Purification and biochemical characterization of beta-glucosidase from Daldinia eschscholzii

Abstract

An extracellular beta-glucosidase (EC 3.2.1.21) was purified from culture filttate of the wood decaying fungus Daldinia eschscholzii grown on 1.0% (wt/vol) carboxymethyl-cellulose by using ammonium sulfate precipitation, ion-exchange hydrophobic interaction, and gel filtration chromatography with 6.28% yield and 50.23 purification fold. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and with a molecular weight of 65,747 Da estimated by MALDI-TOF spectrometry, has pI 8.55 obtained using isoelectric focusing. The enzyme catalyzes the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (PNPG) as the substrate, the K[subscript m] values of 1.52 mM, and V[subscript max], values of 3.21 U/min/mg of protein. Glucose and glucono-delta-lactone inhibited beta-glucoisdase competitively, with K[subscript i] values of 0.79 mM, and 4.72 muM, respectively. 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 50ํC. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose, and gentiobiose, but did not hydrolyze lactorse, sucrose, Avicel, and o-nitrophenyl-beta-D-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca[superscript 2+], Co[superscript 2+], Mg[superscript 2+], Mn[superscript 2+], glycerol, DMSO, DTT, and EDTA, and strongly inhibited by Hg[superscript 2+]. beta-Glucosidase was chemical by modified by EDC in the presence of glycinamide as nucleophile under various conditions to study role of carboxy groups in the catalytic mechanism of this enzyme. beta-Glucosidase was inactivated by the binding of one mol of EDC per mol of the enzyme with a second-order rate constant of 6.73x10[superscript -2] mM/min. Glucono-delta-lactone as a competive inhibitor, partly protected the active-site carboxy group against chemical modification, with k[superscript d] of 70.74x10[superscript -2] mM. The enzyme catalyzed the synthesis of alkyl-glucosiedes in the presence of primary secondary and tertiary alcohol with PNPG by transglucosylation. The internal amino acid sequences of D. eschscholzii beta-glucoisdase have similarity to the sequence of the family 3 beta-glucosyl hydrolase.