NAD-dependent formate dehydrogenase (EC 1.2.1.2), was isolated from the methanol-utilizing yeast Crrridida methylica. Two purification techniques for the enzyme from the crude yeast extract have been developed: a twostep procedure, involving a sequential application of DEAE-cellulose ion-exchange chromatography and Sephadex G-200 gel filtration, and a single-step procedure, preparative isoelectric focusing in a granulated gel layer. The enzyme proved to be electrophoretically homogeneous. I t consisted of two identical subunits with a relative molecular mass of 46000, each containing one -SH group related to manifestation of the catalytic activity. The Michaelis constant was 1 lop4 M for NAD and 1.3 .M for formate. Formate dehydrogenasc was inhibited with p-chlormercuribenzoate, iodoacetamide, dithionitrobenzoatc, cyanide and azide.NAD-dependent formate dehydrogenase, catalyzing the oxidation of formate to COz, has been discovered in many microorganisms utilizing methanol and other C1 compounds as carbon sources [I -41. The homogeneous prcparations of NAD-dependent formate dehydrogenase were obtained from the methylotrophic yeasts Kloeckevu sp. 2201 [5] and Candida hoidinii [6] and their physico-chemical properties studied.In view of elaboration of the cofactor regeneration system based on NAD-dependent dehydrogenases from methylotrophic microorganisms, we isolated and compared formate dehydrogenases from the methylotrophic bacterium Acl7ro-tnohartrr parvulus [7,8] and the methylotrophic yeast Cmdida methqdica [9].The present paper describes the conditions of biosynthesis, isolation and some properties of NAD-dependent formate dehydrogenase from Candida mrthylica.
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