Purification and characterization of an intracellular β-glucosidase from Botrytis cinerea

The filamentous fungus Botrytis cinerea, grown on a cellobiose medium, was found to produce three intracellular β-glucosidases (GluI, II, and III). These enzymes were visualized by activity staining after separation by native polyacrylamide gel electrophoresis. GluI, which represents 95.5% of the total β-glucosidase activity, was purified to homogeneity by ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase estimated by gel filtration was 350 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 7 with citrate-phosphate buffer and 6.5 with phosphate buffer. The enzyme was active against glycosides with (1 → 4)-β, (1 → 2)-β, and (1 → 4)-α linkage configuration. The β-glucosidase was competitively inhibited by glucose and by d -gluconic-acid-lactone, and a glucosyl transferase activity was observed in the presence of ethanol.