Manganese-Sensitive Elastin-Like Polypeptide Modified Superoxide Dismutase(SOD) And Its Double Enzyme System Research

Manganese superoxide dismutase (sod) is a antioxidant metal enzymes, which widely exists in various organisms, its expression quantity and enzyme activity be affected by the concentration of manganese ion, moderate concentration of manganese ions can improve the enzyme activity; In addition, that the elastin-like polypeptide is embedded in a manganese binding sequence, can promote elastin-like polypeptide develop a phase transition in the purification process. Specifically, elastin-like polypeptide is composed of repeat pentapeptide sequence (VPGXG), has the nature of the reversible phase transition with temperature, it still has the nature when it fusion an another protein, so elastin-like polypeptide can be used as purification tag, a manganese binding sequence is F1C2G3D4G5A6N7D8C9G10, then using recursive directional connection technology (RDL) to build manganese-sensitive elastin-like polypeptides, then fuse superoxide dismutase (sod).This particular sequence, combines with manganese ion and then form hydrophobic structure, enhance the hydrophobicity of ELP, improve its purification effect; In addition, the appropriate concentration of manganese ion can promote the enzyme activity of SOD, so this experiment adopts MnCl2 to purifiy SOD.Paper based on superoxide dismutase (sod) and catalase build double enzyme system were studied. SOD can produce H2O2, hydrogen peroxide enzyme can decompose the H2O2, remove the product inhibition in the process of SOD enzyme catalysis effect, improve SOD activity. This article selects the Ssp DnaE fracture contains peptide, respectively with SOD and CAT in the same carrier built two express box, at the same time, in order to avoid the two enzymes after splicing structure change, this experiment selects the rigid connection (EAAAK)5 interval sequence. Will build a carrier to express of success, and nickel column purification, finally to determine enzyme activity, In this test the catalytic efficiency of double enzyme system is twice than the same amount of SOD single enzyme catalytic efficiency. In addition, by intein-mediated SOD and CAT splicing in vitro to measure efficiency, build SOD-InteinC respectively and the CAT-InteinN carrier, into e. coli DH5a characteristic, identification, plasmid and transformed BL21 competent, expression, the use of nickel column purification, dialysis, in vitro splicing, electrophoresis intuitive reflect a result, the results showed 1 MMDTT, pH7.5,25? cradle cultivating 24 h, SOD and CAT 3:1 for double enzyme in vitro splicing is the best condition.