Purification and some properties of beta-glucosidase from Aspergillus niger IBT-90.
beta-glucosidase (EC 3.2.1.21) was isolated from the culture filtrate of Aspergillus niger IBT-90. The crude extracellular enzyme preparation was fractionated by six step purification procedure, (NH4)2SO4 precipitation, gel filtration on Bio-Gel P-10 and P-100, an ion-exchange chromatography on DEAE Bio-Gel A, yielding beta-glucosidase with an isoelectric point at pH 4.05. The enzyme was found to be a dimer with an apparent molecular weight of approximately 200 kDa as determined by size exclusion chromatography. It is composed of two apparently identical subunits of about 100 kDa (determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis). A. niger IBT-90 beta-glucosidase contains 33% carbohydrates. It is most active towards cellobiose at pH 4.8 and 65 degrees C. The enzyme sequentially splits off glucose units from non reducing ends of collodextrins. Kinetic studies on cellobiose and salicin hydrolysis, in concentration from 0.1 to 5.0 mM, resulted in non-linear Lineweaver-Burk and Hanes plots, whereas p-nitrophenyl-beta-D-glucopiranoside (pNPG) did not induce this type of effect. No metal ion is required for the enzyme catalytic activity. Hg2+ and N-bromosuccinimide (NBS) are its strong inhibitors. Glucono-delta-lactone and glucose are competitive inhibitors of the enzyme and glucono-delta-lactone is more potent of the two.
Galas
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Institute of Technical Biochemistry, Technical University of Lódź, Poland.
Romanowska
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D001234:Aspergillus niger / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002475:Cellobiose / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D002241:Carbohydrates; D004791:Enzyme Inhibitors; D002475:Cellobiose; D001617:beta-Glucosidase