Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus

An extracellular beta -glucosidase from Thermoascus aurantiacus was purifie d to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chrom atography. The enzyme was a homotrimer, with a monomer molecular mass of 12 0 kDa; only the trimer was optimally active at 80 degreesC and at pH 4.5, A t 90 degreesC, the enzyme showed 70 % of its optimal activity. It was stabl e at pH 5.2 and at temperatures up to 70 degreesC for 48 h, but stability d ecreased above 70 degreesC and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl beta -D-glucosides and cello-oligosaccharides, a nd was specific for substrates with a beta -glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing en d of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest K-m towards p-nitrophenyl beta -D-glucoside (0.1137 mM) and the highest k(cat) towards cellobiose and beta,beta -trehalose (17052 min(-1)). It released one glucose unit at a time from the non-reducing end of cello- oligosaccharides. and the rate of hydrolysis decreased with an increase in chain length. Glucose and D-delta -gluconolactone inhibited the beta -gluco sidase competitively, with K-i values of 0.29 mM and 8.3 nM respectively, w hile methanol, ethanol and propan-2-ol activated the enzyme. The enzyme cat alysed the synthesis of methyl, ethyl and propyl beta -D-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either gluc ose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols fav oured the latter reaction. The stereochemistry of cellobiose hydrolysis rev ealed that beta -glucosidase from T. aurantiacus is a retaining glycosidase , while N-terminal amino acid sequence alignment indicated that it is a mem ber of glycoside hydrolase family 3.