Study On The Immobilization Of Lactase On Magnetic Microspheres

Magnetic polymer microsphere is a novel functional material that has been developed recently and it is a kind of organic-inorganic compound nanoparticle, which has magnetism and can be separated and oriented easily under applied magnetic field. As the carrier of enzyme, cell and drug, it can be widely applied in many fields, such as bio-engineering, cytology, biomedical science and so on. Magnetic chitosan microspheres have attracted considerable attention as they display very important significance and value in theoretical and application aspects. For example, they can be applied as carrier of biomacromolecules and cells because of functional -NH₂ and -OH of chitosan encapsulating magnetic particles. Whilst, owing to the magnetism of the microspheres, separation efficiency is increased in the presence of outer magnetic field, which leads to achieve a cheap and convenient separation in many aspects. In this paper, lactase immobilization was prepared using magnetic chitosan microspheres as the carrier materials. Immobilized lactase can be repetitive-used. Not only the consumption of enzyme was reduced, but also automation management becomes possible.This thesis mainly includes three aspects as follows:Fe₃O₄ magnetic particles was synthesized by the chemical co-precipitation, using FeCl₂, FeCl₃ and NaOH. The average diameter, the dispersion stability and the relative size of magnetic of Fe₃O₄ magnetic particles were tested and characterized. The synthetic conditions of Fe₃O₄ magnetic particles were determined through orthogonal design and the optimum experimental conditions as follows: nFe²⁺/nFe³⁺ was 2:1, pH was11, curing temperature was 90℃and the amount of PEG was 40ml. Under this condition, the average diameter of Fe₃O₄ particles was 78nm, the dispersion stability was the best and the relative size of magnetic was the strongest . It could be seen from scanning electron microscope that Fe₃O₄ crystal particles were nanometer.Magnetic chitosan microspheres (M-CS) were prepared by reverse phase suspension process using Fe₃O₄ as core, paraffin as dispersed medium, Span-80 as emulsifier, and glutaraldehyde as cross-linking agent and the shape, the average diameter, the dispersion stability and the relative size of magnetic were characterized. The crosslinking time, reaction temperature, the concentration of chitosan, mFe₃O₄ / mCS, the amount of glutaraldehyde and the amount of dispersed medium were discussed, which effected the performance of M-CS. M-CS had strong magnetism under external magnetic field and good suspended stability under the natural condition. M-CS was spheroidal and Fe₃O₄ magnetic particles was parceled in microspheres. Obvious core-shell structure could be seen in the particles and its surface was smooth. The diameter was between 1μm-15μm, which was good for dispersion and magnetic separation.In this paper, lactase immobilization was prepared using magnetic chitosan microspheres(M-CS) as the carrier materials and the amount of lactase., the characteristic of M-CS, the effects of pH value, the effects of pH value and immobilizing time on the activity in the whole immobilize process were studied. As compared with free enzyme, the immobilized lactase had wider applicable range of pH; The optimum temperature of free enzyme was 40℃, but that for immobilized enzyme was improved to 45℃. Therefore, the temperature tolerance of immobilized enzyme had boosted up. The Km value of immobilized lactase was lower than that of free lactase, which meaned that the immobilized lactase had higher affinity towards the substrate; After 5 cycles of operation, the activity of immobilized enzyme remained over 65% initial activity; In addition, immobilization improved the stability of lactase.