Fractionation of the cellulolytic enzymes produced by a species of Monilia; purification and properties of an extracellular beta-D-glucosidase.

Extracellular cellulolytic enzymes produced by a species of Monilia could be fractionated by chromatography on SP-Sephadex, Con A-Sepharose, and cellobiose-Sepharose. These methods did not separate the beta-D-glucosidases (beta-D-glucoside glucohydrolases, EC 3.2.1.21) from the cellulases and xylanases within a single purification step. Fractionation by isoelectric focusing on a flat-bed granulated gel gave all of the beta-D-glucosidase activity in a single zone isoelectric at pH 8-9. The beta-D-glucosidase could be further purified to homogeneity by column isoelectric focusing at pH 8.0-10.5, and gel filtration on Biogel P-100. The purified beta-D-glucosidase showed optimal activity at pH 4-5 and 50 degrees, was isoelectric at pH 8.87, and had a molecular weight of 46,600. SDS-Polyacrylamide-gel electrophoresis demonstrated that the beta-D-glucosidase was not dissociated into subunits and, hence, consisted of a single polypeptide chain. The enzyme is considered a glycoprotein, as it binds to Con A-Sepharose. The beta-D-glucosidase hydrolyzed (1----2)-, (1----4)-, and (1----6)-beta-D-glucosidic linkages but not cellulose. Nitrophenyl beta-D-glucopyranosides and beta-D-xylopyranosides were also degraded. The beta-D-glucosidase was competitively inhibited by D-glucose (Ki 0.67 mM).