Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe. Purification and characterization of intracellular beta-glucosidase.
Intracellular beta-glucosidase was extracted from the mycelium of Th. aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity. Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM. With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C. beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins. Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).
D000596:Amino Acids / Q000032:analysis; D001203:Ascomycota / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002384:Catalysis; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005959:Glucosidases / Q000302:isolation & purification*; D006023:Glycoproteins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D013696:Temperature; D000520:alpha-Glucosidases / Q000032:analysis / Q000302:isolation & purification; D001617:beta-Glucosidase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D006023:Glycoproteins; D005959:Glucosidases; D000520:alpha-Glucosidases; D001617:beta-Glucosidase