Cloning of a thermostable xylanase from Actinomadura sp S14 and its expression in Escherichia coli and Pichia pastoris

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.