Purification, Characterization And Immobilization Of Soybean Hull Peroxidase

One of the bean shells comprehensive utilizations is to extract Soybean Hull Peroxidase in view of the readly available raw materials and low cost, and the product could be used widely.Firstly, the enzyme activity was determined by the colorimetric method. Then the factors to affect the activity were analysised using the single factor experiments, namely, temperature, pH, and the concentrations of guaiacol, hydrogen peroxide and enzyme. Followed by optimization of the factors using design-expert software, results showed the optimal conditions to detemine the activity of Soybean Hull Peroxidase were at 60℃, pH=6.0, guaiacol and hydrogen peroxide dosage is 24μL and 19μL, respectively. Then, bean shells were smashed and added phosphate buffer at the ratio of 1:6, the mixture placed in the oscillating water bath for extration at 30℃and 150r/min for 24h, followed by filtration with the eight-gauzes, the supernatant obtained after centrifugation at 4700r/min for 30min as the crude enzyme solution stored at 4℃. Adding 10% ammonium sulfate powder in certain amount of enzyme solution, dissolved completely, the mixture stored at 4℃overnight. The second day, to collect supernatant after centrifugation at 4000r/min for 15min, follwed by adding ammonium sulfate powder to 90%, dissolved completely, the mixture stored at 4℃overnight. Next day, precipitation obtained from centrifugation at 4000r/min for 15min was dialysed using 0.02M phosphate buffer solution for 48h. Enzyme powder was obtained using the method of freeze-dried for 48h. Soybean hull peroxidase was extracted and purified by Sephadex G-75 column chromatography, followed by collecting the component with high activity. Based on the SDS-PAGE gel electrophoresis, the molecular weight of soybean hull peroxidase is about 40KD. By analysis of the enzymatic properties enzyme, the optimum temperature and pH of soybean hull peroxidase is 30℃and 6.0, respectively. In addition the activity could be stable at 20-60℃, and pH4-7. By Lineweave Burk method using double-reciprocal plots, the results of the apparent Km values for soybean hull peroxidase on guaiacol and hydrogen peroxide were 1.94 and 0.41, respectively. Therefore, soybean hull peroxidase could maintain activity at a wide range of temperature and pH. The soybean hull peroxidase adsorpted by Fe3O4, and then embedded by gelatin, the final cross-linked processed with glutaraldehyde, was used to analyse immobilization of the enzyme. Then the enzyme activity was measured and enzyme immobilization efficiency was calculated. The optimization immobilization program was that Fe3O4 dosage is 1g, gelatin concentration is 10%, crosslinking time is 40min, and the enzyme immobilization efficiency embedded under these conditions is 86%. In the research, the soybean hull peroxidase was extracted, purified and analysed the enzymatic properties and immobilized. The program was feasible and optimal, not only for soybean hull was salvaged, but also laid a good foundation for further research.