Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process
Penicillin G acylase (PGA) from <i>Escherichia coli</i> was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH...
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MDPI AG
2022-11-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/27/21/7587 |
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| author | Thays N. da Rocha Roberto Morellon-Sterlling Javier Rocha-Martin Juan M. Bolivar Luciana R. B. Gonçalves Roberto Fernandez-Lafuente |
| author_facet | Thays N. da Rocha Roberto Morellon-Sterlling Javier Rocha-Martin Juan M. Bolivar Luciana R. B. Gonçalves Roberto Fernandez-Lafuente |
| author_sort | Thays N. da Rocha |
| collection | DOAJ |
| description | Penicillin G acylase (PGA) from <i>Escherichia coli</i> was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support. |
| first_indexed | 2024-03-09T18:47:42Z |
| format | Article |
| id | doaj.art-23c5769a185d412c9398a99f02eb6143 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T18:47:42Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-23c5769a185d412c9398a99f02eb61432023-11-24T06:06:42ZengMDPI AGMolecules1420-30492022-11-012721758710.3390/molecules27217587Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization ProcessThays N. da Rocha0Roberto Morellon-Sterlling1Javier Rocha-Martin2Juan M. Bolivar3Luciana R. B. Gonçalves4Roberto Fernandez-Lafuente5Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, 28049 Madrid, SpainDepartamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, 28049 Madrid, SpainDepartment of Biochemistry and Molecular Biology, Faculty of Biology, Complutense University of Madrid, José Antonio Novais 12, 28040 Madrid, SpainFQPIMA Group, Chemical and Materials Engineering Department, Faculty of Chemical Sciences, Complutense University of Madrid, Complutense Ave., 28040 Madrid, SpainChemical Engineering Department, Campus do Pici, Federal University of Ceará, Bloco 709, Fortaleza CEP 60440-900, CE, BrazilDepartamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, 28049 Madrid, SpainPenicillin G acylase (PGA) from <i>Escherichia coli</i> was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support.https://www.mdpi.com/1420-3049/27/21/7587enzyme immobilization/stabilizationheterofunctional supportsmultipoint covalent attachmentimmobilization optimizationmulti-step immobilizationvinyl sulfone supports |
| spellingShingle | Thays N. da Rocha Roberto Morellon-Sterlling Javier Rocha-Martin Juan M. Bolivar Luciana R. B. Gonçalves Roberto Fernandez-Lafuente Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process Molecules enzyme immobilization/stabilization heterofunctional supports multipoint covalent attachment immobilization optimization multi-step immobilization vinyl sulfone supports |
| title | Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process |
| title_full | Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process |
| title_fullStr | Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process |
| title_full_unstemmed | Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process |
| title_short | Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process |
| title_sort | immobilization of penicillin g acylase on vinyl sulfone agarose an unexpected effect of the ionic strength on the performance of the immobilization process |
| topic | enzyme immobilization/stabilization heterofunctional supports multipoint covalent attachment immobilization optimization multi-step immobilization vinyl sulfone supports |
| url | https://www.mdpi.com/1420-3049/27/21/7587 |
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