Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 beta-xylosidase from Geobacillus stearothermophilus with dual activity of beta-xylosidase and xylanase (XynB2(Y509E)) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 degrees C, and pH 8.5. Under these (most effective) conditions, XynB2(Y509E)-CLEAs retained 92.3% of their original beta-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 degrees C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the K-m value of XynB2(Y509E)-CLEAs obtained was slightly higher than that of free XynB2(Y509E) (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.