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]; Wang, Yong-Hong[1,2]; Zhang, Si-Liang[1,2]; Zhuang, Ying-Ping[1,2]; Yuan, Zhong-Yi[1,2]

机构：[1] State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; [2] National Research Center for Biotechnology [Shanghai], East China University of Science and Technology, Shanghai 200237, China

年份：2008

卷号：24

期号：6

起止页码：789

外文期刊名：World Journal of Microbiology and Biotechnology

收录：EI(收录号：20081811233150)

语种：英文

外文关键词：Enzyme activity - Fermentation - Gel permeation chromatography - pH effects - Purification

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) 2SO4 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°C, respectively. The enzyme was stable at pH 7.0-9.0 and was easy to inactivate in acid solution (pH &le 5.0). The temperature stability was up to 45°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 μM and V max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. ? 2007 Springer Science+Business Media B.V.