| Summary: | Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 β-xylosidase from <i>Geobacillus stearothermophilus</i> with dual activity of β-xylosidase and xylanase (XynB2<sup>Y509E</sup>) 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 °C, and pH 8.5. Under these (most effective) conditions, XynB2<sup>Y509E</sup>-CLEAs retained 92.3% of their original β-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 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the <i>K</i><sub>m</sub> value of XynB2<sup>Y509E</sup>-CLEAs obtained was slightly higher than that of free XynB2<sup>Y509E</sup> (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.
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