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

Abstract

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 were 6.5–7.5 and 50–55°C, respectively. The enzyme was stable at pH 6.0–10.0 and up to 50°C. The apparent Km values for formate and NAD+ were calculated to be 5.0mM and 0.036mM, respectively.