Nitrate reductase from yeast: cultivation, partial purification and characterization

Several yeast strains were assayed for occurence of nitrate reductase after growth in a defined medium with nitrate as the sole nitrogen source, Candida boidinii DSM 70026, showing the highest specific activity, was further investigated. The procedures for yeast fermentation and nitrate reductase purfication are described in detail. Nitrate reductase from this yeast was characterized as NAD(P)H: nitrate oxidoreductase (E.C.1.6.6.2). The enzyme activity with NADH (NADPH) was highest at pH 7.0 (7.1) and 30° C (25° C). The values of Km determinations with NADH/NADPH were both 4 × 10−4 mol/l; values for the substrate inhibition constant (Ki) were 6 × 10−4 mol/l. The molecular mass of the native enzyme was estimated by gel permeation chromatography to be approximately 350 kDa.