Efecto de iones en la actividad de la α-L-fucosidasa de Thermotoga maritima empleada en la síntesis de oligosacáridos fucosilados
Human milk is the fundamental food for the development and growth of newborns, which is composed of a great variety of nutritional and bioactive compounds, among which are the human milk oligosaccharides (HMOs). This complex family of carbohydrates contains the fucosylated oligosaccharides (FucOs) a...
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| Formato: | tesis de maestría |
| Estado: | Versión publicada |
| Fecha de publicación: | 2024 |
| País: | México |
| Recursos: | Universidad Autónoma Metropolitana |
| Repositorio: | Repositorio Institucional de la UAM Iztapalapa |
| Idioma: | español |
| OAI Identifier: | oai:bindani.izt.uam.mx:8623hz647 |
| Acesso em linha: | https://doi.org/10.24275/uami.8623hz647 |
| Access Level: | acceso abierto |
| Palavra-chave: | info:eu-repo/classification/LEM/Leche materna info:eu-repo/classification/LEM/Oligosaccharides info:eu-repo/classification/LEM/Breast milk info:eu-repo/classification/LEM/Oligosacáridos info:eu-repo/classification/cti/6 |
| Resumo: | Human milk is the fundamental food for the development and growth of newborns, which is composed of a great variety of nutritional and bioactive compounds, among which are the human milk oligosaccharides (HMOs). This complex family of carbohydrates contains the fucosylated oligosaccharides (FucOs) as the group of greatest interest, because they participate in numerous crucial physiological processes important for the health of infants. In the present work, the effect of different ions (Ca2+, Mg2+, Mn2+, and Cu2+) on the activity of α-L-fucosidase from Thermotoga maritima (FUC-Tm) was evaluated, with the aim of increasing the yield of FucOS synthesis through the transfucosylation reaction. In the first instance, the effect of the aforementioned ions on the hydrolytic activity of FUC-Tm was evaluated using CaCl2, MgCl2, and MnCl2 concentrations of between 0.25 and 1.5 M and CuSO4 concentrations of 1 and 10 mM. The concentrations evaluated had an increase in hydrolytic activity of 173% in the presence of CaCl2 at 1 M concentration, 138% with 1 M MgCl2, and only 53% with 0.75 M MnCl2. On the other hand, in the presence of Cu2+ the enzyme activity was inhibited using CuSO4 concentrations of 1 and 10 mM. Since the hydrolytic activity of the enzyme was higher when CaCl2 was used, it was decided to evaluate its effect on the synthesis of FucOS, using p-nitrophenyl-α-L-fucopyranoside (pNPFuc) as donor substrate and lactose as acceptor substrate. A maximum yield of 55.24% was obtained with 0.5 M CaCl2 and water activity (aw) of 0.976, likewise, these conditions represented a FucOS production two times higher than that obtained in the absence of said ion, indicating that the presence of Ca2+, together with the decrease in aw favored transfucosylation. Subsequently, an in silico study on the interaction between Ca2+ and FUCTm was performed through a molecular dynamics (MD) simulation, both in the absence and presence of the ion. No changes were observed at the global level in the enzyme structure, according to the root mean square standard deviation (RMSD) and radius of gyration (Rg) values. However, the results of the root of the squared standard fluctuation (RMSF) revealed that Ca2+, can induce a change in the stability of the enzyme catalytic pocket by decreasing the fluctuation of residues that influence the active site cavity such as Asp190, increasing its accessibility for binding to fucose, which helps to explain the increase in enzyme activity when the ion is present. Finally, with the MD simulation of the enzyme-substrate complex, a difference in the stability of the complex between pNP-Fuc and FUC-Tm in the presence and absence of Ca2+ was observed, reflecting a lower binding energy, suggesting the formation of a more stable enzyme-substrate complex when calcium ion is present, agreeing with results obtained in vitro. |
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