Novel superparamagnetic sanoparticles for trypsin immobilization and the application for efficient proteolysis

• Magnetic carboxymethyl chitosan nanoparticles (short as Fe3O4 (PEG + CM-CTS)).
• Lysozyme would adopt a more compact conformation state after immobilization.
• Immobilized trypsin (short as IT).
• IT could be used for fast and efficient BSA digestion under very facile processes.
• IT exhibited 76.3% of the initial activity after re-uses for six times.

Immobilization of trypsin onto the superparamagnetic carboxymethyl chitosan (Fe3O4 (PEG + CM-CTS)) nanoparticles was studied. FTIR and fluorescence spectroscopy data demonstrated that the Fe3O4 (PEG + CM-CTS) nanoparticles 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. The results of kinetic parameters (Michaelis constant, Km) with regards to the free trypsin (FT) and immobilized trypsin (IT) were 23.1 and 24.1 mg/mL separately, implying that IT has less affinity to the BAEE used as the substrate. However, the MALDI-TOF MS analysis indicated that, IT could be used for fast and efficient Bovine Serum Albumin (BSA) digestion under very facile processes, thanks to the easy manipulation of the magnetic nanoparticles (MNs), as well as the greatly reduced digestion time (from 12 h to 15 min). IT exhibited a sound stability after re-uses for six times, with 76.3% of the initial activity thereof still retained, thus making it more attractive in the application fields. These results are expected to open up a great potential use of such Fe3O4 (PEG + CM-CTS) nanoparticles as a superior nanosupport for trypsin immobilization.