Properties of NADH oxidase fromLactobacillus delbrueckii sspbulgaricus

Lactobacillus delbrueckii spp bulgaricus produces substantial amounts of hydrogen peroxide, yet the enzymology of this process has not been explored. We have partially purified a NADH oxidase (EC 1.6.99.3) from the soluble cell fraction (cytosol) of L delbrueckii ssp bulgaricus, prepared by sonication of the microorganisms in a 0·1 M Tris buffer at pH 7·2. The cytosol was treated with agarose beads containing attached FAD residues, which served to bind the enzyme. The enzyme was eluted with a 0·01 M Tris buffer at pH 7·2 containing 10 μM FAD. This preparation had 0·2 mg ml−1 protein and showed a single major band in a polyacrylamide gel electrophoresis system with a molecular weight of near 30 kDa. Gel filtration chromatography gave a molecular weight of 25 kDa. This enzyme preparation consisted of 4–6 subunits with a molecular weight of 5–6 kDa each, which were held together by FAD. No EDTA-sensitive component was required for activity. The enzyme used β-NADH, but not α-NADH or β-NADPH, to produce stoichiometric amounts of H2O2. The apparent Km with respect to NADH, using production of NAD as a criterion, was 534 μM. The enzyme was active at 4°C and was quite thermostable. Its pH optimum was around pH 7·2. The metabolic function of NADH oxidase in lactobacilli remains obscure, though the H2O2 it generates may have a profound effect on the viability of other organisms.