Role of carbohydrate content on the properties of galactose oxidase from Dactylium dendroides.

The stability of intracellular, extracellular, and deglycosylated forms of galactose oxidase was compared with respect to the denaturing effects of heat, pH, and guanidine hydrochloride. The highly glycosylated forms were found to be more stable to pH and thermal inactivation. All forms were reversibly denaturated by guanidine hydrochoride, but the extent was dependent on the carbohydrate content. Deglycosylation did not affect the affinity of the enzyme for dihydroxyacetone and galactose. Exposure of different forms of galactose oxidase to proteases like pronase and trypsin resulted in a rapid degradation of the glycoenzymes with the formation of stable products. After pronase digestion of intra- and extracellular forms of galactose oxidase catalytic species were isolated by gel filtration. The species (61 and 42 kDa) isolated from pronase-digested extracellular enzyme lost their ability to oxidize primary alcohols. Species (67 and 46 kDa) obtained from the intracellular enzyme kept the specificity of the original enzyme. Active pronase-derived peptides (42 and 46 kDa, respectively) had a higher carbohydrate content than the inactive ones.