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 Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg 2+ . The internal amino acid sequences of D. eschscholzii β‐glucosidase have similarity to the sequences of the family 3 β‐glucosyl hydrolase.