Preparation And Activity Evaluation Of Antioxidant Protein From Haliotis Discus Hannai Ino

Haliotis discus hannai Ino is highly valued seafood,which rich in protein,fat,the human body essential trace elements and has an impressive range of uses with high nutritional value.Synthetic antioxidants are widely used as food preservatives,thus the preparation of natural antioxidant protein has become a research hotspot,which may bring huge commercial potentiality to food additives and cosmetics industry.In this study,H.discus hannai Ino was used as a raw material.Through the construction of eukaryotic expression vector,in vitro expression,optimized its expression and purification condition,and evaluated the activity of the obtained protein.This study will provide a reference for development of food additives and cosmetics auxiliary materials.The main research contents and conclusions are listed as follows:1.Constructed of eukaryotic expression vector of Haliotis discus hannai Cu/Zn-SOD and TPX2,recombinant expressed,optimized expression and purification conditions.The optimal expression condition of Cu/Zn-SOD was incubation with 0.5%methanol for 24 h at 28 ?,under pH6,and TPX2 was incubation with 0.5%methanol for 72 h at 28 ?,under pH7.2.SOD activity in different concentration of Cu2+ was compared to evaluate its thermal stability and antioxidant capacity.At the same time,TPX2 antioxidant capacity also was determinated.The highest SOD activity showed in 1%concentration of Cu2+,and maintained 50%under 80 ?.The ability of scavenging free radicals of both Cu/Zn-SOD and TPX2 were higher than Vc,the scavenging hydroxyl free radical ability of Cu/Zn-SOD and TPX2 were 3 and 9 times higher than Vc,while scavenging superoxide anion free radical ability of Cu/Zn-SOD was 16 times of Vc.3.The MTS/PMS assay was used to evaluate the effect on cellular toxicity and antioxidant activity.Whether the recombinant protein caused hematopoietic hemolysis was also studied.The results showed that 100 ?g/mL Cu/Zn-SOD and TPX2 were safe on L929 and L02 cells,960 ?g/mL Cu/Zn-SOD and 720 ?g/mL TPX2 did not cause cell hemolysis.Both have protective effect on cell oxidative damage caused by H2O2.Thus,H.discus hannai Ino Cu/Zn-SOD and TPX2 are safe antioxidants,which can be serve as the auxiliary materials of food additives and cosmetics.