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

ABSTRACT: 1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000-48000 (C1), 30000-35000 (C2), 15000-18000 (C3), 10000-11000 (C4) and 4800-5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. 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. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2-C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight beta-glucosidase (component B1, mol.wt. 350000-380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000-47000) predominated in older filtrates. 9. Intermediate molecular species of beta-glucosidase (B2, mol.wt. 170000-180000; B3, mol.wt. 83000-87000) were also found. 10. Cellulases C2-C5 acted in synergism with C1, particularly in the presence of beta-glucosidase.

SUBMITTER: Umezurike GM

PROVIDER: S-EPMC1186335 | biostudies-other | 1979 Jan

REPOSITORIES: biostudies-other

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Project description:1. Whereas only beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced by the tropical fungus Botryodiplodia theobromae Pat. (I.M.I. 115626; A.T.C.C. 26123) in young cultures containing D-cellobiose as carbon source, lower-Mr forms (B, C and D) were found in older cultures when the pH had drifted from the initial value of pH 6.2 to pH 7.9. 2. The Michaelis constants (Km) of the various molecular forms of the enzyme were 0.30 +/- 0.03 mM-, 0.26 +/- 0.01 mM-, 0.20 +/- 0.02 mM- and 0.16 +/- 0.01 mM-o-nitrophenyl beta-D-glucopyranoside for beta-glucosidase forms A (Mr 320,000), B (Mr 160,000), C (Mr 80,000) and D (Mr 40,000) respectively. 3. Only beta-glucosidase D showed substrate inhibition. 4. Only L-arginine was found as the N-terminal residue, and beta-glucosidase A contained 31.7 +/- 0.6 mol of N-terminal L-arginine/mol of the enzyme. 5. Storage of purified beta-glucosidase A under mildly alkaline conditions caused its dissociation into the lower-Mr forms, whereas adjustment of the pH of a solution of beta-glucosidase A to pH 12.0 with 1 M-NaOH led to complete inactivation on incubation at 40 degrees C for 1 h and to the release of 25.2 +/- 1.5 mol of inorganic phosphate/mol of the enzyme. 6. O-Phospho-L-serine was isolated from the acid-hydrolysis product of beta-glucosidase A but not from that of beta-glucosidase D. 7. Reduction and carboxamidomethylation of the various forms of beta-glucosidase gave only one enzymically inactive protein with an Mr of 10,000-11,000. 8. After partial succinylation (3-carboxypropionylation) of beta-glucosidase D at pH 5.0 and removal of the precipitated protein formed, the supernatant solution contained beta-glucosidase components similar to the other molecular forms (A, B and C) and an aggregate (beta-glucosidase Xs) that gave a positive result in the alkaline hydroxylamine test, whereas N-succinylated beta-glucosidase D, an aggregate (form Xp) that behaved like beta-glucosidase Xs and traces of forms A, B and C were found by gel filtration of the solution of the precipitate solubilized at neutral pH (7.0-7.7). 9. These observations are discussed in terms of the proposed octameric structure of beta-glucosidase A based on the result of electron microscopy [Umezurike (1975) Biochem. J. 145, 361-368].

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

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