An improved method for the determination of NADH oxidase in the presence of NADH peroxidase in lactic acid bacteria

The complexity of the coupled NADH oxidaseââ"šÂ¬Ã¢â‚¬Å“NADH peroxidase enzyme system in lactic acid bacteria makes it difficult to simultaneously determine the individual levels of both these enzymes spectrophotometrically. This study describes an improved assay to accurately determine low concentrations of NADH oxidase from enzyme suspensions containing NADH oxidase and NADH peroxidase. For the standardisation of the assay, pure NADH oxidase and NADH peroxidase were mixed in various proportions and the percentage recovery was estimated by both the currently available assay as well as by the improved assay reported in this study. The recovery of NADH oxidase using the currently available assay ranged from as low as âË" Ã¢â‚¬â„¢200% to as high as +102% as against 90ââ"šÂ¬Ã¢â‚¬Å“102% in the improved assay. The recovery percentage of NADH peroxidase ranged from 91% to 112% in both assays. The slopes of NADH oxidation by cell-free extracts of six lactic acid bacteria were also measured by both assays for the estimation of NADH oxidase and NADH peroxidase levels. The improved assay can further distinguish between NADHââ"šÂ¬Ã¢â‚¬Å“H2O oxidase and NADHââ"šÂ¬Ã¢â‚¬Å“H2O2 oxidase and was successfully applied to identify the type of NADH oxidase in the lactic acid bacteria tested.