芽孢杆菌植酸酶phy(ycD)基因在大肠杆菌中的异源表达与纯化  

Heterologous expression and purification of the phytase phy(ycD) gene from Bacillus subtilis in Escherichia coli

作　　者：王茜[1] 卢文亮[1] 姚明泽[1] 胡风云[2] 付月君[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 [2]山西省人民医院神经内科,太原030012

出　　处：《应用与环境生物学报》2014年第2期295-299,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31372199,31272100);国家高技术研究发展计划(“863”计划)项目(2012AA020809);山西省回国留学人员科研项目资助~~

摘　　要：为研究中性植酸酶的性质,将芽孢杆菌来源植酸酶基因phy(ycD)构建到大肠杆菌表达载体pET-28a(+)上,进行诱导表达,利用Ni-NAT和Superdex-75纯化后,对重组植酸酶r-phy(ycD)的性质进行分析.结果显示,该酶是Ca2+依赖性酶,Ca2+最适浓度为1 mmol/L,酶比活为4.7 U/mg,酶促反应的最适pH为6.5,在中性条件下稳定,最适反应温度为45℃,K m值和V max值分别为0.213 mmol/L和5.55 U/mg.Zn2+、Ni2+、Ba2+和Ag2+对酶促反应均有显著抑制作用.本研究表明,Ca2+对该类酶的活性和热稳定性起着关键性作用,因此在表达和纯化过程中需要在培养基和纯化所需的各类缓冲液中添加Ca2+.Phytase gene phy （ycD） from Bacillus subtilis was cloned into a pET-28a（＋） expression vector and expressed in Escherichia coli. The phytase phy（ycD） was purified by Ni-NAT and Superdex-75. This phytase was a Ca2＋ dependent enzyme, with optimum enzymatic Ca2＋ concentration 1 mmol/L. Specific activity of this phytase was 4.7 U/mg. The phytase was stable in neutral environment with optimum pH 6.5 and optimum reaction temperature 45 ℃. Zn2＋, Ni2＋, Ba：＋ signifcantly inhibited the phytase activity. The Km and Vmax values were 0.213 mmol/L and 5.55 U/mg, respectively. Ca2＋ plays a very important role on its activity and thermostability, therefore Ca2＋ should be added to the solution during the expression and purification.

关 键 词：中性植酸酶 基因表达 纯化 酶学性质 

分 类 号：Q936[生物学—微生物学] Q786