Purification and properties of an acid β-xylosidase from Penicillium sclerotiorum

Abstract

The β-xylosidase from Penicillium sclerotiorum was purified to homogeneity by a rapid and inexpensive procedure involving ammonium sulphate fractionation and molecular exclusion chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed two bands with an estimated molecular mass of 97 and 42 kDa, respectively. Electrophoretical homogeneity was observed under non-denaturing PAGE conditions. These results indicate that this protein has a dimeric structure. The molecular mass of the native enzyme estimated by molecular exclusion chromatography was 144 kDa. The enzyme is a glycoprotein with a 56.4% carbohydrate content. The pH and temperature optima were 2.5 and 60°C, respectively. The enzyme remained stable over a pH range from 2.0 to 7.0 and at temperatures up to 60°C for 375 min. All divalent cations tested, except for Hg2+, inhibited β-xylosidase activity, especially at a concentration of10 mM. The purified enzyme was also sensitive to denaturing agents SDS and EDTA and was activated by thiol-containing reducing agents. The Michaelis–Menten constant for p-nitrophenyl-β-D-xylopyranoside was 0.78 mM, and the maximum reaction velocity was 0.51 μmole of p-nitrophenol min-1 mg-1 of protein. This is the first report on the purification and characterization of a β-xylosidase from P. sclerotiorum, which has potential applications in a number of biotechnological processes, such as animal feed, juice and wine industries.