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Horseradish peroxidase (horseradish peroxidase) is extracted from a horseradish peroxidase (E, C, 1.11.1.7), a glycoprotein of a molecular weight of 44000D, sugar content was 18%. Its molecule contains a heme prosthetic group, the porphyrin ring protoporphyrin center ions Fe (II). Readily available raw materials, low commodity prices, the role of a wide range of substrates, and become one of the most widely used enzyme peroxidase. Used as a catalyst in organic synthesis, the catalytic mechanism for the peroxide cycle process, aniline and its derivatives, phenols, and so has a high catalytic efficiency. In the enzyme immunoassay, the HRP having luminescent substrate, thus established chemiluminescence immunoassay been widely applied in clinical. In recent years, HRP in organic synthesis, biosensors, food analysis, biomedical detection, fluorescence analysis, sewage treatment and other aspects of the more prominent. In particular, rapid progress in the the immunodiagnostics surgery, biosensors, lignin degradation and industrial \Subject to horseradish peroxidase substrate, p-phenylenediamine and peroxide hydrogen, 25% dioxane solution, pH, temperature, substrate concentration , the preliminary study of the general characteristics of the horseradish peroxidase catalyzed oxidation reactions, and determination of the kinetic parameters of a part of the enzymatic reaction, and the reaction product characterized. The results showed that the HRP poor thermal stability, pH adapt a narrow range, the catalytic efficiency of the organic phase, and enzyme inactivation undesirable characteristics. Therefore, in order to improve the organic phase enzyme catalytic efficiency, water phase enzyme thermal stability as a starting point, the combination of the two technologies of molecular imprinting and biological imprinting reach horseradish peroxidase blot. Substrate phenylenediamine induced enzyme conformation, to obtain a natural-blot enzyme (NIP), and added the acylating agent preparation of maleic anhydride acetylation blot enzyme (DIP), freeze-dried after adding the crosslinking agent and initiator for UV polymerization , ligand After the polymerization was eluted with dioxane, and finally dried to obtain the desired crosslinking blot enzyme polymer (CLIPS), namely the aqueous phase bio-blot enzyme. In order to investigate the three enzymes (natural blot enzyme, the acetylation imprinting enzyme, crosslinked imprinting enzymatic nature of the polymer), the research design is as follows: (1) examine the imprinted enzyme catalytic activity in the organic phase, and the pH values ??were investigated. temperature, water content, and the apparent Km of four factors blot enzyme catalytic activity. Organic phase with free enzyme at the same time for comparison; (2) Investigation of acetylation blot enzyme catalytic activity in the aqueous phase, and the pH values ??were investigated, and the catalytic activity of the enzyme in the aqueous phase three factors on temperatures, apparent Km acetylation blot impact, while the free enzyme, with the water phase comparison; (3) Discussion crosslinked imprinting the catalytic activity of the enzymes of the polymer in the aqueous phase, respectively, the effects of pH, temperature, the apparent Km of the three factors crosslinking blot enzyme polymer In the catalytic activity of the aqueous phase, while the aqueous phase free enzyme for comparison. The results show that the three enzymes pH stable range and optimum pH remain unchanged; blot activity is improved about 10 times, an apparent Km value is reduced by about 30-fold; enzyme thermal stability significantly increased after crosslinking, 80 ℃ The enzyme activity is still very high. However, the catalytic efficiency is still low. Shows that bio-imprinting technology features in the modified enzyme, it is effective to improve the organic phase catalytic efficiency of the enzymatic reaction. Crosslinked imprinting the enzyme polymer catalytic efficiency is still low, is expected to improve from the following biological blot enzyme catalytic efficiency: imprinting template choose to be appropriate; may reduce the particle size of the imprinted particles to obtain soluble The catalyst; enzyme catalytic mechanism of research needs to be further in-depth.
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