摘要
目的:对重组腺苷蛋氨酸合成酶催化L-蛋氨酸(Met)和三磷酸腺苷(ATP)合成腺苷蛋氨酸的反应条件进行研究。方法:将重组腺苷蛋氨酸合成酶制成粗酶液,对其酶促反应条件进行研究和优化,摸索反应的最适pH值、最适反应时间及最适酶量,解决产物抑制问题和产物分解问题。根据试验摸索的条件进行450L生物反应罐的中试放大。结果:重组腺苷蛋氨酸合成酶催化Met和ATP合成的反应最适pH值为7.0,反应时间为8h,选择硼酸钠-硫酸缓冲液作为反应的缓冲系统,确定了反应液中酶液的最佳比例为1∶10,解决产物抑制问题的对甲苯磺酸钠的用量为9%,将镁离子和钾离子的盐酸盐形式改为硫酸盐形式,从而保证了最小限度的产物分解。中试放大结果:三批收率分别达到86.8%、87.1%和88.4%。结论:建立了重组腺苷蛋氨酸合成酶酶促反应的方法,为酶促法合成腺苷蛋氨酸大规模生产奠定了基础。
Objective： To study the enzyme catalysis conditions of recombinant S-adenosylmethionine synthetase on L-Met and ATP. Methods： Recombinant S-adenosylmethionine synthetase was used to prepare crude enzyme. Meanwhile, the enzyme catalysis conditions underwent constant optimization in order to find out the most suitable pH value, reaction time and enzyme quantity, and solutions of product inhibition and decomposition problems were also found. Augmentation in 450L bio-reactor was then carried out under the suitable conditions. Results： The most suitable pH value and reaction time acquired through study were 7.0 and 8 h respectively. Sodium borate-sulphuric acid buffer solution was employed as the buffer system. It was confirmed that the best ratio of enzyme and solution was 1：10, while the quantity of paratoluenesulfonic acid sodium salt was 9% in order to solve product inhibition. Transferring magnesian ion and potassium ion from hydrochloride form to sulphate form guaranteed minimum product decomposition. During the augmentation, the yield rate of three batches reached 86.8%, 87.1% and 88.4% respectively. Conclusion： Enzyme catalysis method of recombinant S-adenosylmethionine synthetase is established, laying the foundation for mass production of ademetionine via enzyme catalysis.
出处
《中国医药导报》
CAS
2009年第16期28-30,共3页
China Medical Herald
基金
北京市高成长企业自主创新科技专项
关键词
重组腺苷蛋氨酸合成酶
腺苷蛋氨酸
酶促反应
Recombinant S-adenosylmethionine synthetase
Ademetionine
Enzyme catalysis
