Study On The Property And Immobilization Of Superoxide Dismutase

Immobilized enzyme technique is an important component of modern bioengineering and biotechnology. The application of immobilized enzymes has been widely used in the fields of industry, medicine, analytical and separation techniques, chemical synthesis, and so on. Superoxide dismutase(SOD) was a type of enzyme that has metal ion and was found by American scientists Mccord and Fridovich in 1969 from ox red blood cells, which was also the only enzyme whose substrate is free radical. It takes an important part in keep the balance of body's free radicals creation and clearance for he can clear O2.ˉin the body. This study prepare the immobilized SOD ,tests its activity and study on its physico-chemical property systematically.In this thesis, the studies on the preparation, properties and application of immobilized superoxide dismutase have been investigated.The immobilization of SOD and its activityIn this study, the chitin was treated to be chitosan, which was mixed with glutaraldehyde and amino-group combined with the aldehyde-group becoming a activated carrier. The SOD was mixed and agitated with this carrier for hour and was given sucking filtration, cleaning and put into fridge for storage. The pyrogallol autoxidation method was used to test the activity of the immobilized enzyme with the result of 333U.g-1 and the recovery was 86.32%.The comparison of two methods of activity tests The traditional method of pyrogallol autoxidation was to use the O2.ˉgradually produced in the autoxidation and the SOD can clear the O2.ˉ,thus, the SOD can be measured by the inhibition of the sample. In this study, advanced method of pyrogallol autoxidation that use vitamin C as a terminating agent was also used, it has no differences in statistics on contrary with the traditional one but also has its rare dominance that several samples can be tested at the same time.The chemical and physical properties of the immobilized enzymeThe storage stability, pH stability and temp stability was studied. The half-life of the enzyme was 43.8 day showed by testing activity once a day. The very best of the pH condition was 6 by testing series of pHs, while the best temperature was 45℃in a series of different temperatures. The relative activity of the immobilized enzyme was 0.7012 after used for the first time and 0.5172 for the second time, setting original activity was 1.The dynamics of the immobilized enzymeIt was the first time in country that study on the dynamics of the immobilized enzyme and testing of the Km of the immobilized SOD and original SOD solution. The Km was 0.18mmol/L of immobilized enzyme and was little higher than 0.16mmol/L that the Km of SOD solution. This was the signal that the immobilization had a little bad influence on the affinity of the enzyme and the substrate.