Purification and Characterization of a Thermostable β-Xylosidase from Thermomonospora fusca

Xylan-degrading enzymes, including β-xylosidase (EC 3.2.1.37), were induced when Thermomonospora fusca was grown at 50 ° 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 °. The enzyme was stable at room temperature at pH 6.0-8.0; it had a half-life of 8 h at 65 °, and of 1.5 h at 70 °. 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 (Ki 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.