Improved preparation of immobilized trypsin on superparamagnetic nanoparticles decorated with metal ions

The superparamagnetic carboxymethyl chitosan nanoparticles (Fe3O4 (PEG + CM-CTS) NPs) which prepared by chemical coprecipitating were treated with Cu(II) and Zn(II) ions solutions to obtain the novel immobilized metal affinity magnetic nanoparticles (IMANs), short as IMAN @ Cu(II) NPs and IMAN @ Zn(II) NPs. Immobilization of trypsin onto the obtained IMANs, as a result of metal affinity interaction was confirmed by Fourier Transform Infrared (FTIR) spectroscopy. FTIR data demonstrated that the IMAN @ Cu(II) NPs and IMAN @ Zn(II) NPs were capable of preventing the trypsin unfolding. Due to the large specific surface area and excellent dispersibility, the adsorption equilibrium of trypsin onto the nanoparticles was achieved quickly within 30 min, and adsorption equilibrium of trypsin onto the IMANs fitted well with the Langmuir model. The results of kinetic parameters (Michaelis constant, Km) showed that, both immobilized trypsin, compared with free trypsin, have higher affinity to the substrate, furthermore, they could be used for fast and efficient Bovine Serum Albumin (BSA) digestion under very facile processes, as well as the greatly reduced digestion time (from 12 h to 15 min) which confirmed by MALDI-TOF MS analysis. Both immobilized trypsin exhibited a sound stability. Considering that the studied IMANs possess the advantages of high efficiency, cost-effectiveness and lack of negative effect on trypsin bioactivity, such IMANs may hold potential applications in immobilization and stabilization of trypsin.