Purification and characterization of the oxygen-thermostable hydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum camini(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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Aeropyrum camini that was isolated from a deep-sea hydrothermal vent chimney, possessed two hydrogenases (161 and 85kDa) in its soluble fraction. The 85-kDa hydrogenase was purified to homogeneity using several chromatography columns. The specific activities of the purified hydrogenase were: 14.8μmol methyl viologen^<ox>/mg/min for hydrogen oxidation, and 14.6μmol methyl viologen^<red>/mg/min for proton reduction. The oxygen stabilities of hydrogenases that were purified from A. camini and the hydrogen thermophilic bacterium Persephonella hydrogeniphila, were compared. The hydrogenase purified from P. hydrogeniphila completely lost its activity following a 96-h exposure to atmosphere; however, the A. camini hydrogenase maintained 75% of its initial activity, even after a 168h of atmospheric exposure. A. camini hydrogenase showed a half-life of 48h at 90℃, while P. hydrogeniphila hydrogenase showed complete denaturation after a 30min incubation at the same temperature. Nine residues of the N-terminal amino acid sequence of A. camini hydrogenases (MARLLMIPGT) correspond to the protein sequence encoded by the hypothetical soluble hydrogenase subunit gene (APE2423) from A. pernix strain K1. A. camini hydrogenase has a high thermostability and is very tolerant to oxygen; therefore, it may be used for actual H_2 production.