Biochemistry and Biotechnology
Wiley InterScience Backfile Collection 1832-2000
Process Engineering, Biotechnology, Nutrition Technology
A β-glucosidase (E.C. 18.104.22.168) was isolated from the culture filtrate of fungus Trichoderma reesei QM 9414 grown in continuous culture with biomass retention. The crude extracellular enzyme preparation was fractionated by a three-step purification procedure [chromatography on Fractogel HW-55 (S) and Bio-Gel A 0.5 plus final preparative isoelectric focusing] to yield three β-glucosidases with isoelectric points at pH 8.4, 8.0, and 7.4. Only one enzyme (pi 8.4) met the stringent criterion of being homogeneous according to titration curve analysis. This enzyme was then characterized not to be a glycoprotein, although the native protein contained 35% carbohydrate (as glucose). It was found to have an apparent molar mass of 7 × 104 g/mol (SDS-PAGE), exhibited its optimum activity towards cellobiose at pH 4.5 and 70°C (30 min test), and lost less than 3% activity at 50°C over a period of 7 h. The KM values towards cellobiose and p-nitrophenyl-β-D-glucopyranoside were determined to be 0.5mM and 0.3mM, respectively. The enzyme hydrolyzed cellodextrins (cellotriose to cellooctaose) by sequentially splitting off glucose units from the nonreducing end of the oligomers. The extent of the observed transfer reactions varied with the initial substrate concentration. No enzyme activity towards microcrystalline cellulose or carboxymethylcellulose could be detected. The classification of the enzyme as β-glucosidase or exo-β-1,4-glucan glucohydrolase is discussed with respect to the exhibited hydrolytic activities.
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