Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic alkaliphile

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain o f Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and p urified by chromatography on DEAE-Sepharose and hydroxyapatite. The purifie d enzyme appears to consist of a single polypeptide component with an appar ent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH wi th the formation of H2O2 and exhibited a specific activity of 46, mu mol NA DH min(-1) (mg protein)(-1). NADPH did not serve as a substrate for the enz yme. The K-m for NADH was estimated to be 0.05 mM. The enzyme exhibited a p H dependence for activity, with a pH optimum at approximately 9.5. The enzy me required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl.