β-Glucosidase fromChalaraparadoxaCH32: Purification and Properties

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Abstract
The hyphomycete Chalara paradoxa CH32 produced an extracellular β-glucosidase during the trophophase. The enzyme was purified to homogeneity by ion-exchange and size-exclusion chromatography. The purified enzyme had an estimated molecular mass of 170 kDa by size-exclusion chromatography and 167 kDa by SDS−PAGE. The enzyme had maximum activity at pH 4.0−5.0 and 45 °C. The enzyme was inactivated at 60 °C. At room temperature, it was unstable at acidic pH, but it was stable to alkaline pH. The purified enzyme was inhibited markedly by Hg2+ and Ag2+ and also to some extent by the detergents SDS, Tween 80, and Triton X-100 at 0.1%. Enzyme activity increased by 3-fold in the presence of 20% ethanol and to a lesser extent by other organic solvents. Purified β-glucosidase was active against cellobiose and p-nitrophenyl-β-d-glucopyranoside but did not hydrolyze lactose, maltose, sucrose, cellulosic substrates, or galactopyranoside, mannopyranoside, or xyloside derivatives of p-nitrophenol. The Vmax of the enzyme for p-NPG (Km = 0.52 mM) and cellobiose (Km = 0.58 mM) were 294 and 288.7 units/mg, respectively. Hydrolysis of pNPG was inhibited competitively by glucose (Ki = 11.02 mM). Release of reducing sugars from carboxymethylcellulose by a purified endoglucanase produced by the same organism increased markedly in the presence of β-glucosidase. Keywords: β-Glucosidase; Chalara paradoxa; purification, kinetics

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