Hydrogen Peroxide Production and Oxidation of Ferrous Iron by Lactobacillus delbrueckii ssp. bulgaricus

Properties of NADH oxidase fromLactobacillus delbrueckii sspbulgaricus

“…NADH was determined spectrophotometrically at 340 nm in a Perkin-Elmer Lambda 2 instrument (Ueberlingen, Germany), from which NAD concentration could be calculated. Hydrogen peroxide was determined using Sigma Corp kit no 352, as described previously (Kot et al 1996). With high concentrations of NADH (eg 1000 lM), a lag period was often observed when velocities were determined, and, in addition, (product) concentration tended to decline H 2 O 2 after reaching a maximum.…”

“…Chemical sources and D( [ )-lactate assays were as described previously (Kot et al 1996). Protein determinations were done either by spectrophotometry at 280 nm (column chromatography eluates) or by the Lowry et al (1951) reaction.…”

“…In some situations, it was sufficient to measure the hydrogen peroxide produced or NADH utilised after a single reaction time, eg 20 min ; in other cases, velocity was determined by measuring one or both products at 1 min intervals following the addition of the enzyme solution to NADH. Both hydrogen peroxide and NADH (ergo NAD) were estimated as previously described (Kot et al 1996). A typical assay was performed as follows : to a vial containing, eg 1É28 lmols of solid NADH (product 340È101, Sigma Corp, St Louis, MO, USA) in a water bath at 37¡C were added 5 ml of the enzyme solution (puriÐed preparation, or for example, 1 : 2 dilution of the cytosol).…”

“…L actobacillus delbrueckii ssp bulgaricus (ATCC 11842) was obtained and grown as previously described (Kot et al 1996). The soluble cell fraction was prepared as follows : bacterial cells in the post-logarithmic stage of growth were harvested from the growth medium by centrifugation at 6000 ] g for 10 min at 4¡C.…”

“…(Collins and Aramaki 1980) and in L actobacillus delbrueckii spp bulgaricus (Kot et al 1996), and, most likely, in many other lactic acid bacteria. Surprisingly, the properties of NADH oxidases of this type from lactobacilli remain largely unknown, most likely because they were said to be unstable (Condon 1987).…”