Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics
Laccases (E.C. 1.10.3.2) are glycoproteins widely distributed in nature. Their structural conformation includes three copper sites in their catalytic center, which are responsible for facilitating substrate oxidation, leading to the generation of H<sub>2</sub>O instead of H<sub>2&l...
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MDPI AG
2023-10-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/28/21/7263 |
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| author | María P. C. Mora-Gamboa María C. Ferrucho-Calle Leidy D. Ardila-Leal Lina M. Rojas-Ojeda Johan F. Galindo Raúl A. Poutou-Piñales Aura M. Pedroza-Rodríguez Balkys E. Quevedo-Hidalgo |
| author_facet | María P. C. Mora-Gamboa María C. Ferrucho-Calle Leidy D. Ardila-Leal Lina M. Rojas-Ojeda Johan F. Galindo Raúl A. Poutou-Piñales Aura M. Pedroza-Rodríguez Balkys E. Quevedo-Hidalgo |
| author_sort | María P. C. Mora-Gamboa |
| collection | DOAJ |
| description | Laccases (E.C. 1.10.3.2) are glycoproteins widely distributed in nature. Their structural conformation includes three copper sites in their catalytic center, which are responsible for facilitating substrate oxidation, leading to the generation of H<sub>2</sub>O instead of H<sub>2</sub>O<sub>2</sub>. The measurement of laccase activity (UL<sup>−1</sup>) results may vary depending on the type of laccase, buffer, redox mediators, and substrates employed. The aim was to select the best conditions for rGILCC 1 and rPOXA 1B laccases activity assay. After sequential statistical assays, the molecular dynamics proved to support this process, and we aimed to accumulate valuable insights into the potential application of these enzymes for the degradation of novel substrates with negative environmental implications. Citrate buffer treatment T2 (CB T2) (pH 3.0 ± 0.2; λ<sub>420nm</sub>, 2 mM ABTS) had the most favorable results, with 7.315 ± 0.131 UL<sup>−1</sup> for rGILCC 1 and 5291.665 ± 45.83 UL<sup>−1</sup> for rPOXA 1B. The use of citrate buffer increased the enzyme affinity for ABTS since lower <i>K<sub>m</sub></i> values occurred for both enzymes (1.49 × 10<sup>−2</sup> mM for rGILCC 1 and 3.72 × 10<sup>−2</sup> mM for rPOXA 1B) compared to those obtained in acetate buffer (5.36 × 10<sup>−2</sup> mM for rGILCC 1 and 1.72 mM for rPOXA 1B). The molecular dynamics of GILCC 1–ABTS and POXA 1B–ABTS showed stable behavior, with root mean square deviation (RMSD) values not exceeding 2.0 Å. Enzyme activities (rGILCC 1 and rPOXA 1B) and 3D model–ABTS interactions (GILCC 1–ABTS and POXA 1B–ABTS) were under the strong influence of pH, wavelength, ions, and ABTS concentration, supported by computational studies identifying the stabilizing residues and interactions. Integration of the experimental and computational approaches yielded a comprehensive understanding of enzyme–substrate interactions, offering potential applications in environmental substrate treatments. |
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| publishDate | 2023-10-01 |
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| spelling | doaj.art-75b37e94433b4412a7f7912b8bb7d9f02023-11-10T15:08:25ZengMDPI AGMolecules1420-30492023-10-012821726310.3390/molecules28217263Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular DynamicsMaría P. C. Mora-Gamboa0María C. Ferrucho-Calle1Leidy D. Ardila-Leal2Lina M. Rojas-Ojeda3Johan F. Galindo4Raúl A. Poutou-Piñales5Aura M. Pedroza-Rodríguez6Balkys E. Quevedo-Hidalgo7Laboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaLaboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaLaboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaDepartamento de Química, Universidad Nacional de Colombia, Bogotá 111321, ColombiaDepartamento de Química, Universidad Nacional de Colombia, Bogotá 111321, ColombiaLaboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaLaboratorio de Microbiología Ambiental y Suelos, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaLaboratorio de Biotecnología Aplicada, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaLaccases (E.C. 1.10.3.2) are glycoproteins widely distributed in nature. Their structural conformation includes three copper sites in their catalytic center, which are responsible for facilitating substrate oxidation, leading to the generation of H<sub>2</sub>O instead of H<sub>2</sub>O<sub>2</sub>. The measurement of laccase activity (UL<sup>−1</sup>) results may vary depending on the type of laccase, buffer, redox mediators, and substrates employed. The aim was to select the best conditions for rGILCC 1 and rPOXA 1B laccases activity assay. After sequential statistical assays, the molecular dynamics proved to support this process, and we aimed to accumulate valuable insights into the potential application of these enzymes for the degradation of novel substrates with negative environmental implications. Citrate buffer treatment T2 (CB T2) (pH 3.0 ± 0.2; λ<sub>420nm</sub>, 2 mM ABTS) had the most favorable results, with 7.315 ± 0.131 UL<sup>−1</sup> for rGILCC 1 and 5291.665 ± 45.83 UL<sup>−1</sup> for rPOXA 1B. The use of citrate buffer increased the enzyme affinity for ABTS since lower <i>K<sub>m</sub></i> values occurred for both enzymes (1.49 × 10<sup>−2</sup> mM for rGILCC 1 and 3.72 × 10<sup>−2</sup> mM for rPOXA 1B) compared to those obtained in acetate buffer (5.36 × 10<sup>−2</sup> mM for rGILCC 1 and 1.72 mM for rPOXA 1B). The molecular dynamics of GILCC 1–ABTS and POXA 1B–ABTS showed stable behavior, with root mean square deviation (RMSD) values not exceeding 2.0 Å. Enzyme activities (rGILCC 1 and rPOXA 1B) and 3D model–ABTS interactions (GILCC 1–ABTS and POXA 1B–ABTS) were under the strong influence of pH, wavelength, ions, and ABTS concentration, supported by computational studies identifying the stabilizing residues and interactions. Integration of the experimental and computational approaches yielded a comprehensive understanding of enzyme–substrate interactions, offering potential applications in environmental substrate treatments.https://www.mdpi.com/1420-3049/28/21/7263ABTSacetate and citrate bufferlaccasesmolecular docking and dynamics |
| spellingShingle | María P. C. Mora-Gamboa María C. Ferrucho-Calle Leidy D. Ardila-Leal Lina M. Rojas-Ojeda Johan F. Galindo Raúl A. Poutou-Piñales Aura M. Pedroza-Rodríguez Balkys E. Quevedo-Hidalgo Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics Molecules ABTS acetate and citrate buffer laccases molecular docking and dynamics |
| title | Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics |
| title_full | Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics |
| title_fullStr | Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics |
| title_full_unstemmed | Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics |
| title_short | Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics |
| title_sort | statistical improvement of rgilcc 1 and rpoxa 1b laccases activity assay conditions supported by molecular dynamics |
| topic | ABTS acetate and citrate buffer laccases molecular docking and dynamics |
| url | https://www.mdpi.com/1420-3049/28/21/7263 |
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