Facile Synthesis and Evaluation of Size-tunable Immobilized Laccase-mediator Microreactor

A series of immobilized laccase-mediator microreactor (LMMR) was prepared in a one-pot process by simultaneously immobilizing Trametes versicolor laccase and acetylacetone (AA) into size-tunable chitosan copper-polyacrylamide hydrogel beads. The polymerization was induced by a laccase-AA-persulfate ternary initiating system and was finished within the chitosan beads at room temperature. The preparation conditions for the LMMR were optimized by an orthogonal array design. The method developed in this work, for the first time, realized the co-immobilization of laccase and mediator in microreactors of tunable size and mechanic strength. Experimental results from scanning electron microscopy and nitrogen adsorption-desorption analysis indicate that the resulting LMMR had a core-shell structure. Chitosan copper served as the mechanical shell, whereas polyacrylamide hydrogel was the core of three-dimensional network. Throughout the hydrogel beads, there were abundant mesopores of size in the range of 2 similar to 8 nm. The microreactor beads could endure a 20 N pressure in the axial direction, ensuring the structural integrity in the practical application in wastewater. The loading efficiency of laccase in the microreactor reached up to 93.5%. As compared with the free laccase, the LMMR showed better storage stability and higher tolerance to changes in solution pH and temperature. In the enzymatic conversion of malachite green (MG), benefited from the mediation effect of the immobilized AA, the LMMR still worked after 17 cycling runs (12 h for each cycle), which was 3-fold longer than that of a free laccase-mediator system. The successful recovery of both laccase and mediator is promising to reduce the cost for the application of laccase in wastewater treatment and might be helpful to cut down the secondary pollution from free laccase mediators. These results demonstrate that this novel one-pot synthesis was a useful strategy in the immobilization of laccase. The LMMR has a great potential in large-scale application for dyeing effluent treatment.