Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus.
An extracellular beta-glucosidase from Thermoascus aurantiacus was purified to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120 kDa; only the trimer was optimally active at 80 degrees C and at pH 4.5. At 90 degrees C, the enzyme showed 70% of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70 degrees C for 48 h, but stability decreased above 70 degrees C and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl beta-d-glucosides and cello-oligosaccharides, and 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 end 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-glucosidase competitively, with K(i) values of 0.29 mM and 8.3 nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl beta-d-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that beta-glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.
Parry
N J
NJ
Food Materials Science Division, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
Beever
D E
DE
Owen
E
E
Vandenberghe
I
I
Van Beeumen
J
J
Bhat
M K
MK
D000438:Alcohols / Q000494:pharmacology; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology*; D002475:Cellobiose / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000378:metabolism; D004795:Enzyme Stability; D005942:Gluconates / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006031:Glycosylation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D007783:Lactones; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D000438:Alcohols; D004187:Disaccharides; D005942:Gluconates; D007783:Lactones; D011134:Polysaccharides; D002475:Cellobiose; D001617:beta-Glucosidase; D005947:Glucose; C010730:beta-glucono-1,5-lactone