| Enzyme immobilization is the process that a free enzyme is anchored on an insoluble carrier. The key to the process is to reach a high immobilization yield, catalytic activity and easy recovery by appropriate design of the carriers and choose the favorable approach methods. This work chose angiotensin converting enzyme (ACE) as target molecules and focused on design and preparation of a series of functional materials with highly active multiple-points. The enzyme were immobilized on these solid supports by covalent attachment and cross-linking methods. And then, the reactor was prepared by filling the anchored enzyme powders in a plastic volume and finally coupled to the HPLC. Thus, an on-line system was established for screening the enzyme inhibitor.There are four parts in this thesis.1. PrefaceThe preface was introduced from three aspects including summary of immobilized enzyme, immobilization methods and preparation of the functional carriers. In this section, the work focuses on the discussion and comparison of the primary preparation methods and several immobilized enzyme reactors.2. Preparation of the magnetic Fe3O4/chitosan composite microspheres and research of the immobilized enzymeMagnetic chitosan composite microspheres as immobilized enzyme affinity support were synthesized and characterized by FT-IR spectra, XRD, SEM and TEM. The results indicated that the carrier was nearly spherical in shape and owned core-shell structure, and the properties of the aminated chitosan derivation were improved significantly as compared with chitosan. Then, angiotensin converting enzyme (ACE) was immobilized on the nanocomposite microspheres by covalent attachment, and the extent of immobilization and enzyme activities were measured. When the glutaraldehyde was 2.5%, and 200μL of EDC/NHS (v:v/1:1), the immobilization yield (IY) reached up to 78.25% which improved nearly 30% as comparison with that in literature, and the relative analyst activity retained more than 70% over 30 d. Finally, kinetic parameters were also determined as 454.5 and 416.7 mmol-1 for Km and 31.36,46.67 mmol-1 for Vmax in the case of free and immobilized enzymes, respectively.3. The studies on preparation and comparison of other functional supports and molecular imprinted polymer of products.In order to solve the conflicts between the immobilization yield and relative activities, functional materials with other groups were prepared in this chapter. Specifically, three advanced materials, amine-functionalized magnetic nanospheres, L-cys functionalized magnetic nanospheres and carboxylated multi-walled carbon nanotubes (MWCNT), were synthesized. And these functional supports were characterized by FT-IR, XRD, TEM, EDS, Raman and size analysis. Additionally, ACE was covalently attached on these carriers. By comparing the immobilization yield (IY) and relative catalytic activity, MWCNT was selected as an excellent carrier, the IY of the MWCNT support reached 68.2% and its relative activity kept 80.7% after 30 d under the optimum conditions. In addition, to increase the signal intensity, the molecular imprinted polymer (MIP) of products was prepared and characterized. And its properties were also determined which indicated that the adsorption content of 200mg MIP of HA was 0.39μM, and the eluent volume was 3.5mL, and the RSD was 1.0%(n=7).4. On-line analysis of the immobilized enzyme reactor combined with HPLCAccording to the design, the immobilized enzyme reactor was combined with HPLC for on-line screening of the inhibitors. Under the optimum chromatographic conditions, the immobilized enzyme reactor was proved to be reusable. Herein, Captopril as an inhibitor was injected to the reactor, and the result showed that R is 60%, and RSD is 13%(n=7). However, R decreased to 62%, and the RSD was 4.7%(n=7) when the reactor was combined with MIP of HA. And IC50 was 2.1×10-8M which falls in the range as reported in literatures. |
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