Purification and characterization of a thermostable xylanase from a thermophilic fungus <i>Thermoascus aurantiacus</i>

The xylanase was purified from the culture filtrate of a thermophilic fungus Thermoascus aurantiacus C436 using a single chromatographic step on SP-Sephadex C50 and was homogeneous based on denaturing polyacrylamide and isoelectric focusing gels.

Abstract

A thermostable endo-β-D-xylanase (1,4-β-D-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a thermophilic fungus Thermoascus aurantiacus C436, using a single chromatographic step on SP-Sephadex C50. The purified preparation was homogeneous based on denaturing polyacrylamide and isoelectric focusing gels. The xylanase had a subunit molecular mass of 32 000 daltons, isoelectric point at pH 7.1, apparent K m and V max of 0.17% (w/v) xylan and 61.3IU/mg protein, respectively, at 50 °C. The pH and temperature optima for xylan hydrolysis were pH 5.1 and 80 °C, respectively. The xylanase retained full activity following incubation at 60 °C for 97 h or 70 °C for 24 h. At 80 °C, the half-life of the enzyme was 54 min. The xylanase was not affected by copper sulfate, zinc sulfate, calcium chloride, cobalt chloride, barium chloride, magnesium sulfate, and EDTA at concentrations of 2 mM. Mercury chloride at 2 mM concentration abolished all xylanase activity, while lead acetate at the same concentration reduced xylanase activity by approximately 25%. From the initial hydrolysis products of xylan, the xylanase was deduced to hydrolyse xylan through an endo-acting mechanism.