Preparation And Performance Optimization Of Magnetic Fe₃O₄-chitosan Nanoparticles For Cellulase Immobilization

In this study,the preparation of nano magnetic immobilized cellulase and its performance optimization were studied.The parameters in the immobilization of cellulase onto Fe₃O₄-chitosan were optimized based on the principle of Box-Behnken design,one of the response surface methodology,and the physical and chemical properties of immobilized cellulase which was synthesized under the optimal immobilization condition were studied.The variations of immobilized cellulase in hydrolysis process were explored.Finally,the research experiments of repeated cellulase hydrolysis and cold storage were carried out.The detailed results of the whole research work could be followed:By chemical co-precipitation method,the Fe²⁺and Fe³⁺were mixed in a ratio of 1:2 at a certain temperature with lye as a precipitating agent.The nano Fe₃O₄ was obtained from co-precipitation of Fe²⁺and Fe³⁺.The nanoparticles were characterized by XRD,TEM and VSM.It was showed that nanoparticles had regular lattice structured,and the particle size was in the range of 12-30 nm,with good superparamagnetic at room temperature.Chitosan-coated magnetic nanoparticles were gained by adopting physical methods.With FT-IR,XRD,TEM and VSM characterization,it was confirmed that chitosan coated successfully onto the particles and it did not change the lattice structure of Fe₃O₄.Particle size did not change significantly,and the superparamagnetic was not affected.The immobilized process of cellulase was optimized by Box-Behnken design.The optimum conditions for immobilization process were enzyme dosage of 9.29,temperature of 30.6 ℃,pH value of 5.4 and glutaraldehyde concentration of 0.14%（w/v）with a maximum predicted standard enzyme recycle rate of 68.3%.The standard enzyme recycle rate in the validation experiment was 69.2%,which was in accordance with the predicted value.Immobilized cellulase was used in hydrolysis experiments under optimal condition.Compared with free cellulase,it was found that the optimum pH of immobilized cellulase was 5.0,and the optimum temperature was 50 ℃.The hydrolysis bath experiments of immobilized cellulase were carried out with 100 mL CMC solution at 50 ℃,48 h.Immobilized cellulase could be reused 5 times,while the highest yield was close to 70%,average yield of55%.Immobilized cellulase reduced the cost of enzymes,improved economic efficiency.