Purification and Characterization of Membrane-bound Hydrogenase from Hydrogenobacter thermophilus Strain TK-6, an Obligately Autotrophic, Thermophilic, Hydrogen-oxidizing Bacterium.
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- ISHII Masaharu
- Department of Biotechnology, the University of Tokyo
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- TAKISHITA Seiichi
- Department of Biotechnology, the University of Tokyo
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- IWASAKI Toshio
- Department of Biochemistry and Molecular Biology, Nippon Medical School
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- PEERAPORNPISAL Yuwadee
- Department of Biology, Chiangmai University
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- YOSHINO Jun-ichiro
- Department of Biotechnology, the University of Tokyo
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- KODAMA Tohru
- Faculty of Textile Science and Technology, Shinshu University
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- IGARASHI Yasuo
- Department of Biotechnology, the University of Tokyo
Bibliographic Information
- Other Title
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- Purification and Characterization of Membrane-bound Hydrogenase from<i>Hydrogenobacter thermophilus</i>Strain TK-6, an Obligately Autotrophic, Thermophilic, Hydrogen-oxidizing Bacterium
- Purification and characterization of membrane-bound hydrogenase froim Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium
- Purification and characterization of membrane-bound hydrogenase from Hydrogenobacter thermophilus TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium
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Abstract
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.<br>
Journal
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- Bioscience, Biotechnology, and Biochemistry
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Bioscience, Biotechnology, and Biochemistry 64 (3), 492-502, 2000
Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Details 詳細情報について
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- CRID
- 1390001206474702592
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- NII Article ID
- 110002679941
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- NII Book ID
- AA10824164
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- COI
- 1:CAS:528:DC%2BD3cXit1Gnsb0%3D
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- ISSN
- 13476947
- 09168451
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- NDL BIB ID
- 5334276
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- PubMed
- 10803945
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- PubMed
- CiNii Articles
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- Abstract License Flag
- Disallowed