Immobilization and Characterization of Trypsin on TiO2 Nanoparticles Activated with Crosslinkers

The immobilization of trypsin (TRP) on the amine-functionalized silica coated TiO2 nanoparticles (ASTiNPs) with/without different crosslinkers (1,4-phenylene diisothiocyanate (PDC), 1,3-phenylene diisothiocyanate (MDC), glutaraldehyde (GA) has been studied. The ASTiNPs and modified ASTiNPs with the crosslinkers were characterized by FTIR and TEM. When considered the specific activity of immobilized TRP on ASTiNPs, GA-bound TRP showed the higher specific activity. Loading capacity was higher when PDC used as crosslinker. Optimum concentration of the crosslinkers for the TRP immobilization was determined as 20.8 µM of PDC, 5.2 µM of MDC and 1.5 v/v of GA. The direct-bound TRP showed 5% of its initial activity after the four cycles while the GA-bound TRP sustained 7% of its initial activity after the seven cycles and MDC and PDC-bound TRP sustained 7% and 11% of its initial activity after the ten cycles, respectively. The digestion of the Cyt C with immobilized TRP was evaluated by LC-MS/MS analysis. The immobilized TRP on ASTiNPs with crosslinkers showed the higher digestion efficiency of Cyt C when compared to the immobilized TRP on ASTiNPs without crosslinker. Consequently, the PDC-bound TRP on the ASTiNPs gave the better result of digestion efficiency, loading capacity, catalytic activity and reusability than the others.