Optimized immobilization of peracetic acid producing recombinant acetyl xylan esterase on chitosan coated-Fe₃O₄ magnetic nanoparticles

Recombinant acetyl xylan esterase (rAXE) from Aspergillus ficcum, which mediated the production of peracetic acid (PAA), was covalently immobilized with magnetic Fe₃O₄-chitosan nanoparticles (Fe₃O₄-CSN) using glutaraldehyde. Fe₃O₄-CSN were prepared by co-precipitation of Fe²⁺ and Fe³⁺ ions in ammonia solution followed by coating of chitosan with sodium tripolyphosphate (TPP), and were characterized by FESEM, SEM, FTIR and XRD. The immobilization rAXE on Fe₃O₄-CSN was optimized using response surface methodology (RSM) by examining immobilization cross-linking time, enzyme concentration, and glutaraldehyde (GA) concentration. Based on the experimental values the predicted variables for the maximum immobilization of rAXE in terms of specific activity (0.042 U) were found to be 13.65 μg of rAXE protein and 0.265% of GA concentrations, where the optimum cross-linking time was 11.33 h. The immobilized rAXE onto Fe₃O₄-CSN nanoparticles shows better stability at thermal and pH ranges than soluble free rAXE. The immobilized rAXE was stable for around 90% of activity in the aqueous phase, whereas it retains only 60% of its activity in the semi-aqueous phase after 10 cycles of reuse.