Preparation Of BSA-Coated Cerium Oxide Artificial Enzyme And Its Catalytic Activity Study

Natural enzyme has been widely applied in food and biomedicine or other fields which can improve the quality of the food, include flavor regulation effect and adjust its color, the practical value of natural enzyme is very important and cannot be ignored. However natural enzyme exists many defects such as the thermal instability, the poor stability, source raw material is limited, expensive and so on, those defects led to its applications are limited widely, then the introduction of the nano artificial enzymes can eliminate these limitation and expand its range of application. In this paper, with one-step synthesis the nanomaterials of BSA-CeO2, then study and evaluation its intrinsic simulation enzyme activity. This dissertation has carried out the following research work:1. Results show that with bovine serum albumin (BSA) as protective agent, BSA-CeO2 nanocomposites were prepared by a one-step mixing method at room temperature. The as-prepared materials were then characterized by UV visible spectra, transmission electron microscope (TEM), Fourier transform infrared spectrum (FT-IR), powder X-ray Diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and thermogravimetric analysis (TGA). These conventional methods clearly confirmed that the formation of BSA-CeO2 nanocomposites, the results of TEM show that the average particle size of BSA-CeO2 nanoparticles is 2.29 nm. The XRD analysis revealed fluorite cubic structure for BSA-CeO2. In the condition of neutral solutions, by zeta potential, confirming the presence of a negative charged polymer coating for BSA-coated CeO2 nanoparticles (ζ=-25 mV). XPS data indicates the presence of a mixed valence state in BSA-CeO2, giving the corresponding binding energy peaks for Ce3+ and Ce4+, suggesting that the BSA coating does not affect its mixed valence state, and the Ce3+ concentration is approximately 44%.2. The steady-state kinetic assays of the oxidase-like activity of BSA-CeO2 nanomaterials show the value of Km is 0.0544 mM, and compared with acting on the same substrate of HRP has lower Km value, indicating that BSA-CeO2 have stronger affinity to the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). Unlike nature enzymes, the BSA-CeO2 nanomaterials have strong robustness. Incubated by different temperature, the oxidase-like activity of nanomaterials showed good stability, even the temperature is as high as 95 ℃, still kept more than 60% activity. Incubated at a range of values of pH (1-12), and showed that the catalytic oxidation of TMB in the presence of BSA-CeO2 nanomaterials was much faster in acidic solutions than in neutral or basic solutions. The possible reasons as following:(1) with the decrease of the solution pH, enhancing the capacity of the oxidation of Ce4+, so as to make the oxidase-like activity stronger in acidic solutions. (2) the influence of acidity on the substrate TMB and led to the TMB amine salt produced which is the final oxidation product of TMB. (3) the catalytic oxidate activity is related to the surface charges of BSA-CeO2 nanomaterials. Research and analysis results reveal that BSA-CeO2 nanomaterials possess effectively intrinsic oxidase-like activity. Also, we can conclude that the oxidase-like activity of BSA-CeO2 nanomaterials has a broad range of the applicable scope at varies temperatures and pH values compared with nature enzymes.3. Based on the principle of xanthine oxidase coupling reaction system and WST-1, confirming the SOD mimetic activity of BSA-CeO2 nanomaterials. When the nanomaterials concentration is 5 μM, the corresponding enzymatic activity is 11.975 U/mL. The thermal studies have shown that the change of inhibition ratio slightly and have little fluctuation at temperature from 4 ℃ to 65 ℃, almost remain at around 50%; When the temperature at 75 ℃, the inhibition ratio began to decrease, at 95 ℃, there was still retained inhibition ratio of about 25%. These results showed that under the treatment of high temperature, the SOD mimetic activity of BSA-CeO2 nanomaterials were still maintained and had the effectively inhibition ratio. Acid-base stability study results revealed that there is not too much affect on the SOD mimetic activity of BSA-CeO2 nanomaterials at different pH. In summary, our results indicate that BSA-CeO2 nanomaterials possess intrinsic superoxide dismutase (SOD) mimetic activity. And the SOD mimetic activity of BSA-CeO2 nanomaterials has good thermal stability and effective enzyme-like catalysis over a wide range of temperatures and pH values.