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Glycerol : NAD+ 2-oxidoreductase (EC 1.1.1.6) was purified to homogeneity from the non-methylotrophic yeast Candida valida H 122. Results of electrophoresis in polyacrylamide gels, gel filtration and ultracentrifugation were compatible with the enzyme’s consisting of two subunits with a molecular weight 38000. No heterogeneity was observed by isoelectric focusing. The pH optima were10·0 for glycerol oxidation and 7·5 for dihydroxyacetone reduction. The K m values for glycerol, dihydroxyacetone, NAD+ and NADH were 5·8 10−2 m, 7·7 10−4 m, 1·4 10−4 m and 4·8 10−5 m, respectively. 1,2-Propanediol also served as substrate in the forward and dl-glyceraldehyde in the reverse reaction. The oxidation product of glycerol was identified as dihydroxyacetone. An ordered bi-bi mechanism was deduced from product-inhibition studies. Of several anions and cations tested, pyrophosphate and ammonium ions stimulated the dehydrogenating activity the most. 2-Mercaptoethanol, ethylene glycol and Tris inhibited activity; an inhibition was also observed by phosphorylated coenzymes and substrates. The purified enzyme, which is labile at low concentrations, was stabilized by the addition of neutralized filtrate from the culture liquid.
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