Novel H_2-oxidizing [NiFeSe]hydrogenase from Desulfovibrio vulgaris Miyazaki F(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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[NiFeSe]hydrogenases are promising biocatalysts in H_2-based technology due to their high catalytic activity and O_2- stability. Here, we report purification and characterization of a new membrane-associated [NiFeSe]hydrogenase from Desulfovibrio vulgaris Miyazaki F ([NiFeSe]_<DVMF>). The [NiFeSe]_<DVMF> was composed of two subunits, corresponding to a large subunit of 58.3 kDa and a small subunit of 29.3 kDa determined by SDS-PAGE. Unlike conventional [NiFeSe] hydrogenases having catalytic bias toward H_2-production, the [NiFeSe]_<DVMF> showed 11-fold higher specific activity of H_2-oxidation (2444 U/mg) than that of H_2-production (217 U/mg). At the optimal reaction temperature of the enzyme (65℃), the specific activity of H_2-oxidation could reach up to 21,553 U/mg. Amperometric assays of the [NiFeSe]_<DVMF> clearly indicated that the enzyme had a remarkable O_2-stability. According to the amino acid sequence alignment, the conserved cysteine residue at position 281 in medial cluster of other [NiFeSe]hydrogenases was specifically replaced by a serine residue (Ser281) in the [NiFeSe]_<DVMF>. These results indicate that the [NiFeSe]_<DVMF> can play as a new H_2-oxidizing and O_2-stable biocatalyst, along with providing helpful insights into the structure-function relationship of [NiFeSe] hydrogenases.
公益社団法人日本生物工学会の論文
2013-04-00