Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i>
Gabriela Romero,
Lellys M. Contreras,
Carolina Aguirre,
Jeff Wilkesman,
Josefa María Clemente-Jiménez,
Felipe Rodríguez-Vico,
Francisco Javier Las Heras-Vázquez
Affiliations
Gabriela Romero
Center for Environmental, Biological and Chemical Research, Experimental Faculty of Sciences and Technology, University of Carabobo, Valencia 2001, Venezuela
Lellys M. Contreras
Center for Environmental, Biological and Chemical Research, Experimental Faculty of Sciences and Technology, University of Carabobo, Valencia 2001, Venezuela
Carolina Aguirre
Centro de Investigación en Biodiversidad y Ambientes Sustentables (CIBAS), Department of Environmental Chemistry, Faculty of Sciences, Universidad Católica de la Santísima Concepción, Casilla 297, Concepción 4090541, Chile
Jeff Wilkesman
Center for Environmental, Biological and Chemical Research, Experimental Faculty of Sciences and Technology, University of Carabobo, Valencia 2001, Venezuela
Josefa María Clemente-Jiménez
Department of Chemistry and Physics, University of Almeria, Building CITE I, Carretera de Sacramento s/n, La Cañada de San Urbano, 04120 Almería, Spain
Felipe Rodríguez-Vico
Department of Chemistry and Physics, University of Almeria, Building CITE I, Carretera de Sacramento s/n, La Cañada de San Urbano, 04120 Almería, Spain
Francisco Javier Las Heras-Vázquez
Department of Chemistry and Physics, University of Almeria, Building CITE I, Carretera de Sacramento s/n, La Cañada de San Urbano, 04120 Almería, Spain
Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 β-xylosidase from Geobacillus stearothermophilus with dual activity of β-xylosidase and xylanase (XynB2Y509E) 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, XynB2Y509E-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 Km value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.
