Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis alpha-L-arabinofuranosidase Axh43A indicated that U1 was alpha-L-Araf-(1 -> 2)-[alpha-L-Araf-(1 -> 3)]-D-Xylp and U2 was alpha-L-Araf-(1 -> 2)-D-Xylp, suggesting that rXyn10C had strong activity towarda xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an alpha-1,2- and alpha-1,3-linked arabinose-substituted xylose unit in both the -1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite - 1. This is the first report of a xylanase capable of producing alpha-L-Araf-(1 -> 2)-[alpha-L-Araf-(1 -> 3)]-D-Xylp from highly arabinosylated xylan. (C) 2015 Elsevier Inc. All rights reserved.