A thermophilic xylan-degrading Actinomadura sp. 514 was isolated from compost in Thailand. Hemicellulase activities such as endo-1,4-beta-xylanase, beta-xylosidase and alpha-arabinofuranosidase were induced with xylan-containing agriculture wastes and oat spelt xylan. The gene encoding xylanase consisting of 687 bp was cloned from Actinomadura sp. S14. The deduced amino acid sequence contained a signal peptide of 41 amino acids and a probable mature xylanase of 188 amino acids. An open reading frame (xynS14) corresponding to a mature xylanase was expressed in Escherichia colt and Pichia pastoris. The specific activity of purified XynS14 (P. pastoris) was 2.4-fold higher than XynS14 (E. colt). Both XynS14s showed the same basic properties such as optimal pH and temperature (pH 6.0 and 80 degrees C) and stability in a broad pH range (pH 5.0-11.0) and at high temperatures up to 80 degrees C. Both XynS14s showed approximately the same substrate specificity and K(m) values toward various xylans, but XynS14 (P. pastoris) showed higher V(max) and K(cat) than XynS14 (E. colt). Higher specific activities of XynS14 (P. pastoris) may be due to protein-folding in the host. Purified XynS14 showed more endo-1,4-beta-xylanase activity on xylan and xylooligosaccharides than on xylotriose. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.