1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration ; it resulted in a yield of 40 %.2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol.3 . Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80000.4. Amino acid composition and some other physico-chemical properties of the enzyme were studied.5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.1 1 mM for NAD and 15 mM for formate (pH 7.0, 37 "C).6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.Dehydrogenases are of great importance for microorganisms when taking part in the redox reactions of various functional groups of organic compounds. The perspective of their use in various biotechnological processes (fine organic synthesis, analysis of different metabolites and energetics) gave rise to the interest in the enzymes involved in the redox reactions. The systematic investigation of the structure, mechanism of action and stability of the enzymes of this class is critical for their development as highly effective bioorganic catalysts.At present close attention is being paid to the problem of the enzymatic oxidation of methanol. The last stage of this process is catalyzed by NAD-dependent formate dehydrogenase, observed in the majority of methylotrophic microorganisms [l -81. Since formate dehydrogenase is a part of multienzyme systems it can be used for a number of practical purposes: for example, for the development of an EKJTW. Formate dehydrogenase (EC 1.2.1.2).NADH regeneration system or for the production of hydrogen from organic fuels, etc. In the recent years the interest in formate dehydrogenase has been promoted also by the study of the metabolism of methylotrophic microorganisms.The practical irreversibility of the reaction of formate oxidation to CO2 enables high degrees of substrate conversion to be achieved. The process offers a number of technological advantages: one of the products of the reaction evolves in gaseous form, also formate does not inhibit conjugated enzyme systems and can be easily separated from the end products.The isolation of a strain of methylotrophic bacteria which was an active producer of NAD-dependent formate dehydrogenase [9], enabled us to develop an effective method of purification of this enzyme a...

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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.

The reaction of copper with benzyl bromides in hexamethylphosphoramide has been studied. The kinetic and thermodynamic parameters of the reaction have been obtained. Hammett plots of log (k/k o ) vs the substituent constant σ gave good correlations (ρ = 0.15, S ρ = 0.02, r = 0.954). The structure of the organic group has little effect on the rate of reaction of benzyl bromide with copper. In the absence of atmospheric oxygen, the oxidative dissolution of copper occurred by the mechanism of single-electron transfer with the formation of 1,2-diphenylethane and copper(I) complexes. The stereochemistry and intermediates compound was also investigated.The reaction mechanism is discussed. C 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: [296][297][298][299][300][301][302][303][304][305] 2005