Synthesis of papain nanoparticles by electron beam irradiation - A pathway for controlled enzyme crosslinking

Crosslinked enzyme aggregates comprise more stable and highly concentrated enzymatic preparations of current biotechnological and biomedical relevance. This work reports the development of crosslinked nanosized papain aggregates using electron beam irradiation as an alternative route for controlled enzyme crosslinking. The nanoparticles were synthesized in phosphate buffer using various ethanol concentrations and electron beam irradiation doses. Particle size increase was monitored using dynamic light scattering. The crosslinking formation by means of bityrosine linkages were measured by fluorescence spectra and the enzymatic activity was monitored using Na-Benzoyl-dl-arginine p-nitroanilide hydrochloride as a substrate. The process led to crosslinked papain nanoparticles with controlled sizes ranging from 6 to 11nm depending upon the dose and ethanol concentration. The irradiation atmosphere played an important role in the final bioactivity of the nanoparticles, whereas argon and nitrous oxide saturated systems were more effective than at atmospheric conditions in terms of preserving papain enzymatic activity. Highlighted advantages of the technique include the lack of monomers and crosslinking agents, quick processing with reduced bioactivity changes, and the possibility to be performed inside the final package simultaneously with sterilization.