Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus.
beta-Glucosidase from the fungus Thermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps--ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, beta-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80 degrees C. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl beta-D-glucopyranoside as substrate, Km values of 1.17 +/- 0.35 and 1.38 +/- 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.
de Palma-Fernandez
E R
ER
Centro Universitário de Rio Preto, São Paulo, Brazil.
Gomes
E
E
da Silva
R
R
D001203:Ascomycota / Q000201:enzymology*; D001938:Brazil; D002118:Calcium / Q000494:pharmacology; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D012834:Silver / Q000494:pharmacology; D014462:Ultrafiltration; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D012834:Silver; D001617:beta-Glucosidase; D002118:Calcium