| Natural peroxidase has some disadvantages such as complex separation process,not easy to preserve,unstable and easy to denature.Various mimic enzymes with high peroxidase catalytic activity and good stability have been used to replace natural peroxidase,which is widely used in food,medicine and disease detection.The reverse micelle system can avoid direct contact between the enzyme and the surrounding organic solvent and protect the catalytic activity and stability of the enzyme.In recent years,several studies have shown that appropriate microwave conditions can enhance enzyme activity.In this thesis,a methanobactin-copper?Mb-Cu?complex with mimic enzyme activity of peroxidase was dissolved in reverse micelle system.Microwave assisted enzymatic catalysis was used to simulate Mb-Cu mimic enzymatic reaction in order to find a new method to enhance Mb-Cu mimic enzymatic activity.The main research contents are as follows:Methanoxidin?Mb?produced by Methylosinus trichosporium OB3b was combined with copper coordination to synthesize the Mb-Cu mimic enzyme with peroxidase activity.The synthesis of the Mb-Cu mimic enzyme was determined by UV-visible spectrum analysis.The cationic CTAB,anionic AOT and non-ionic Brij35 surfactants were used to prepare the reverse micelles system.With rhodamine B as a probe,the results showed that critical micelle concentration of CTAB was 3?10-44 mol/L,and the critical micelle concentration of Brij35 was2?10-44 mol/L,critical micelle concentration of AOT was 3?10-44 mol/L.For CTAB,AOT and Brij35,the average conductivity of the three reverse micelles increased by increasing the saturated solubilizing water content and no turning point was found.This indicated that the reverse micelle systems were stable.The study on the particle size of reverse micelles showed that the reverse micelles were not affected by time once they had been formed and stabilized.Cationic type reverse micelles of CTAB,anion type reverse micelles of Brij35 and non-ion type reverse micelles of AOT were prepared and three reverse micelles containing Mb-Cu were used for catalytic reaction.Ultraviolet-vis spectrum analysis showed that CTAB,Brij35 and AOT reverse micelles could all simulate the catalytic reaction of the mimic enzyme Mb-Cu.The optimum concentration and water content of each surfactant in the reverse micelle system were studied.The pH,KCl concentration and H2O2 concentration of the reaction system under the optimum concentration and water ratio were optimized.After optimization,it was found that the three kinds of reverse micelles had the same optimum conditions but different enzyme activities.The optimum conditions was that the pH 8,the KCl concentration 0.03mol/L and the H2O2concentration 9×10-44 mol/L.The mimic peroxidase activities of Mb-Cu in the reverse micelles were 3.593×10-44 mol/L·s?1.356×10-44 mol/L·s?2.176×10-44 mol/L·s,respectively.Cationic CTAB reverse micelles have the best catalytic performance for Mb-Cu complexes.Finally,the Microwave-assisted mimic peroxidase catalyzed reaction by Mb-Cu in CTAB reverse micelle was studied.Ultraviolet-vis spectrum analysis showed that microwave could further accelerate the catalytic reaction in Mb-Cu containing reverse micelles.The microwave power,microwave time and microwave temperature of the reaction system were studied.By using the response surface analysis,combined with practical operations,the microwave temperature was adjusted to 56?,the microwave power was 500 W,and the reaction time was2 min.Under these conditions,the average peroxidase activity of the Mb-Cu was 5.267?10-33 mol/L·s,which was about 15 times larger than that of the mimic enzyme without microwave assistance.It is proved that microwave can promote the mimic enzyme Mb-Cu catalysis in reverse micelle system. |
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