Ersearch On Preparation Of Magnetic Nano-carriers And Its Immobilization Of Cholesterol Oxidase

In recent decades,with the improvement of people's living lever,some diseases caused by excessive cholesterol intake have seriously harmed human health.Controlling and reducing the intake of cholesterol in diets is crucial for lowering cholesterol levels and preventing cardiovascular diseases.The cholesterol oxidase(COD)method degrades the cholesterol in foods without affecting the flavor of the food.And it does not interfere with the metabolism of human body with only a little side effect.It has become one of the most effective and simple methods for reducing cholesterol in foods.As a kind of catalyst,cholesterol oxidase has the characteristics of high catalytic efficiency,mild reaction conditions,and high specificity.However,it also has problems such as being easily deactivated and difficult to recover.Enzyme immobilization technology can solve this problem very well,and the enzyme can enhance its stability and reusability after being immobilized.In a wide variety of carriers,magnetic carriers have become an important immobilization carrier due to their unique advantages.The dissertation prepared two kinds of magnetic immobilized enzyme carriers,and carried out research on magnetic nanoparticles immobilized cholesterol oxidase.The main contents include:1.Magnetic Fe3O4@SiO2 and Fe3O4@PDA magnetic carriers were prepared using the prepared Fe3O4 magnetic nanoparticles as the core,and the immobilization of cholesterol oxidase was realized.After a series of characterization analyses,the results showed that the two immobilized cholesterol oxidases had a particle size of about 50 nm,a relatively uniform particle size,and a relatively large specific surface area.2.The optimal conditions for the immobilization of cholesterol oxidase on two kinds of carriers were studied.The results showed that the optimal conditions for Fe3O4@SiO2@COD were as follows:5 mg/mL of cholesterol oxidase,immobilization temperature 30?,pH=7.5,immobilization reaction time 14 h.The optimal conditions of Fe3O4@PDA@COD were as follows:2 mg/mL of cholesterol oxidase,temperature 30?,pH=8.5,immobilization reaction for 3 h.3.By analyzing the enzymatic properties of the three enzymes,the optimal reaction temperatures of free enzyme,Fe3O4@SiO2@COD and Fe3O4@PDA@COD were 35 ?,50? and 45?,respectively.The optimum reaction pH was 7.5,7.5,and 8.5,respectively.After two immobilized enzymes were stored in a refrigerator at 4? for one week,Fe3O4@SiO2@COD retained 74%of the enzyme activity,and Fe3O4@PDA@COD retained 71%of the enzyme activity.While the free enzyme only maintained 15%of the enzyme activity;Fe3O4@SiO2@COD retained 56%of the relative enzymatic activity after 7 times of repeated use of the two immobilized enzymes,and Fe3O4@PDA@COD could maintain a relative enzyme activity of 44%.By Km analysis,it was found that the affinity of the enzyme to the substrate was increased after the enzyme was immobilized.4.Through the cholesterol egg yolk cholesterol degradation experiment,the results showed that the degradation rate of Fe3O4@SiO2@COD on cholesterol of egg yolk was 57.31%,and the degradation rate of Fe3O4@PDA@COD was 56.95%.Both of the immobilized enzymes have a good effect of degrading egg yolk cholesterol and are suitable for industrial production applications.