The pH induced conformational and structural changes of Xyl II have been investigated from the alkalophilic thermophilic Bacillus sp. using kinetic, circular dichroism and fluorescence spectroscopy studies. The systematic studies on the folding and stability of cellulase-free xylanases are important, since their biotechnological applications require them to function under extremes of pH and temperature. The Trp fluorescence and the kinetic constants were found dependent on the pH. Above pH 8, the enzyme exhibited unfolding transitions as revealed by a red shift in the emission maximum as well as decreases in the fluorescence intensity. Circular dichroism studies revealed a decrease in the CD ellipticity at 222 nm at pH 9 and 10. The reduced catalytic activity of Xyl II at alkaline pH is correlated to the pH induced unfolding and ionization or protonation of key protein residues. The pH profile of Xyl II showed apparent pK values of 5.5 and 7 for the free enzyme and 5.6 and 6.7 for the enzyme-substrate complex. The abnormally high pK of 6.7 indicated the participation of a carboxyl group present in a non-polar environment. The pH dependence of inactivation kinetics of Xyl II with Woodward's reagent K corroborates evidence for the presence of a catalytically important carboxyl residue. The sequence alignment studies of Xyl II, in combination with kinetic and chemical modification data provide strong evidence for the participation of Asp94 in the catalytic function. The Xyl II produced from an alkalophilic source, was stable at pH 10 with a t(1/2) of 24 h. However, the enzyme exhibited pH optimum at near neutral values, which can be explained by the ionization and microenvironment of the active site residues. (C) 2001 Elsevier Science Inc. All rights reserved.