文献类型：期刊文献

英文题名：Purification and properties of Saccharomyces cerevisiae S-adenosylmethionine synthetase expressed in recombinant Pichia pastoris

作者：Zhou, Jin[1,2];Chu, Ju[1,2];Wang, Yong-Hong[1,2];Zhang, Si-Liang[1,2];Zhuang, Ying-Ping[1,2];Yuan, Zhong-Yi[1,2]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]E China Univ Sci & Technol, Natl Res Ctr Biotechnol Shanghai, Shanghai 200237, Peoples R China

年份：2008

卷号：24

期号：6

起止页码：789

外文期刊名：WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000255259200008)】；

语种：英文

外文关键词：intracellular; Pichia pastoris; properties; purification; SAM-synthetase

摘要：An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)(2)SO4 fractionation (30-60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 degrees C, respectively. The enzyme was stable at pH 7.0-9.0 and was easy to inactivate in acid solution (pH <= 5.0). The temperature stability was up to 45 degrees C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and L-methionine in vitro with a K-m of 120 and 330 mu M and V-max of 8.1 and 23.2 mu mol/mg/min for L-methionine and ATP, respectively.