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| Constitutive Expression,Purification and Identification of S-Adenosylmethionine Synthetase |
| Faculty of Life Science, Northwestern Polytechnical University, Xi’an 710072, China |
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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)。
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Received: 01 December 2009
Published: 25 March 2010
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