p-Xylosidase production was maximal for the mutant Pichia stipitis NP54376 grown on xylan as the sole carbon source. P-Xylosidase was purified from culture supernatant by (NH4)(2)SO4 precipitation and a hydrophobic interaction chromatography on phenyl sepharose. Optima of pH and temperature were 5.0 and 50 degrees C, respectively. The enzyme was inhibited by 2-mercaptoethanol (100%) and Fe3+ (80%), and moderately affected by Cu2+, Ag+, NH4+ and Mg2+ and SDS. The purified xylosidase hydrolyzed xylobiose and xylo-oligosaccharides and it did not exhibit activity against cellulose, starch, maltose and cellobiose. 2.5 g l(-1) glucose repressed P-xylosidase activity in the NP54376 strain. The K-m and V-max values on p-nitrophenyl-p-xylopyranoside were 1.6 mM and 186 mu mol p-nitrophenyl min(-1) mg(-1) protein, respectively. Analysis of the hydrolysis products by HPLC indicated that the major hydrolysis product is xylobiose in all the carbon sources tested. (C) 2006 Elsevier Ltd. All rights reserved.