A β-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65°C and pH 5.6–6.3. The stereochemistry of the hydrolysis of p-nitrophenyl β-D-xylopyranoside was followed by 1H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.
