Coenzyme regeneration catalyzed by NADH oxidase from Lactococcus lactis

l Lactococcus lactis was aerobically grown in a bioreactor to produce NADH oxidase, an enzyme used for NAD(+) regeneration. The enzyme was isolated and purified from the cells that were harvested at the end of exponential phase of growth. The influence of temperature, pH and oxygen on enzyme activity was investigated. The enzyme was kinetically characterized at different pH values and in different buffers. It was found that Michaelis constants for oxygen are very low (the highest was 4.5 mu mol dm(-3) at pH 8.0). NADH oxidase was tested as a regenerating enzyme in a model system of L-methionine oxidation catalyzed by L-phenylalanine dehydrogenase from Rhodococcus sp. When NADH oxidase from L lactis was used for NAD(+) regeneration, 100% L-methionine conversion was achieved, while without regeneration of NAD(+) it was estimated to be about 28%. The operational stability of NADH oxidase was followed and it was found that enzyme activity decay occurs. The operational stability decay rate constant, k(d), was estimated to be 8.0 x 10(-5) min(-1). (C) 2014 Elsevier B.V. All rights reserved.