AbstractAn extracellular xylanase was purified from the mesophilic fungus Penicillium rolfsii c3-2(1) IBRL. After three consecutive purification steps, the extracellular cellulase-free xylanase was successfully purified to homogeneity with a recovery yield of 24%. A single protein band of 35 kDa was detected by SDS-PAGE, which had an optimum catalytic activity at pH 5.0 and 50 °C. This purified enzyme was stable at pH 5 to 7, thermostable up to 55 °C, and retained up to 83% of its activity after 4 hours of pre-incubation. A kinetic study yielded estimated Km and Vmax values of 5.73 mg/mL and 691.6 µmol/min/mg, respectively. Thin layer chromatography experiments showed that the purified xylanase was capable of hydrolyzing xylotriose, xylotetraose, xylopentaose, and xylohexaose but not xylobiose, suggesting it is an endo-xylanase. Enzymatic hydrolysis of oil palm trunk residues by commercial enzymes supplemented with the purified xylanase showed a considerable increase in total sugar conversion compared with the commercial enzymes alone, suggesting that xylanase is a key enzyme in the hydrolysis of oil palm trunk residues.