Preparation And Characterization Of Hybrid Magnetic Cross-linked Enzyme Aggregates

The magnetic nanomaterials had drawn attention to public as a kind of green materialin recent years. The magnetic nanomaterials had many advantages as the carrier of enzymeimmobilization because of its good properties, such as large specific surface area andmangnetic responsiveness. The functionalized magnetic nanoparticles have been used asthe carrier for enzyme immobilization in previous reports. However, there were littlepapers concerned in nonfunctionalized magnetic nanoparticles. In this study, a hybridmagnetic cross-linked enzyme aggregates was preparped successfully by combining cross-linkedenzyme aggregates with nonfunctionalized magnetite nanoparticles. Phenylanine ammonialyase from Rhodotorula glutinis and lipase from Candida sp. were made as modelenzymes, respectively. Hybrid magnetic cross-linked enzyme aggregates of phenylanineammonialyase (HM-PAL-CLEAs) and hybrid magnetic cross-linked enzyme aggregates ofCandida sp. lipase (HM-CSL-CLEAs) were prepared. Moreover, the characteristics ofHM-PAL-CLEAs and HM-CSL-CLEAs were also investigated.1) Preparation and characterization of Fe3O4magnetic nanoparticlesFe3O4magnetic nanoparticles was prepared by coprecipitation, and thecharacterization was examined by using scanning electron microscope (SEM), X-raydiffraction (XRD), fourier transform infrared spectroscopy (FTIR), and vibrating samplemagnetometer (VSM). The results showed that Fe3O4magnetic nanoparticles were of niceround shape and homogeneous, and diameter was about30nm. Magnetic saturationinduction of Fe3O4magnetic nanoparticles was70.865emu/g. The XRD and FTIRdiagrams indicated that the magnetic nanoparticles were Fe3O4crystals.2) Preparation and enzymatic characteristics of HM-PAL-CLEAsThe preparing conditions of HM-PAL-CLEAs were optimized by response surfacemethodology. Enzymatic characteristics of HM-PAL-CLEAs were investigated. Theresults showed that the optimal conditions of HM-PAL-CLEAs were nanoparticlesconcentration at33.45mg/ml, enzyme concentration at2.10mg/ml, saturation ofammonium sulfate concentration at55%, and glutaraldehyde concentration at0.05%.Under optimized conditions, the maximal activity recovery and immobilized efficiency ofHM-PAL-CLEAs were respectively44%and96%. Compare to free enzyme, stability andreusability of HM-PAL-CLEAs were significantly enhanced. 3) Preparation and enzymatic characteristics of HM-CSL-CLEAsThe preparing conditions of HM-CSL-CLEAs were optimized by Surface Responsemethodology and one-variable-at-a-time method. Enzymatic characteristics ofHM-CSL-CLEAs were investigated The optimum conditions were nanoparticlesconcentration at35mg/ml, enzyme concentration at1.82mg/ml, ammonium sulfateconcentration at2.41M, and glutaraldehyde concentration at0.21%. In the best level, theactivity recovery and immobilized efficiency of HM-CSL-CLEAs were respectively40%and62%, respectively. Compare to free enzyme, thermal stability, storage stability andreusability of HM-CSL-CLEAs were significantly enhanced.