【作者】 刘树滔； 李仁宽； 赖庆安； 陈菁； 饶平凡；

【Author】 Liu Shu-tao Li Ren-kuan Lai Qing-an Chen Jing Rao Ping-fan (Institute of Biotechnology,Fuzhou University,Fuzhou,Fujian 350002,China)

【机构】 福州大学生物工程研究所；

【摘要】 猪胃蛋白酶作为重要的消化酶,广泛应用于农产品蛋白质水解加工以及医药与养殖工业中。本文尝试利用巴斯德毕赤酵母表达体系大量制备猪胃蛋白酶原A。首先用化学法合成猪胃蛋白酶原A(pepsinogen A,pA)基因,并构建重组质粒pGAPZα-pA;然后利用化学转化技术,将该基因整合到毕赤酵母X-33菌株的染色体上,构建表达菌株Pp-pA。该菌株在摇瓶培养条件下经甲醇诱导分泌表达的猪胃蛋白酶原A酶活就达到5250U/L。发酵液上清经过DEAE离子交换色谱和Sephacryl S200凝胶过滤色谱分离,得到纯化倍数为96.7的电泳纯pA。活化pA后获得比活达到52800 U/g的猪胃蛋白酶。更多还原

【Abstract】 High-level expression by Pichia pastoris for porcine pepsinogen A(pA),one kind of important protease both in industrial application and academic research,was reported in this paper.The DNA encoding pA was inserted into XhoⅠand XbaⅠsites of the vector pGAPZα.After transformation,the clonies of P.pastoris were screened for expression of pA based on enzyme activity of its active form, pepsin.The recombinant pA was purified by DEAE ion-exchange and Sephacryl S200 gel filtration chromatography.Homogeneity was confirmed by SDS-PAGE,the specific activity of pepsin was about 52800 U/g;the expression level was 5250 U/L.These results indicated that porcine pepsinogen A was high-level expressed,and could be efficiently purified.更多还原