This is the first report describing the cloning, expression, and characterization of a putative thermostable, cellulase-free xylanase (XYN) from the thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum DSM 571. The temperature and pH values for optimal enzyme activity of XYN were found to be 65 degrees C and pH 6.5, respectively. The XYN activity was apparently enhanced by Co2+, Mn2+, and Tween 60 and significantly inactivated by Al3+, Cu2+, Zn2+, and SDS. The K-m and V-max values of XYN for the hydrolysis of beechwood xylan were 2.1 mg/ml and 222.1 U/mg, respectively. The k(cat) values of XYN for beechwood xylan at the optimal temperature and pH values were 481.4 s(-1). XYN represents an attractive candidate for use in the large-scale production of xylooligosaccharides (XOs) from forest residues because it is an endo-xylanase capable of degrading xylan.