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

Abstract

Crosslinked enzyme aggregates comprise more stable and highly concentrated enzymatic preparationsof current biotechnological and biomedical relevance. This work reports the development of crosslinkednanosized papain aggregates using electron beam irradiation as an alternative route for controlledenzyme crosslinking. The nanoparticles were synthesized in phosphate buffer using various ethanol con-centrations and electron beam irradiation doses. Particle size increase was monitored using dynamiclight scattering. The crosslinking formation by means of bityrosine linkages were measured by fluores-cence spectra and the enzymatic activity was monitored using Na-Benzoyl-dl-arginine p-nitroanilidehydrochloride as a substrate. The process led to crosslinked papain nanoparticles with controlled sizesranging from 6 to 11 nm depending upon the dose and ethanol concentration. The irradiation atmosphereplayed an important role in the final bioactivity of the nanoparticles, whereas argon and nitrous oxidesaturated systems were more effective than at atmospheric conditions in terms of preserving papain enzy-matic activity. Highlighted advantages of the technique include the lack of monomers and crosslinkingagents, quick processing with reduced bioactivity changes, and the possibility to be performed inside thefinal package simultaneously with sterilization.