The Study And Application Of Enzyme Biochromatography For The Screening Of Aminopeptidase N Inhibitors

Aminopeptidase N (APN) is closely related to the growth of primary and secondary tumors and the creation of new blood vessel, even is contributed to the proliferation and differentiation of tumor cells. The invasion and metastasis of tumors can be effectively blocked by the inhibition of APN activity. APN has become to be an attractive target of research on anticancer. Enzyme biochromatography is a biochromatography, which takes enzymes for chromatographic stationary phase, integrates the separation and screening of active components and can directly screen specific components from mixtures. This paper studied on the preparation of new APN stationary phase and the establishment of APN biochromatography with APN as target, providing a new screening model for anticancer drug research. This paper contains four chapters, The main research work and achievements are summarized as follows:1. APN was prepared by tissue extraction with porcine kidney as raw materials and purified by salt out and column chromatography. Then the biological chemistry and related properties of APN were studied. The results showed that APN was well separated and purified and the specific activity was 10.08 U/mg, which can satisfy the requirement of experiment. The optimal temperature of APN was 50℃, and it was relatively stable between 50～55℃. The optimal pH was 7.8, which was weakly alkaline. The IC50 of APN inhibitor Bestatin was 10.35μM. The Km of APN was 0.55 mM, with L-leucine-p-nitroanilide as substrate.2. Monolithic silica columns were prepared, clad and modified before used as the carrier. The conditions of the immobilization of APN and the properties of immobilized APN were studied. The results showed that the optimum conditions to immobilize APN were:pH 7.8, the quantity of enzyme was 40 ml and the reaction time was 6 h. After immobilization, the optimal pH of immobilized APN was also7.8, the stability limits of pH was expanded. The optimal temperature of immobilized APN was 55℃. The stability of immobilized APN was increased.3. The biochromatography screening model of APN was established, and was validated by Bestatin.35 new compounds were screened by this screening model. The results showed that in this screening model, dose-response relationship of Bestatin to APN activity was observed, the inhibition rate of Bestatin to APN activity was depend on the concentration of Bestatin, proved the screening model was feasible. The screening results provided effective information for further compounds structure modification.