PURIFICATION AND CHARACTERIZATION OF AN INTRACELLULAR BETA-GLUCOSIDASE FROM BOTRYTIS-CINEREA

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