黑曲霉糖化酶基因在酿酒酵母中的表达

摘要

从黑曲霉eDNA文库中筛选出糖化酶基因，并在酿酒酵母中进行表达．阳性克隆在发酵培养基中培养60h后，产生的糖化酶酶活力达到峰值为4．3U／mL．测定结果显示其糖化酶大小为1908bp，编码636个氨基酸残基组成的蛋白质．酶学性质分析显示该酶的最适反应温度为50℃，pH为5．0．经柱分离纯化其发酵上清液后，SDS-PAGE电泳方法，测得它的分子量大约为70kD，且条带清晰．%An expression cDNA library was constructed from aspergillus niger, then glucoamylase gene was isolated and efficiently expressed in S. cerevisiae. The results showed that the maximum activity of glucoamylase was 4.2 U/mL when cultivated for 60 h. Sequence analysis revealed that glucoamylase had 1 908 bp,which encodes a putative polypeptide of 636 amino acids. The enzyme proerties showed that the optimum pH and temperature was 5.0 and 50 ℃, the expressed protein was purified from the fermented supernatant using DEAE clumon and determined by SDS-PAGE. The results of SDS-PAGE also showed that the molecular weights of the enzyme was 70 kDa.