Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm.
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.
Karnchanatat
Aphichart
A
Biotechnology Programme, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
Petsom
Amorn
A
Sangvanich
Polkit
P
Piaphukiew
Jittra
J
Whalley
Anthony J S
AJ
Reynolds
Colin D
CD
Sihanonth
Prakitsin
P
D000645:Ammonium Sulfate / Q000737:chemistry; D001203:Ascomycota / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D014934:Wood / Q000382:microbiology*; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D005656:Fungal Proteins; D008670:Metals; D001617:beta-Glucosidase; D000645:Ammonium Sulfate