RESUMEN
Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis ß-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin-protein interaction. Circular dichroism shows that saponin changes the ß-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of ß-galactosidase with the artificial substrate o-nitrophenyl-ß-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.
Asunto(s)
Kluyveromyces/enzimología , Saponinas/farmacología , beta-Galactosidasa/efectos de los fármacos , Calorimetría , Dicroismo Circular , Cinética , Nitrofenilgalactósidos/metabolismo , Corteza de la Planta/química , Estructura Secundaria de Proteína , Quillaja/química , Espectrometría de Fluorescencia , beta-Galactosidasa/metabolismoRESUMEN
Tannins are compounds with antinutrient properties that hinder food digestibility, prejudicing human and animal nutrition. This work aimed to evaluate the negative effects of tannic acid on Kluyveromyces lactis ß-galactosidase catalytic activity and correlate these changes with the protein structure. ß-Galactosidase activity decreased in the presence of tannins, which caused changes to the structure of the enzyme, as demonstrated by circular dichroism. It was verified that tannin binds to the protein by a static mechanism. Additionally, isothermal titration calorimetry suggested that tannic acid modified the molecular interaction between ß-galactosidase and o-nitrophenyl-ß-d-galactoside, reducing their affinity and prejudicing the protein activity. This study helps to understand the effects of tannins on the ß-galactosidase structure and how they are related to the enzyme catalytic activity. The alterations in the conformation and activity of the enzyme should be taken into consideration when dairy products are consumed with tannin-rich food.
