Purification and characterization of a β‐glucosidase from Trichoderma reesei

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. Copyright © 1987, Wiley Blackwell. All rights reserved