Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from Scytalidium thermophilum

The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic beta-D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level Of beta-D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial beta-xylosidase induced by xylan was purified using a procedure that included heating at 50degreesC, ammonium sulfate fractioning (30-75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified beta-D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60degreesC and 5.0, respectively. beta-D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for I h at 60degreesC and has half-lives of 11 and 30 min at 65degreesC in the absence or presence of calcium, respectively. The purified beta-D-xylosidase hydrolyzed p-nitrophenol-beta-D-xylopyranoside and p-nitrophenol-beta-D-glucopyranoside, exhibiting apparent K-m and V-max values of 1.3 mM, 88 mumol min(-1) protein(-1) and 0.5 mM, 20 mumol min(-1) protein(-1), respectively. The purified en zyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true beta-D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most beta-xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum beta-xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.