Covalent immobilization of trypsin onto modified magnetite nanoparticles and its application for casein digestion.

The immobilization method consists of the production magnetite nanoparticles (Fe3O4) by solvothermal treatment of FeCl3 and sodium acetate (NaAc) in the presence of ethylene glycol. Subsequently, the surface of magnetite nanoparticles was modified with a well-known polyphenol tannic acid. Trypsin was covalently immobilized on the tannic acid modified magnetite nanoparticles after exposing the modified nanoparticles to pH 9.4. Then, tryptic digestion of casein by free and immobilized trypsin was carried out for 13h and 1h, respectively. TGA curves, FTIR spectra, and magnetization curves demonstrated the decent amount of trypsin immobilization without compromising the enzyme activity. Digestion efficiency of casein was investigated using liquid chromatography-mass spectrometry (LC-MS/MS) technique. LC-MS chromatograms confirmed the efficient digestion of casein by immobilized trypsin compared to free trypsin owing to prevention of autohydrolysis. Also, the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed the satisfactory digestion of casein by immobilized trypsin.