Purification and Characterization of a NAD⁺-Dependent Formate Dehydrogenase Produced by Paracoccus sp.

抄録

A facultative formate-utilizing bacterium, Paracoccus sp. strain 12-A, with a high specific activity (11.6 units/mg) of NAD⁺-dependent formate dehydrogenase, was isolated from sewage. From a crude extract of the cells, the enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. The enzyme protein amounted to 15% of intracellular soluble proteins in strain 12-A. The enzyme had a molecular weight of approximately 100, 000 by gel filtration with Sephadex G-150 and 49, 000 by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 5.4. The pH and temperature optima ware 6.5-7.5 abd 50-55°C, respectively. The enzyme was stable at pH 6.0-10.0 and up to 50°C. The apparent K_m values for formate and NAD⁺ were calculated to be 5.0 mM and 0.036 mM, respectively.