Properties of NADH oxidase from Lactobacillus delbrueckii ssp bulgaricus

Lactobacillus delbrueckii spp bulgaricus produces substantial amounts of hy drogen 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 ce ll fraction (cytosol) of L delbrueckii ssp bulgaricus, prepared by sonicati on 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 t o bind the enzyme. The enzyme was eluted with a 0.01 M Tris buffer at pH 7. 2 containing 10 mu M FAD. This preparation had 0.2 mg ml(-1) protein and sh owed a single major band in a polyacrylamide gel electrophoresis system wit h a molecular weight of near 30 kDa. Gel filtration chromatography gave a m olecular weight of 25 kDa. This enzyme preparation consisted of 4-6 subunit s 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 bet a-NADH, but not alpha-NADH or beta-NADPH, to produce stoichiometric amounts of H2O2. The apparent K-m with respect to NADH, using production of NAD as a criterion, was 534 mu M. The enzyme was active at 4 degrees C and was qu ite thermostable. Its pH optimum was around pH 7.2. The metabolic function of NADH oxidase in lactobacilli remains obscure, though the H2O2 it generat es may have a profound effect on the viability of other organisms. (C) 1998 Society of Chemical Industry.