Purification and characterization of NADH dehydrogenase from Bacillus megaterium

Abstract

NADH dehydrogenase of Bacillus megaterium was isolated from the sonicate soluble fraction. The enzyme was purified approximately 61-fold by a combination of ammonium sulfate fractionation and column chromatography on DEAE-Sephadex and hydroxyapatite. The purified enzyme has an apparent molecular weight of 42 000 as determined by SDS-polyacrylamide gel electrophoresis and activity staining for NADH-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) oxidoreductase. The enzyme is specific for NADH and has a pH optimum of 7.5–7.8. The apparent Km values for NADH are 15.7, 34.8, and 69.2 μM as determined for NADH-DCIP (dichlorophenol–indophenol), NADH-ferricyanide, and NADH-MTT oxidoreductases. FAD is the prosthetic group of the enzyme. NAD+ acts as a competitive inhibitor. The inhibition studies suggest that NADH dehydrogenase is the primary electron donor of the NADH oxidase system. Localization studies and inhibition studies together indicate that the NADH oxidase is a complex of membrane-bound enzymes and coenzymes.Key words: NADH dehydrogenase, NADH oxidase, Bacillus megaterium, purification, characterization.