Preparation Of Chitosan Magnetic Microspheres And Study On Magnetic Microspheres Immobilized Trypsin

The polymer magnetic microspheres are a new type of function material with wide applications in the fields of biochemical, cytological and biomedical engineering that have been developed over the past two decades. Chitosan magnetic 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 addition, Chitosan magnetic microspheres can be widely used in fine chemical industry, environment monitoring, immobilization of enzyme, targeted medicine, immunoassay and cell isolation.This paper mainly includes two aspects as follows:(1) Chitosan magnetic microspheres were prepared by reverse phase suspension process using Fe/C as core, paraffin as dispersed medium, Span-80 as emulsifier, and epichlorohydrin as cross-linking agent and characterized by TEM, AFM, Zeta potential-diameter analyzer and IR. Whilst, the amount of active functions on the surface of microspheres were also measured. For the big microspheres the contents of -NH₂ and -OH were 63.9 umol/g (3.85×10¹⁶ -NH₂/g) and 168 umol/g (10.1×10¹⁶ -OH/g), respectively, and for the small microspheres the contents of amidocyanogen and hydroxy were 13.0μmol/g (7.8×10¹⁵-NH₂/g) and 25.2μmol/g (15.2×10¹⁵ -OH/g), respectively. We had investigated the adsorption capacities of chitosan magnetic microspheres, which were activated by glutaraldehyde and modified by Cibacron Blue F3G-A, respectively, for bovine serum albumin. The results indicated that the adsorbances of large and small size chitosan magnetic microspheres activated by glutaraldehyde were 24.6 and 46.3mg/g, respectively, and the capacities of the microspheres modified by Cibacron Blue F3G-A were 56.7 and 66.2mg/g, respectively, corresponding to the concentration of bovine serum albumin was 1.50mg/mL. (2) Immobilized enzymes share many merits in comparison with active enzymes. There have been lots of reports on immobilized enzyme with chitosan as carrier. In this paper, Immobilized trypsin was prepared using chitosan magnetic microspheres as the support and glutaraldehyde as the cross-linking agent and the effects of pH value, glutaraldhyde concentration and the amoun't of trypain on the activity in the whole immobilize process were studied. Indicating that big diameter immobilized trypsin display the optimum activity at pH 6.5-7.5, temperature 80℃and K_m values 7.08 mg/mL and small diameter immobilized trypsin display the optimum activity at pH 6.5～7.5, temperature 80℃and K_m values 6.49 mg/mL and in comparison with free trypsin, the immobilized trypsin have better stability in thermal, pH storage and ethyl alcohol, which indicate immobilized trypsin have better properties and can be applied in more areas than free trypsin, the large size magnetic immobilized trypsin has better stability and the higher activity recovery yield than the small one and the large size immoblized magnetic enzyme can be applied in industrialization.