Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities
Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities
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DOI：
10.1128/aem.71.5.2777-2781.2005
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发表时间：
2005-05-01
影响因子：
4.4
通讯作者：
Soucaille, P
中科院分区：
文献类型：
--
作者：
Girbal, L;von Abendroth, G;Soucaille, P
以乙酰丁酸梭菌ATCC 824为研究对象，研究了其铁氢酶的同源高表达，以及莱茵衣藻和斜生栅藻HydA1铁氢酶的异源表达。通过两步柱层析分离得到了三种链状标签H标记的高比活力的纯Fe氢酶。纯化的海藻氢酶放氢速率约为700mU·molH~(-2)min(-1)mg(-1)，而乙酰丁酸菌(HydA(Ca))的HydA在吸氢方向上表现出最高的活性(5,522 mU·molH~(-2)min(-1)mg(-1))。此外，还报道了动力学参数和底物专一性。电子顺磁共振(EPR)分析表明，硫素氧化的HydA(Ca)蛋白具有典型的菱形EPR信号，这是含铁氢酶的氧化H簇的典型特征。
Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag H-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 mu mol H-2 min(-1) mg(-1), while HydA from C. acetobutylicum (HydA(Ca)) shows the highest activity (5,522 mu mol H-2 min(-1) mg(-1)) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydA(Ca) protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.