Biosynthesis, isolation and properties of NAD‐dependent formate dehydrogenase from the yeast Candida methylica

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Abstract
NAD-dependent formate dehydrogenase (EC 1.2.1.2), was isolated from the methanol-utilizing yeast Candida methylica. Two purification techniques for the enzyme from the crude yeast extract have been developed: a two-step procedure, involving 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. It 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 .cntdot. 10-4 M for NAD and 1.3 .cntdot. 10-2 M for formate. Formate dehydrogenase was inhibited with p-chlormercuribenzoate, iodoacetamide, dithionitrobenzoate, cyanide and azide.