Purification and Properties of Glycerol Dehydrogenase from Candida valida

Summary: 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 were 10.0 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.