Purification and characterization of thermostable alpha-galactosidase from Aspergillus terreus (GR).
An extracellular thermostable alpha-galactosidase producing Aspergillus terreus (GR) strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 degrees C, respectively, for artificial substrate pNPalphaGal. alpha-Galactosidase from A. terreus (GR) is found to be thermostable, as it was not inactivated after heating at 65 degrees C for 40 min. The K (m) for pNPalphaGal, oNPalphaGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg(2+), Ni(2+), Ca(2+), Co(2+), and Mn(2+) had no effect on enzyme activity, but Ag(+), Hg(2+), and Cu(2+) have inhibited complete activity.
Shankar
S K
SK
Department of Biochemistry, Gulbarga University, Karnataka, India.
Dhananjay
S K
SK
Mulimani
V H
VH
D001230:Aspergillus / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D055550:Protein Stability; D013379:Substrate Specificity; D013696:Temperature; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D004791:Enzyme Inhibitors; D008670:Metals; D000519:alpha-Galactosidase