A novel facultative thermophilic bacterium capable of producing rare ketoses from polyols was isolated from soil and identified as Bacillus pallidus Y25. We have employed this isolated strain for the production Of L-xylulose, a rare ketopentose, from xylitol by a resting cell reaction at 50 degrees C. After optimization of culture conditions and reaction states, L-xylulose was produced at up to 85% yield when 2% xylitol was utilized. The key enzyme responsible for the interconversion of xylitol and L-xylulose was purified to homogeneity and identified as NAD-dependent xylitol dehydrogenase (XDH-Y25), with a molecular mass of 28 kDa as determined by SDS-PAGE. XDH-Y25 is most active in glycine-NaOH buffer (pH 10) at 40 degrees C and its substrate specificity is restricted to D-threitol, xylitol and D-iditol. This suggested that the enzyme is specific for polyols that have a hydroxyl group at the C-2 and C-3 positions in the L- and D-sides, respectively, in the Fischer projection. N-terminal amino acid sequencing of XDH-Y25 revealed low homology to any known dehydrogenases. To our knowledge, this is the first report on the characterization of xylitol dehydrogenase from a Bacillus species. (c) 2006 Elsevier Inc. All rights reserved.