Purification and characterization of a β‐glucosidase from Trichoderma reesei

Abstract

A $-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The P-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 f 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 P-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 P-glucosidase contains less than 1 YO by weight of neutral carbohydrate.The P-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl 8-D-glucopyranoside and 4-methylumbelliferyl P-D-glucopyranoside; the values of V / K , for each substrate were determined to be 2.3 x lo4, 6.9 x lo5 and 2.9 x 106 M-' s-' respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The P-glucosidase has an unusually high affinity for D-glucose (Ki = 700 pM).Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the P-glucosidase comprises multiple subsites.One of the best sources of cellulolytic enzymes is the fungus Trichoderma reesei, which possesses the complete array of enzymes required for conversion of cellulose to glucose El]. When grown on cellulose or induced by sophorose (2-0-p-~-glucopyranosyl-a-D-glucopyranose) T. reesei QM9414 elaborates the full complement of cellulolytic enzymes into the extracellular medium [2, 31. The cellulase system comprises 1,4-P-~-glucan 4-glucanohydrolases, 1,4-b-~-glucan cellobiohydrolases and b-glucosidases, which together act sequentially and cooperatively to convert native, crystalline cellulose to oligosaccharides and glucose [2]. Endoglucanases randomly attack internal glycosidic bonds of cellulose chains, thereby producing polymer chain ends and soluble oligosaccharides. Cellobiohydrolases cleave cellobiosyl residues from the ends of cellulose chains. P-Glucosidases catalyze the hydrolysis of cellobiose, which is inhibitory to the depolymerizing enzymes, and oligosaccharides to glucose.

Application ofTrichoderma reeseiCellulase and Xylanase Promoters through Homologous Recombination for Enhanced Production of Extracellular β-Glucosidase I

“…The mass of alkaline BGLI from T. reesei isolated by different authors has varied from 71 to 76 kDa because of different levels of glycosylation and partial proteolysis, 21,22,38) in good agreement with our findings (Fig. 4).…”

“…To overcome this disadvantage, cellulase preparations of T. reesei must be supplemented with -glucosidase from other sources to increase the rate and extent of cellulose hydrolysis. Among the several -glucosidases in T. reesei culture supernatant, the most abundant (and best studied) is -glucosidase I (BGLI), 21,22) which belongs to the glycosyl hydrolase family 3.…”

Enzymatic Properties and Intracellular Localization of the Novel Trichoderma reesei β-Glucosidase BGLII (Cel1A)

“…However, some data suggest that the extracellular and a major part of the cell-wall-bound activities could be due to the same enzyme (12,26). The gene bgl1 (3), encoding an extracellular ␤-glucosidase, has been isolated from T. reesei, and most probably it encodes the extracellular protein isolated previously (5). The corresponding BGLI enzyme has sequence similarity with other ␤-glucosidases belonging to family 3 of the glycosyl hydrolases (3).…”

A novel microplate‐based screening strategy to assess the cellulolytic potential of Trichoderma strains

“…Moreover, despite the presence of a strong phosphate buffer and relatively high initial pH (6.2), the final pH in shake flasks was 3.3-3.7, potentially affecting b-glucosidase stability (Chirico and Brown, 1987). To verify these limitations, the potential high-cellulolytic producing Ba8/86 strain was tested in a different medium (M2) and compared with the reference strain T. reesei Rut-C30.…”