活为28.86 IU/mL.表明糖化酶基因已在酿酒酵母S78中成功表达并能有效分泌到细胞外.%In order to express the glucoamylase gene efficiently with Saccharomyces cerevisiae S78 as a host strain and to further expand the application of glaA gene in industrial production. The glaA gene was cloned from Aspergillus niger by RT-PCR,and the sequence removed the natural signal peptide coding region was recombined in-to the downstream of the yeast expression vector pVT102 U/αADH1 strong promoter and fused with the sequence ofα factor secretion peptide signal. The recombinant expression vector was transformed into Saccharomyces cerevisiae S78 strain by PEG/LiAc method. The selected transformants were spread on soluble starch plates and the expression of recombinant genes was identified by iodine staining. The yeast transformants of the typical hydrolytic circles were identified and the transformants were inoculated into YPD medium. The molecular weight of the target protein about 80 kDa was detected by SDS-PAGE. The supernatant was detected by DNS method and the highest activity of en-zyme was 28. 86 IU/mL in normal bottle fermentation. It indicated that the glucoamylase gene had been successfully expressed in Saccharomyces cerevisiae S78 and the enzyme could be effectively secreted.
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