新型中性植酸酶在大肠杆菌中的高效表达纯化及酶学性质-食品科学.PDF

※生物工程 食品科学 2012, Vol.33, No.21 153 1 2, * 2 1 路国伟 ，许 伟 ，邵 荣 ，云 志 (1.南京工业大学化学化工学院，江苏 南京 210009 ；2.盐城工学院化学与生物工程学院，江苏 盐城 224051) 摘 要：运用基因工程技术，将克隆到的淀粉液化芽孢杆菌Bacillus amyloliquefaciens DSM 1061新型中性植酸酶基 因(GenBank登录号为HM747163)构建到表达载体pET22b( ＋)上，并在E. coli BL21(DE3) 中进行高效表达。电泳结果 表明该酶分子质量约为42kD ，目的蛋白占大肠杆菌可溶性蛋白30%左右。利用Ni-NTA琼脂糖凝胶进行亲和纯化， K 酶学性质研究表明，其最适温度为60℃，最适pH值为7.0，最大酶比活力为15U/mg。60℃时以植酸钠为底物的 m值 为0.30mmol/L 。 淀粉液化芽孢杆菌；高效表达；中性植酸酶；酶学性质 Expression of a New Neutral Phytase in Escherichia coli and Its Purifi cation and Enzymatic Characterization , 1 2 * 2 1 LU Guo-wei ，XU Wei ，SHAO Rong ，YUN Zhi (1. College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009, China； 2. School of Chemical and Biological Engineering, Yancheng Institute of Technology, Yancheng 224051, China) Abstract phyc Bacillus amyloliquefaciens ：A new neutral phytase gene ( ) from DSM 1061 (GenBank: HM747163) was cloned into expression vector pET22b(+). The phytase was expressed in Escherichia coli BL21(DE3). The molecule weight of the phytase protein was about 42 kD determined by SDS. The expressed phytase was about 30% of the total soluble protein of E. coli. The recombinant protein was purifi ed by 6 ×His-Tagged Protein Purifi cation Kit. Optimal pH value and temperature of the phytase were 7.0 and 60 ℃, respectively. Under th