Separation And Purification Cu/Zn-SOD From Newborn Bovine Blood And Prepatation Of （HEP/CHI）₅-SOD Using Layer-by-Layer Self-assembly Technique

Super oxide dismutase is an important antioxidant enzymes to remove superoxide anion free radical in living body, and has good antioxidation function. However SOD is easy to be inactivatjed and have a short half life period. In order to improve its stability, we usually adopt the method of physical adsorption/embedding and chemical modification. This study uses thermal denaturation extraction method to extract Cu/Zn-SOD from newborn bovine blood, and uses layer by layer self-assembly technology, heparin and chitosan as material to modifiy the SOD which was separated and purified from newborn bovine blood. The stability of (HEP/CHI) 5-SOD was also studied. The results are as follows.(1)Single factor analysis method was used in the preparation of layer by layer self assembly microcapsules (LBL-MC). The adsorption time, concentration of ChI, HEP concentration and EDTA amount were analyzed. The results show that optimal embedding time is 15min, HEP and ChI optimal coating concentration is 2 g/L, EDTA optimal concentration is 30mgCaCO3 microspheres added 0.75ml0.4 mol/L EDTA, namely with EDTA and calcium ion molar ratio of 1:1.(2)The thermal stability, acid and alkali stability of (HEP/CHI)5-SOD was investigated. In the thermal stability test, the results show that at 70 ℃, relative activity of natural SOD and modified SOD was little different. However, when the temperature was increased to 85℃, modified SOD relative activity can also keep at 84.5%, but the natural enzyme relative enzyme activity dropped to 25.9%, the relative enzyme activity of modified SOD has been increased to 3.3 times as compared with natural SOD. The reason is that formation of heparin chitosan microcapsules can effectively protect the SOD secondary bonds. SOD molecular space structure has not been damaged. In acid and alkali stability analysis, the results showed that the natual SOD relative enzyme activity in acid alkali environment decreased significantly, whereas the modified SOD in pH 3～11 relative enzyme activity can keep in more than 80%. The acid-base resistance property of the modified SOD has been raised 2 times that natural SOD. It shows that the (HEP/CHI)5 can protect the active center of SOD, reduce the influence of acid and alkali environment on the active center, and improve the stability of SOD in acid and alkali environment.(3)In the study of antitrypsin hydrolysis ability, the results show that after trypsin hydrolysis 240min modified SOD relative enzyme activity remained over 77%, but natural SOD relative enzyme activity has dropped to 29%, on the antitrypsin hydrolysis ability. Modified SOD enzyme activity has increased 2.7 times than natural SOD. It is proved that the heparin chitosan shell can protect SOD from trypsin hydrolysis.