| 摘要 |
A method for producing and purifying hydrogenase is provided to produce the highly concentrated hydrogenase by culturing Thiocapsa roseopersicina in an acetate-added culture medium and purify the hydrogenase with stable activity from a culture material of the Thiocapsa roseopersicina. A method for producing and purifying hydrogenase comprises the steps of: (a) treating fungi bodies obtained by centrifuging a culture solution obtained by culturing Thiocapsa roseopersicina with ultrasonic wave to lyse the fungi bodies; (b) precipitating the fungi body lysate using 60% ammonium sulfate to obtain an inactive hydrogenase fraction; (c) heat-treating the fraction at a temperature of 55-65 deg.C for 15-25 minutes; (d) subjecting the fraction to an anion exchange chromatography using KCl as a solvent; (e) subjecting the fraction to a hydrophobic interaction chromatography using a 2M ammonium sulfate-added potassium phosphate buffer solution(50mM, pH 7.0); (f) subjecting the fraction to an anion exchange chromatography using 20mM Tris-HCl buffer solution as a solvent; (g) subjecting the fraction to a gel filtration chromatography using a phosphate buffer(50 mMpH 7.0) including 150 mM KCl; and (h) subjecting the fraction to a gel electrophoresis using 10% polyacrylamide gel.
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