Summary
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 K m determinations with NADH/NADPH were both 4 × 10−4 mol/l; values for the substrate inhibition constant (K i) were 6 × 10−4 mol/l. The molecular mass of the native enzyme was estimated by gel permeation chromatography to be approximately 350 kDa.
Similar content being viewed by others
References
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brown CM, MacDonald-Brown DS, Meers JL (1974) Physiological aspects of microbial inorganic nitrogen metabolism. Adv Microb Physiol 11:1–52
Campbell WH, Smarelli J (1978) Purification and kinetics of higher plant NADH: nitrate reductase. Plant Physiol 61:611–616
Choudary PV, Deobagkar DN, Rao GR (1986) Partial purification and properties of assimilatory nitrate reductase of the food yeast Candida utilis. Microbios 47:135–147
De La Rosa MA, Diez J, Vega JM, Losada M (1980) Purification and properties of assimilatory nitrate reductase (NAD(P)H) from Ankistrodesmus braunii. Eur J Biochem 106:249–256
Funkhouser EA, Ackerman R (1976) Nitrate reductase from Chlorella vulgaris. Eur J Biochem 66:225–228
Guerrero MG, Gutierrez M (1977) Purification and properties of the NAD(P)H: nitrate reductase of the yeast Rhodotorula glutinis. Biochim Biophys Acta 482:272–285
Gromes R, Johannssen W (1990) Bestimmung des Nitratgehaltes in Wasserproben mit einem halbenzymatischen Test. GIT Fachz Lab 34:759–761
Hewitt EJ (1975) Assimilatory nitrate-nitrite reduction. Ann Rev Plant Physiol 26:73–100
McGeehan JK (1987) Particles suitable for tabletting into diagnostic reagents. European patent application no. O 238951
Renosto F, Ornitz DM, Peterson D, Segel JH (1981) Nitrate reductase from Penicillium chrysogenum. J Biol Chem 256:8616–8625
Rivas J, Guerrero MG, Panaque A, Losada M (1973) Characterization of the nitrate reducing system of the yeast Torulopsis nitratophila. Plant Sci Lett 1:105–113
Zauner E, Dellweg H (1983) Purification and properties of the assimilatory nitrate reductase from the yeast Hansenula anomala. Appl Microbiol Biotechnol 17:90–95
Author information
Authors and Affiliations
Additional information
Offprint requests to: R. Gromes
Rights and permissions
About this article
Cite this article
Gromes, R., Schwartz, H., Heinrich, M. et al. Nitrate reductase from yeast: cultivation, partial purification and characterization. Appl Microbiol Biotechnol 35, 491–495 (1991). https://doi.org/10.1007/BF00169755
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00169755