Enhanced activity and stability of papain by covalent immobilization on porous magnetic nanoparticles

Papain enzyme was successfully immobilized by covalent bonding onto biocompatible Fe₃O₄/SF nanoparticles, which were prepared with the soft template of silk fibroin (SF). The optimized immobilization condition is pH6.0, hydrolysis time of 60min, and an enzyme/support ratio of 10.0mg/g. Compared with free papain, the immobilized papain exhibits a high effective activity, broader working pH and temperature. This immobilized papain can be separated from the solution by the external magnetic field for cyclic utilization, and 70% of initial activity was retained after eight consecutive operations while completely loss of proteolytic activity for the free papain. Furthermore, the immobilized papain maintained 85% of their initial activity after being stored for 28days. Kinetic parameters, maximum reaction rate (V_max) and Michaelis constant (K_m) of immobilized papain, were determined as 4.95mg/l·min and 0.23mg/ml, larger than its free counterpart. All the results above indicated that the immobilized papain onto magnetic Fe₃O₄/SF nanoparticles would have potential industrial and medical applications.