Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i>
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 precipit...
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MDPI AG
2021-01-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/2/451 |
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| author | Gabriela Romero Lellys M. Contreras Carolina Aguirre Jeff Wilkesman Josefa María Clemente-Jiménez Felipe Rodríguez-Vico Francisco Javier Las Heras-Vázquez |
| author_facet | Gabriela Romero Lellys M. Contreras Carolina Aguirre Jeff Wilkesman Josefa María Clemente-Jiménez Felipe Rodríguez-Vico Francisco Javier Las Heras-Vázquez |
| author_sort | Gabriela Romero |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T04:34:52Z |
| format | Article |
| id | doaj.art-5cd4b9a4223d4b9c8e0827b9802b2f4d |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T04:34:52Z |
| publishDate | 2021-01-01 |
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| record_format | Article |
| series | Molecules |
| spelling | doaj.art-5cd4b9a4223d4b9c8e0827b9802b2f4d2023-12-03T13:29:49ZengMDPI AGMolecules1420-30492021-01-0126245110.3390/molecules26020451Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i>Gabriela Romero0Lellys M. Contreras1Carolina Aguirre2Jeff Wilkesman3Josefa María Clemente-Jiménez4Felipe Rodríguez-Vico5Francisco Javier Las Heras-Vázquez6Center for Environmental, Biological and Chemical Research, Experimental Faculty of Sciences and Technology, University of Carabobo, Valencia 2001, VenezuelaCenter for Environmental, Biological and Chemical Research, Experimental Faculty of Sciences and Technology, University of Carabobo, Valencia 2001, VenezuelaCentro 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, ChileCenter for Environmental, Biological and Chemical Research, Experimental Faculty of Sciences and Technology, University of Carabobo, Valencia 2001, VenezuelaDepartment of Chemistry and Physics, University of Almeria, Building CITE I, Carretera de Sacramento s/n, La Cañada de San Urbano, 04120 Almería, SpainDepartment of Chemistry and Physics, University of Almeria, Building CITE I, Carretera de Sacramento s/n, La Cañada de San Urbano, 04120 Almería, SpainDepartment of Chemistry and Physics, University of Almeria, Building CITE I, Carretera de Sacramento s/n, La Cañada de San Urbano, 04120 Almería, SpainCross-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.https://www.mdpi.com/1420-3049/26/2/451β-xylosidasethermostabilityCLEAs<i>G. stearothermophilus</i>xylanase |
| spellingShingle | Gabriela Romero Lellys M. Contreras Carolina Aguirre Jeff Wilkesman Josefa María Clemente-Jiménez Felipe Rodríguez-Vico Francisco Javier Las Heras-Vázquez Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i> Molecules β-xylosidase thermostability CLEAs <i>G. stearothermophilus</i> xylanase |
| title | Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i> |
| title_full | Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i> |
| title_fullStr | Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i> |
| title_full_unstemmed | Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i> |
| title_short | Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from <i>G. stearothermophilus</i> |
| title_sort | characterization of cross linked enzyme aggregates of the y509e mutant of a glycoside hydrolase family 52 β xylosidase from i g stearothermophilus i |
| topic | β-xylosidase thermostability CLEAs <i>G. stearothermophilus</i> xylanase |
| url | https://www.mdpi.com/1420-3049/26/2/451 |
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