Utilização de beta-ciclodextrina para aumento da estabilidade cinética e termodinâmica da beta-tripsina

Abstract

Cyclodextrins are cyclic oligosaccharides that have a polar exterior and a nonpolar interior cavity. They are known to have the ability to promote stability, increase solubility, carry and encapsulate substances such as enzymes. Therefore, this work aimed to investigate whether the addition of one of the cyclodextrins, beta-cyclodextrin, in a system containing beta-trypsin causes an increase in the enzymatic, structural and thermodynamic stability of the enzyme. For this, enzymatic activity assays, ultraviolet scanning spectrophotometry, fluorescence scanning spectroscopy and microcalorimetry were carried out. The beta-trypsin:beta-cyclodextrin 1:30 (mass/mass) system with a 10-minute interaction time showed the best cost-benefit ratio (enzymatic activity per value). This system also showed the best storage results (3 hours at 4oC or 25oC). Ultraviolet scanning spectrophotometry was not sensitive to detect global changes between 1:30 and 1:0 ratios. In the fluorescence scanning spectroscopy, the 1:30 sample showed a maximum wavelength 28 nm shorter than the other samples, indicating that the presence of beta-cyclodextrin promoted an increase in the structural stability of the enzyme and in the microcalorimeter test, it was observed an increase of 2 K in the temperature at which 50% of the molecules are denatured, in addition to an increase in the parameters of entropy variation, calorimetric enthalpy variation and in the Van't Hoff enthalpy variation of sample 1:30 compared to sample 1:0, indicating that the presence of beta-cyclodextrin in the protein system promotes an increase in the number of beta-trypsin molecules that are in the native state, making the system thermodynamically more stable. Furthermore, the cooperativity indices and the ratio of calorimetric enthalpy changes to the Van't Hoff enthalpy changes were not altered. Therefore, the structural, thermodynamic and enzymatic parameters of the protein system were improved due to the addition of betacyclodextrin in a 1:30 ratio, indicating that beta-cyclodextrin increased the stability of beta-trypsin.