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

Abstract

An extracellular beta-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-beta-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 beta-glucosidase with a K(i) value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 degrees C. The enzyme was stable at pH 5.0 at temperatures up to 50 degrees C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-beta-d-galactopyranoside. The activity of beta-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. eschscholziibeta-glucosidase have similarity to the sequences of the family 3 beta-glucosyl hydrolase.

Production and Characterization of Highly Thermostableβ-Glucosidase during the Biodegradation of Methyl Cellulose byFusarium oxysporum

“…The result of this study agrees with some earlier reports in which many commercial β -glucosidases have been reported to exhibit optimum activity at acidic pH regions. Singhania and Karnchanatat et al reported similar optimum pH of 5.0 for β -glucosidase from A. niger NII 08121 and Daldinia eschscholzii , respectively [36, 37]. β -glucosidases from various species of Penicillium have been reported to have optimum pH range of 4.0–6.0 [38–40].…”

Characterization of Cellulolytic and Xylanolytic Enzymes of <italic>Bacillus licheniformis</italic> JK7 Isolated from the Rumen of a Native Korean Goat

“…The strong inhibitory effect of Mn 2+ on endoglucanase activity is consistent with previous reports of Bacillus amyloliquefaciens DL-3 and Bacillus flexus (Lee et al, 2008; Trivedi et al, 2011). An inhibitory effect on enzyme activity by metal ions usually suggests the presence of a sulfhydryl group in the active site, where oxidation by the metal ions destabilizes the conformational folding of the enzymes (Karnchanatat et al, 2007). …”

Expression of a Glucose-tolerant β-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae

“…BGL4 Sc was shown to be highly tolerant to glucose inhibition, with a K i value of ∼70 mM. Effective inhibition by glucose is frequently observed on β-glucosidases, whose K i values range from 0.35 to 100 mM when assayed with pNPG as substrate [35]. Recently, it has been reported the identification of glucose-tolerant β-glucosidades from compositing soil fungi, which were active in the presence of 300 mM glucose [12].…”