S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定  

Constitutive Expression,Purification and Identification of S-Adenosylmethionine Synthetase

作　　者：田林奇[1] 牛卫宁[1] 左晓佳[1] 钦传光[1] 

机构地区：[1]西北工业大学生命科学院,西安710072

出　　处：《中国生物工程杂志》2010年第3期61-66,共6页China Biotechnology

基　　金：国家自然科学基金(20802057);中国博士后科学基金(20070411144);西北工业大学青年科技创新基金(W016212);西北工业大学科技创新基金(W016143)资助项目

摘　　要：将大肠杆菌(E.coliK12)S-腺苷甲硫氨酸合成酶(SAMS)基因克隆至质粒pBR322中,获得的重组质粒pBR322-SAMS转入大肠杆菌JM109菌株,构建了能高效组成型表达SAMS的重组菌E.coliJM109(pBR322-SAMS)。将重组大肠杆菌破碎后上清液经20%～40%硫酸铵分级盐析、Phenyl-Sepharose Fast Flow疏水层析和Q-Sepharose Fast Flow离子交换层析,即可得到纯度提高5倍,比活为48.7μ/mg的SAMS,三步纯化的总回收率为62%,纯度达到92%。SAMS表达量为1176μ/L,占到菌体可溶性总蛋白的20%。重组酶的最适反应pH为8.5,4℃下在pH7.5的缓冲液中保温10h酶活性几乎不改变。重组酶反应的最适温度为55℃,酶活性稳定的温度范围为20～35℃。重组酶的KmL-Met为0.22mmol/L,VmaxL-Met为1.07mmol/(L.h),KmATP为0.52mmol/L,VmaxATP为1.05mmol/(L.h)。S-Adenosylmethionine （SAM） which is synthesized from methionine and ATP by S-adenosylmethionime synthetase （SAMS） plays an important role in many biological reactions.SAMS gene which was cloned from the genome of E.coli K12,and the recombinant E.coli JM109（pBR322-SAMS） strain which can constitutively express SAMS was constructed.The productivity of SAMS reached 1 176μ/L,and was 20% of total soluble proteins of recombinant strain.After 20%～40% ammonium sulfate fractionation,the solution was loaded on Phenyl-Sepharose Fast Flow column.The enzyme fraction was absorbed on the column and was eluted as concentration of ammonium sulfate decreased to 0.Subsequently the effluent wad loaded on Q-Sepharose Fast Flow column,and the enzyme was eluted as concentration of KCl increased to 0.3mol/L.After ammonium sulfate fractionation and two column chromatography,the enzyme was enriched 5 times with a 62% activity recovery.The purified enzyme had a specific activity of 48.7μ/mg protein.The purity of SAMS reached 92%.The optimum reactive pH was 8.5,and the recombinant enzyme activity changed little when incubated in the buffer of pH 7.5 on 4℃ for 10 h.The optimum reactive temperature of recombinant enzyme was 55℃,and the recombinant enzyme was more stable on the temperature of 20～35℃.KmL-Met of recombinant SAMS was 0.22mmol/L and VmaxL-Met was 1.07mmol/（ L·h）.KmATP was 0.52mmol/L and VmaxATP was 1.05mmol/（ L·h）。

关 键 词：S-腺苷甲硫氨酸合成酶 大肠杆菌 组成型表达 纯化 酶学性质 

分 类 号：TQ426.97[化学工程]