Study On Purification And Characterization Of Superoxide Dismutase From Larvae Of Zophobas Morio L.

Superoxide dismutase (SOD) is a metalloenzyme which can specifically catalyze superoxide anion to disproportionate.It has been found that SOD is relational with organism anti-decrepit, anti-tumor, nutrition situations, immunity disease, the inflammation and protection and has been widely used in medical, agriculture, food and cosmetic industry. Zophobas morio L.is nutritious and especially rich in protein .However, there is no deep research for this insect. In this paper, we attempted to obtain optimum conditions to extract and pure SOD from larvae of Zophobas morio L.,and to study its characterization.On the basis of single factor experiments, second central composite design was adopted to optimizate the extracting conditions of SOD. The results showed pH, ionic strength of the extracting lipid and ratio of lipid to solid significantly affected the extracting ratio of SOD, and so did quadratic terms and interaction terms of the three.The optimum conditions were as follows: pH of phosphate buffer was 7.43, Ionic strength was 0.26mol·L^-1, Liquid to solid ratio was 34.72:1. SOD specific activity getting from the optimum conditions could reach up to 116.747 U·mg^-1.SOD from Zophobas morio L. was purified by the process of heating, ammonium sulfate precipitation and column chromatography DEAE-Sepharose F.F., Sephadex G-75. The best heating temperature was 65℃. The saturaiton of ammonium sulfate was 50% and then 80%. SOD purified with DEAE-Sepharose F.F. and Sephadex G-75 with high activity was in the first peak of the three protein peaks. The purified SOD which was informed to be electrophoretically pure by PAGE electrophoresis had about 6815.66 units enzymatic activity and the specific activity was 7044.61 units per 1 mg protein and the purified fold of SOD was 68.54.Thermal stability of the SOD was quite good and the best suitable temperature was 40℃. The optimum reaction pH was 6.0. This SOD was sensetive to H₂O₂ andβ-mercaptoethanol. SDS affected the SOD depending on its concentration. Urea had little effect to the SOD. However, if 0.5% EDTA was also added into, the SOD had no activity which indicated that metal cofactor was quite important for activity. The mixture of chloroform and ethanol had little effect to the SOD.4. The SOD from Zophobas morio L. containing copper and zinc had a UV absorption peak at 278nm . Holoenzyme molecular weight and Subunit molecular weight were 42.76kDa and 21.55kDa respectively.