Immobilization of Euphorbia tirucalli peroxidase onto chitosan-cobalt oxide magnetic nanoparticles and optimization using response surface methodology

Euphorbia tirucalli peroxidase (ETP) was immobilized on chitosan beads having magnetic properties for the ease of separation and increasing the reusability of ETP for cost effective assay conditions. The present work reports immobilization of ETP on polymeric support chitosan-cobalt oxide beads subsequently activated with 0.05% cynuric chloride. The magnetic immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM). The immobilized ETP can be reused up to 10 cycles with retention of more than 60% activity. The optimum pH was shifted from 6.0 to 5.5 for soluble ETP to immobilized ETP and optimum temperature from 50 °C and 55 °C for the immobilized ETP. Based on response surface methodology, the optimal immobilization conditions obtained were: enzyme concentration, 2 mg/286 mg beads; optimal pH, 4.93; temperature, 28.88; cynuric chloride concentration, 0.17%; reaction time, 14.4 h, which resulted 74.51% maximum immobilization. The enzyme magnetic nanoparticles could be separated magnetically for easy reuse. Immobilization of ETP onto the magnetic nanoparticles could be useful for biotechnological applications and bioassay due to its reusability and improved stability.