The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

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Abstract
Filtrates from cultures of different ages of B. theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. Five cellulases (C1, C2, C3, C4 and C5) were found and their MW, estimated by gel filtration, were 46,000-48,000 (C1), 30,000-35,000 (C2), 15,000-18,000 (C3), 10,000-11,000 (C4) and 4800-5500 (C5). Cellulase C5 was absent from old culture filtrates. Cellulase C1 had little or no activity on CM[carboxymethyl)-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. The other components (C2-C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the MW of cellulases C2-C5. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. A high MW .beta.-glucosidase (component B1, MW 350,000-380,0000) predominated in filtrates from young cultures, but a low MW enzyme (B4, MW 45,000-47,0000) predominated in older filtrates. Intermediate molecular species of .beta.-glucosidase (B2, MW 170,000-180,000; B3, MW 83,000-87,000) were also found. Cellulases C2-C5 acted in synergism with C1, particularly in the presence of .beta.-glucosidase.