Purification and characterization of glycerol dehydrogenase isoenzymes from Geotrichum candidum.

Abstract

Two glycerol dehydrogenases, GD-I and GD-II, were purified from Geotrichum candidum by precipitation of ammonium sulfate, sequential column chromatography on Blue-Sepharose CL-4B and DEAE-Sepharose CL-6B and gel filtration on Cellulofine GC-700. The purified enzyme preparations were homogeneous upon disc gel electrophoresis. The molecular weights were estimated to be 135,000 for GD-I and 130,000 for GD-II, and their isoelectric points were 5.9 and 6.2, respectively. The specific activity of GD-I was twice that of GD-II. However, their other properties were very similar: Their optimum pHs for the oxidation of glycerol were 10.5, and those for the reduction of dihydroxyacetone were 5.5. The enzyme activities were highly activated by Cl-, but inhibited by PO43−, BO33− and 2-mercaptoethanol. The enzymes showed highest activity for glycerol, but also acted on several analogs of glycerol.