Purification and Characterization of a Thermostable -Xylosidase from Thermomonospora fusca

Abstract

Xylan-degrading enzymes, including β-xylosidase (EC 3.2.1.37), were induced when Thermomonospora fusca was grown at 50 °C in liquid medium containing 0·2% xylan. The intracellular β-xylosidase activity was concentrated and characterized by fast protein liquid chromatography and gel electrophoresis. A zymogram technique was developed to identify β-xylosidase directly on polyacrylamide gels. A single enzyme (168 kDa; pI 4·37) was identified and purified to homogeneity. The consistent detection of a single band on denaturing SDS gels suggested that the enzyme was composed of identical subunits; since the subunit molecular mass was 56 kDa, a trimeric structure is suggested. High activity against p-nitrophenyl β-d-xylopyranoside (pNPX) occurred in the pH range 5·0–9·0 and temperature range 40–60 °C. The enzyme was stable at room temperature at pH 6·0–8·0; it had a half-life of 8 h at 65 °C, and of 1·5 h at 70 °C. The purified enzyme did not exhibit any detectable activity against arabinoxylan, carboxymethylcellulose or p-nitrophenyl β-d-glucopyranoside. The enzyme had a K m of 0·89 mm (pNPX) and was inhibited by d-xylose (K i 19 mm) but not d-glucose. The size of the T. fusca enzyme is in the range reported for the few other bacterial β-xylosidases described, but the acidic nature of the protein and its affinity for the substrate have more in common with some of the monomeric β-xylosidases described in fungi.