Immobilization of trypsin on Fe3O4 nanoparticles which have been functionalized with polyphenols and application of digestion

In this study, by synthesizing of iron oxide magnetic nanoparticles were functionalized with polyphenols and immobilization of trypsin enzyme and by examining digestion of proteins after that aimed to lead in the work to be done. This study was carried out in three separate parts. At the first part, Fe3O4 magnetic nanoparticles were synthesized by the precipitation method and tannic acid was functionalized on the synthesized nanoparticles. Trypsin enzyme was immobilized on modified nanoparticles. pH, temperature, storage stability and thermal stability of the substrate work (kinetic parameters) of the free and immobilized trypsin, and also reuse of immobilized enzyme was studied. Electrostatic interaction of the enzyme at different pH was explained by zeta potential analysis. The structures of synthesized Fe3O4 and its derivative were illuminated by FTIR, XRD, TGA, SEM and VSM analysis. The efficiency on free and immobilized trypsin enzyme of hydrolysis of Bovine Serum Albumin (BSA) and casein protein were studied and the results were also analyzed by LC-MS / MS. The second part is different from the first part, gallic acid was functionalized on Fe3O4 magnetic nanoparticles which synthesized by the precipitation method. All other studies and characterizations were carried out in parallel with the first part. At the third part, Fe3O4 magnetic nanoparticles were synthesized with solvotermal method and tannin was functionalized on the synthesized nanoparticles. Trypsin enzyme was immobilized on modified nanoparticles. The efficiency on free and immobilized trypsin enzyme of digestion of Bovine Serum Albumin (BSA) and casein protein were studied and the results were also evaluated by SDS-PAGE. Also MALDI-TOF MS analysis was used to determine the amount and number of peptides formed after digestion of Bovine Serum Albumin (BSA) of immobilized trypsin enzyme. Trypsin, which specifically cleaves peptidic bonds on C-terminal group of lysine or arginine, is a pancreatic serine endoprotease and is used mostly for protein digestion and peptide mapping.