Purification and characterization of a β‐glucosidase from Trichoderma reesei

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Abstract
A β-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The β-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 β-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 β-glucosidase contains less than 1% by weight of neutral carbohydrate. The β-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl β-d-glucopyranoside and 4-methyl-umbelliferyl β-d-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 × 104, 6.9 × 105 and 2.9 × 106 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 β-glucosidase has an unusually high affinity for d-glucose (Ki= 700 μM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the β-glucosidase comprises multiple subsites.

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