Imobilização de tripsina suína em nanopartículas magnéticas: atividade e estabilidade enzimática

Abstract

Obtaining a nanobiocatalyst based on the direct interaction between superparamagnetic iron oxide nanoparticles (SPIONs) and porcine trypsin was investigated in the present work. Specifically, the physical adsorption of magnetic nanoparticles with trypsin was studied from the modulation of surface charges by controlling the pH of both the enzyme and the supporting nanomaterial. Nanoparticle- enzyme interaction was evaluated by experimental measurements of fluorescence and theoretical modeling. The results obtained showed that the immobilized material, under different pH conditions, showed 66.2, 67.1, and 75.4% of enzyme adsorption to the support at pH 4.0, 7.0, and 8.0, respectively. The enzyme activity recovered in each case was 31.84, 27.56, and 32.28%, respectively, in relation to the initial free trypsin activity. The catalytic efficiency was measured through the reaction involving the conversion of the substrate N-α-Benzoyl-DL-arginine-4-nitroaniline into the product p-nitroaniline monitored by UV-Vis spectrophotometry at a wavelength of 410 nm. The desorption studies showed by the analyzed techniques that after 5 washing cycles it was not possible to determine the desorption rate due to the values obtained being lower than the fluorescence detection limits (0.4 mg.mL -1 ). The reuse of immobilized Trypsin was tested over several cycles in catalytic processes using BapNA as substrate. The catalysis process occurred with an efficiency of approximately 88% until the second cycle, and the immobilized enzyme retained an efficiency of around 40% until the fourth cycle. In addition, the trypsin-nanoparticle interaction was also evaluated by fluorescence spectroscopy and the values of the thermodynamic parameters ∆�� �� = −31.5 kJ mol–1, ∆�� �� = 15.31 kJ mol–1, and ��∆�� �� = 32.99 kJ mol–1��–1 were found, which is consistent with the occurrence of predominantly ionic interactions. Immobilization of porcine trypsin on the surface of SPIONs was also computationally evaluated. The potential energy profile for the adsorption of porcine trypsin on the surface of the material was calculated and showed that the enzyme protonated at pH 8.0 can be more strongly adsorbed on the surface of the nanoparticles. It was also possible to verify that the enzyme adsorption rate increased significantly at pH 8.0, and at this same pH, the adsorption energy profile is more favorable and stable at smaller distances from the surface, indicating that a greater amount of enzyme can be adsorbed over SPIONs.