beta-glucosidase from Chalara paradoxa CH32: Purification and properties

The hyphomycete Chalara paradoxa CH32 produced an extracellular beta-glucos idase during the trophophase. The enzyme was purified to homogeneity by ion -exchange and size-exclusion chromatography. The purified enzyme had an est imated molecular mass of 170 kDa by size-exclusion chromatography and 167 k Da by SDS-PAGE. The enzyme had maximum activity at pH 4.0-5.0 and 45 degree s C. The enzyme was inactivated at 60 degrees C. At room temperature, it wa s unstable at acidic pH, but it was stable to alkaline pH. The purified enz yme 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 increas ed by 3-fold in the presence of 20% ethanol and to a lesser extent by other organic solvents. Purified beta-glucosidase was active against cellobiose and p-nitrophenyl-beta-D-glucopyranoside but did not hydrolyze lactose, mal tose, sucrose, cellulosic substrates, or galactopyranoside, mannopyranoside , or xyloside derivatives of p-nitrophenol. The V-max of the enzyme for p-N PG (K-m = 0.52 mM) and cellobiose (K-m = 0.58 mM) were 294 and 288.7 units/ mg, respectively. Hydrolysis of pNPG was inhibited competitively by glucose (K-i = 11.02 mM). Release of reducing sugars from carboxymethylcellulose b y a purified endoglucanase produced by the same organism increased markedly in the presence of beta-glucosidase.