Purification and Characterization of Membrane-bound Hydrogenase from Hydrogenobacter thermophilus Strain TK-6, an Obligately Autotrophic, Thermophilic, Hydrogen-oxidizing Bacterium
Purification and Characterization of Membrane-bound Hydrogenase from Hydrogenobacter thermophilus Strain TK-6, an Obligately Autotrophic, Thermophilic, Hydrogen-oxidizing Bacterium
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嗜热氢杆菌菌株 TK-6（一种专性自养、嗜热、氢氧化细菌）膜结合氢化酶的纯化和表征
DOI：
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Y. Igarashi
中科院分区：
文献类型：
--
作者：
M. Ishii;Seiichi Takishita;T. Iwasaki;Y. Peerapornpisal;Jun;T. Kodama;Y. Igarashi
从嗜热氢杆菌TK-6菌株（一种专性自养、嗜热、氧化氢的细菌）的细胞中纯化出一种膜结合氢化酶，达到电泳均匀性。在Triton X-100存在下进行好氧增溶和纯化。采用三步色谱法进行纯化；丁基sepharose， Mono-Q和Superose 6，按这个顺序。纯化完成后，总活性得率为6.73%，比活性比膜组分提高21.4倍。纯化后的氢化酶为α2 - β2结构的四聚体，大亚基分子量为60000 Da，小亚基分子量为38000 Da。纯化后的氢化酶直接还原甲硫喹酮，表观Km约为300 μM，周转数约为2900 （min-1）。金属分析和EPR性质表明该酶为[NiFe]-氢化酶之一。此外，还报道了反应的最佳pH和温度、热稳定性和电子受体特异性。最后，建立了嗜热嗜血杆菌TK-6的能量和中枢代谢模型。
A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with α2β2 structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent Km of around 300 μM and with a turnover number around 2900 (min-1). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilus strain TK-6.
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DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
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作者：
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DOI：
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发表时间：
1989
期刊：
The Journal of biological chemistry
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DOI：
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发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
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作者：
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DOI：
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发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
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作者：
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