Soybean Hulls Peroxidase Extraction, Separation, Purification And Immobilization Study

The yield of soybean hull coats was arising year by year at present in our country,but its utilization was inadequate,so it was needed to develop and use of soybean hull coats reasonable to increase benefits of economic and social. Soybean hull peroxidase (SHP)was high activity and it was used broadly in oxidation,so there was significance to study of SHP.The thesis has done a systematic study on the extraction,isolation,purification and immobilization of peroxidase from the soybean hull coats.The mainly results as follows:(1) Soybean hull peroxidase was extracted by two methods which were using PBS buffer and ultrasonic wave,on the basis of single factor experiment, then compared of those methods using response surface analysis to get the optimal condition of the extraction.The optimal conditions of the PBS extraction were as follows:the temperature 38℃,the extraction time 6h,the ratio of solution and soybean hull at 20mL/g,PBS concentration 0.02mol/L,3 times.The enzyme activity was 2608.7U and enzyme specific activity was 89.03U/mg based on the above optimal conditions from 1g soybean hull coats.The optimal conditions of the ultrasonic wave extraction were as follows:ultrasonic power 70 W, the temperature 38℃, the extraction time 41min, the ratio of solution and soybean hull at 20mL/g,PBS concentration 0.02mol/L,3 times. The enzyme activity was 3409.3U and enzyme specific activity was 89.96U/mg based on the above optimal conditions from 1g soybean hull coats.The conclusion showed that the ultrasonic wave extraction was time-saving and high activity.(2)Using ammonium sulfate fractionated precipitation, acetone fractionated precipitation, zinc sulfate impurition and membrane filtration to isolate and purificat the crude SHP that was purified by 29.01,the value of Rz was 1.89.(3)The SHP was immobilized by using alumina adsorption-sodium alginate entrapping-glutaraldehyde crosslinking and macroporous resins adsorption-glutaraldehyde crosslinking, then studied the thermodynamics and dynamics for the adsorption process.The optimal conditions of the alumina adsorption-sodium alginate entrapping-glutaraldehyde crosslinking were as follows:pure enzyme 10mg, alumina 1.5g,the adsorption time lh, sodium alginate concentration 4%, calcium chloride concentration 0.02 mol/L, glutaraldehyde concentration 0.3%, crosslinking time 30min. The yield of immobilized was 77.3% based on the above optimal conditions.The optimal conditions of the macroporous resins adsorption-glutaraldehyde crosslinking were as follows:resin of LX-42C 1g(dry weight), the rate of oscillation 125r/min, the temperature 35℃, the adsorption time 4h, enzyme content given 30mg, glutaraldehyde concentration 0.4%, crosslinking time 1h. The yield of immobilized was 89.47% based on the above optimal conditions.The study of the thermodynamics and dynamics for adsorption process showed that was fit for Langmuir Model, the adsorption process was endothermic and spontaneous reaction, the rate of adsorption had well shown in agreement with quasisecondary kinetin equation.By comparing the characters of free SHP and immobilized SHP to find that the immobilized SHP was used many times and more stable in temperature, pH and storage.