A third and new extracellular xylanase (XynC) was purified from birchwood xylan-induced culture filtrates of the mesophilic fungus Penicillium capsulation. Purified XynC is an acidic protein (pI 2.8), with a relative molecular mass of 22 +/- 0.4 kDa and, unlike XynA and Xyn 3 from the same source, is not glycosylated. pH and temperature optima of 3.8 and 48 degreesC, respectively, were determined for XynC using standard assay conditions. Thermal stability was appreciable at pH 5.0, with an estimated half-life of 25 h at 50 degreesC and retention of full; activity after 2 weeks at 30 degreesC. Substrate specificity studies revealed that XynC was exclusively xylanolytic. Complete inhibition of enzyme activity by N-bromosuccinimide, Hg2+, and carboxylate-specific reagents suggested the presence of essential tryptophan, cysteine and ispartate/glutamate residues. Kinetic parameters were assessed using a range of soluble and insoluble xylans. Apparent K-m values ranged from 7.02 to 31.94 mg ml(-1), while V-max values were highest for the more soluble substrates. High performance anion exchange chromatography (HPAEC) analysis of the products of hydrolysis confirmed that XynC is an endoxylanase (EC 3.2.1.8) and indicated that subs rate solubility and degree of substitution modulate hydrolysis and product formation. (C) 2003 Elsevier Inc. All rights reserved.