S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定

中国生物工程杂志

2010, Vol. 30

Issue (03): 61-66

研究报告

S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定

田林奇,牛卫宁,左晓佳,钦传光^**

西北工业大学生命科学院西安 710072

Constitutive Expression,Purification and Identification of S-Adenosylmethionine Synthetase

Faculty of Life Science, Northwestern Polytechnical University, Xi’an 710072, China

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摘要：

将大肠杆菌(E.coli K12) 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.coli JM109 (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48.7 μ/mg的SAMS，三步纯化的总回收率为62%，纯度达到92%。SAMS表达量为1 176μ/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pH 7.5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，Vmax L-Met为1.07mmol/(L·h)，Km ATP为0.52 mmol/L，Vmax ATP为1.05 mmol/( 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

Abstract:

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)。

Key words: S-Adenosylmethionime synthetase(SAMS) Escherichia coli Constitutive expression Purification Characterization

收稿日期: 2009-12-01 出版日期: 2010-03-25

基金资助:

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

通讯作者: 钦传光 E-mail: qinchg@nwpu.edu.cn

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引用本文:

田林奇牛卫宁左晓佳钦传光. S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定[J]. 中国生物工程杂志, 2010, 30(03): 61-66.

TIAN Lin-Ai, NIU Wei-Ning, ZUO Xiao-Jia, QIN Chuan-Guang. Constitutive Expression,Purification and Identification of S-Adenosylmethionine Synthetase. China Biotechnology, 2010, 30(03): 61-66.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I03/61

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