热稳定性丙酮酸:铁氧还蛋白氧化还原酶异源表达及其在乙酰辅酶A合成中的应用  

Expression and Characterization of a Thermostable Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophile Thermotoga neapolitana and Its Application in Acetyl-Co A Production

作　　者：乐易林 傅毓 倪黎 孙建中 LE Yi-lin;FU Yu;NI Li;SUN Jian-zhong(Biofuels Institute,School of the Environment and Safety Engineering,Jiangsu University,Jiangsu 212013,China)

机构地区：[1]江苏大学环境与安全工程学院,生物质能源研究所,镇江212013

出　　处：《中国生物工程杂志》2020年第3期72-78,共7页China Biotechnology

基　　金：国家自然科学基金(31772529);国家重点科技项目(2018YF0107100)资助项目。

摘　　要：乙酰辅酶A被广泛应用到生物医学研究中,使用TPP代替昂贵的ATP为辅因子合成乙酰辅酶A受到广泛关注。新阿波罗栖热袍菌(Thermotoga neapolitana)来源的丙酮酸:铁氧还蛋白氧化还原酶(Tn PFOR)在大肠杆菌中进行了重组表达,分析了其酶学特性,并探讨了利用嗜热酶(TnPFOR)酶法合成乙酰辅酶A。采用pET-20b(+)载体,将新阿波罗栖热袍菌来源的四亚基组成的嗜热酶(Tn PFOR)在大肠杆菌中进行异源表达;通过热处理和阴离子交换层析法纯化嗜热酶(TnPFOR);重组表达的嗜热酶(TnPFOR)的最适反应温度和pH分别为90℃和6.5,TnPFOR在90℃下孵育1h时保留了50%活性。利用嗜热酶(Tn PFOR),以TPP为辅酶合成了乙酰辅酶A,并探讨了不同温度、丙酮酸钠底物浓度和反应时间对乙酰辅酶A合成的影响。得到的优化条件为:最适反应温度为90℃,丙酮酸钠浓度为1.5mmol/L,反应时间为2min。Pyruvate ferredoxin oxidoreductase(PFOR)catalyzes the synthesis of acetyl-CoA from pyruvate and coenzyme A(CoA)using thiamine pyrophosphate(TPP)as coenzyme.The four subunit-type TnPFOR from T.neapolitana was expressed in Escherichia coli and characterized.The gene of TnPFOR from T.neapolitana was cloned into pET-20 b(+).Tn PFOR was purified by a heat treatment followed by an ion exchange chromatography.The TnPFOR had an optimal condition for its maximum activity at 90℃and p H 6.5 and it was indeed thermostable with a half-life of more than 1 h at 90℃.The application of TnPFOR to catalyze the conversion of pyruvate into acetyl-CoA was also evaluated.The influence of different reaction conditions(reaction temperature,pyruvate concentrations and reaction time)on the synthesis of acetyl-CoA was discussed.The optimal reaction temperature is 90℃,pyruvate concentrations is 1.5 mmol/L and the reaction time is 2 min.

关 键 词：大肠杆菌 重组表达 嗜热酶 嗜热菌 合成乙酰辅酶A 

分 类 号：Q946.5[生物学—植物学]