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Preparation of Dextran Magnetic Microspheres and Study on Their Immobilizing Papain

Author: LiuYao
Tutor: HanDeYan；ZhangHaiLi
School: Central China Normal University
Course: Analytical Chemistry
Keywords: Dextran magnetic microspheres (DMMS) Papain Active radicals Immobilization Stability
CLC: O629.8
Type: Master's thesis
Year: 2007
Downloads: 342
Quote: 5
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Abstract


The natural polymer magnetic microspheres are a new type of functional material with wide applications in biochemical industry, cytology, and biomedical engineering that have been developed over the past two decades. The binding of magnetic particles to biological molecules is due to the special magnetic properties and biological activities of these materials. Dextran magnetic microspheres are recently developed as a new biocompatible magnetic complex, which have high stability and low toxicity for living body. Also owing to the abundant hydroxyl groups on surface, dextran magnetic microspheres can be chosen as immobilizing carrier. Common method, which introduced active functional groups at random positions in dextran, is to cleave the sugar rings with periodates to form magnetic polyaldehyde-dextran, and then conjugated to biomolecules such as proteins, antibodies, peptid, DNA or RNA. As a new type of funtional material, dextran magnetic microspheres have a broad application perspective in the fields of enzyme fixation, immunity testing, cell separation and targeted drug, etc.（1） Dextran magnetic microspheres are prepared through the reverse phase suspension and embedment techniques, with iron nanoparticles as magnetic nuclei and epichlorohydrin （ECH） as cross linker. The main physical properties of size, morphology and microstructer are characterized by means of transmission electron microscopy（TEM）, atomic force microscopy（AFM）, laser grain size analyzer and infrared absorption spectrum（IR）. Also the number of the active radicals on the surface of dextran magnetic microspheres and their absorption capacity for protein are measured. For the two kinds of microspheres with diameter of 615nm and 193nm, the number of surface active radicals are 9.23μmol/g (5.56×1015/mg) and 11.32μmol/g (6.81×l015/mg), the adsorbance for protein are 36.02mg/g (3.19×1014/mg) and 53.62mg/g (4.75×1014/mg) correspondly. The results suggest that the number of surface active radicals satisfy the requirements for detection, and dextran magnetic microspheres are suitable for cell separation, immunity analysis and detection.（2） To further study the properties of dextran magnetic microspheres, papain is chosen as a model enzyme, which is mostly immobilized on the surface of activated magnetic carriers by covalent binding. The influence on the binding protein capacity with varying immobilized conditions is invesgated, and enzymic properties of immobilized papain are studied compared with soluble enzyme, for which casein is chosen as a substrate. For the two kinds of microspheres with diameter of 615nm and 193nm, the results indicate that protein binding capacity are 21.3mg/g and 23.5mg/g, Km of immobilized papain are 0.16mg/mL and 0.12mg/mL, the optimum temperature, thermal stability and storags stabilities of immobilized papain are higher than soluble enzyme, immobilization process does not lead to a great loss of enzyme activity, immobilization enhance papain’s ability to resist protein denaturants and endow it with more higher stability. All these suggest that immobilized enzyme doesn’t change its molecule structure and preserve its catalytic activity, immobilized papain exhibit better environmental adaptability and reusability.All above these indicate dextran magnetic microspheres are a good kind of immobilizing carrier, and have great practical signality and applied value for cell separation, immunity analysis and detection, etc.

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CLC: > Mathematical sciences and chemical > Chemistry > Organic Chemistry > Natural compounds > Enzymes, hormones,
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