大肠杆菌植酸酶在毕赤酵母中的表达与纯化  

Expression and Purification of Phytase appA in Pichia pastoris

作　　者：张耀华[1] 姚明泽[1] 卢文亮[1] 梁爱华[1] 

机构地区：[1]山西大学生物技术研究所、化学生物学与分子工程教育部重点实验室,太原030006

出　　处：《应用与环境生物学报》2012年第4期678-681,共4页Chinese Journal of Applied and Environmental Biology

基　　金：山西省自然科学基金项目(No.2009011059-3);山西省回国留学人员科研项目资助~~

摘　　要：为探讨植酸酶的结构与功能,研究了来源于大肠杆菌的植酸酶基因在酵母中的表达和纯化条件.将含有大肠杆菌植酸酶基因的毕赤酵母工程菌在不同甲醇浓度和不同诱导时间下培养,检测植酸酶的表达情况.结果表明:诱导培养基中一次性添加2.5%的甲醇,诱导96 h后,蛋白浓度达到1.36 mg mL-1,酶活力达到6 530 U mL-1;将在毕赤酵母中表达的植酸酶粗酶液经过硫酸铵盐析、Resource S柱和Superdex-75三步纯化,得到单峰纯的蛋白,比活力为137 280 Umg-1.用圆二色谱分析纯化后的蛋白在pH 5.0和8.0环境中的结构变化,结果显示pH 8.0环境使大肠杆菌植酸酶发生了变性.Phytase （appA） from Escherichia coli was expressed in yeast Pichia pastoris. Different methanol concentrations and induction times were tested to get appA expressed efficiently. The results showed that after induction of 96 h with 2.5% of methanol, the concentration of protein reached 1.36 mg mL-1, specific activity of phytase reached 6 530 U mg-1. The crude phytase expressed in P. pastoris was sequentially purified by ammonium sulfate precipitation, Resource S and Superdex-75. The purified protein had a specific activity of 137 280 U mg-1. Circular dichroism was used to study the secondary structure of the phytase at pH 5.8 and pH 8.0, and the result showed appA denatured at pH 8.0. Fig 3, Ref 16

关 键 词：大肠杆菌植酸酶 毕赤酵母 甲醇浓度 表达与纯化 圆二色谱 

分 类 号：Q556.9[生物学—生物化学] Q936