Purification and biochemical characterization of an extracellular β-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm

An extracellular β-glucosidase was purified from culture filtrates of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p -nitrophenyl-β-d-glucopyranoside (PNPG) as the substrate, with a K m of 1.52 mM, and V max of 3.21 U min mg−1 protein. Glucose competitively inhibited β-glucosidase with a K i value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o -nitrophenyl-β-d-galactopyranoside. The activity of β-glucosidase was stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg2+. The internal amino acid sequences of D. eschscholzii β-glucosidase have similarity to the sequences of the family 3 β-glucosyl hydrolase.