Desenvolvimento de um novo suporte heterofuncional de Fe3O4@DTPMP-GLU para imobilização da lipase Eversa® Transform 2.0

Abstract

This study developed and evaluated a new heterofunctional support based on magnetic magnetite (Fe3O4) nanoparticles functionalized with diethylenetriaminepenta (methylene phosphonic acid) (DTPMP) through the sonochemical method, followed by activation with glutaraldehyde (GLU) for the immobilization of Eversa® Transform 2.0 lipase (ET2). The optimization of the immobilization process was carried out using the Taguchi method, determining the ideal conditions such as reaction time (6 h), ionic strength (5 mM), enzyme load (5 mg of ET2 per gram of support) and temperature (40 °C). The support was characterized structurally and magnetically by X-ray diffraction (XRD), vibrating sample magnetometer (VSM), Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TGA), scanning electron microscopy (SEM) and X-ray fluorescence (XRF), confirming the functionalization of the support and the immobilization of the enzyme. Stability and catalytic performance were evaluated by desorption, reuse, support load capacity, and organic solvent resistance tests. The activity of immobilized ET2 decreased to 54.08% after ten reuse cycles. The immobilization yield was 60.53% ± 1.37, with a relative activity of 92.37% ± 2.26. Among the solvents heptane, hexane, cyclohexane, tetrahydrofuran (THF), and acetonitrile, activity was improved only in the presence of acetonitrile and tetrahydrofuran. Although the support did not promote increased activity at different pHs and temperatures compared to the free enzyme, the results demonstrate its potential as an efficient platform for lipase immobilization, conferring stability and reusability of immobilized ET2. The molecular coupling between the support and ET2 was evaluated through molecular docking, which demonstrated that GLU acts as a functional link between ET2 and the Fe3O4@DTPMP-GLU support, favoring the efficient immobilization of the enzyme.