Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular beta-D-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The beta-D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only beta-D-xylosidase activity, but also alpha-L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS-PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 degrees C, respectively. The enzyme remained stable over a pH range of 3.5-6.5 and up to 50 degrees C for 30 min. The Michaelis constant for p-nitrophenyl beta-D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.