PURIFICATION AND SOME PROPERTIES OF BETA-GLUCOSIDASE FROM THE ECTOMYCORRHIZAL FUNGUS PISOLITHUS-TINCTORIUS STRAIN SMF

A cell-associated beta-glucosidase was purified 152-fold to homogeneity from the ectomycorrhizal fungus Pisolithus tinctorius strain SMF. The apparent molecular weight of the native protein, as determined by size exclusion chromatography, was approximately 450 000. A single band with a molecular weight of 150 000 was obtained after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Thus, the native enzyme may consist of three monomers. The pI of the enzyme was determined to be 3.8 by isoelectric focusing. The enzyme had an optimal pH of 4.0 and an optimal temperature for activity of 65-degrees-C. It showed a high substrate specificity toward aryl-beta-glucosides, such as p-nitrophenyl beta-D-glucopyranoside (PNPG), and beta-1,6 glucosidic linkages. Cellobiose was hydrolyzed at about two-thirds the rate of PNPG. The K(m) for hydrolysis of PNPG was 0.87 mM. Strong inhibitors of the enzyme were aluminum, copper, ethylenediaminetetraacetic acid (EDTA), deoxynojirimycin, gluconic acid, and SDS. Calcium, manganese, and p-hydroxymercuribenzoic acid reduced the activity to a lesser extent. Potassium, mercury, cobalt, dithiothreitol, and glucosamine had no effect on activity. Enzyme activity was slightly increased to 112% in the presence of 1% glycerol. The enzyme was more stable under acidic conditions than under alkaline conditions.