Purification and characterization of a beta-glucosidase from Trichoderma reesei.
A beta-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The beta-glucosidase was purified using two successive DEAE-Sephadex anion-exchange chromatography steps, followed by SP-Sephadex cation-exchange chromatography and concanavalin-A--agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band. Sedimentation equilibrium analysis yielded a molecular mass of 74.6 +/- 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The beta-glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half-cystine and methionine residues. The purified beta-glucosidase contains less than 1% by weight of neutral carbohydrate. The beta-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 X 10(4), 6.9 X 10(5) and 2.9 X 10(6) M-1 S-1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The beta-glucosidase has an unusually high affinity for D-glucose (Ki = 700 microM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the beta-glucosidase comprises multiple subsites.
D000596:Amino Acids / Q000032:analysis; D002384:Catalysis; D002845:Chromatography / Q000379:methods; D004589:Electrophoresis, Disc; D004795:Enzyme Stability; D005959:Glucosidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D003904:Mitosporic Fungi / Q000201:enzymology*; D014242:Trichoderma / Q000201:enzymology*; D001617:beta-Glucosidase / Q000302:isolation & purification*
D000596:Amino Acids; D005959:Glucosidases; D001617:beta-Glucosidase