Cloning And Expression Of A Thermostable Superoxide Dismutase Gene,Extract And Research Of The Superoxide Dismutase Enzyme

Superoxide dismutase(SOD) is a kind of metalloenzyme which is found in animals,plants and microorganisms.It plays an important role in the balance between the producing and cleaning of O.2- ,since it can dismute superoxide into hydrogen peroxide and oxygen through altermate oxidation and reduction of their catalytic metal ions.There are three kinds SODs on the base of different ions: Cu,Zn-SODs, Fe/Mn-SOD,and Ni-SOD.We got three genes of superoxide dismutase from a bacteria of Geobacillus: Fe/Mn-sod,Mn-sod and Cu/Zn-sod.Amino acids'sequences deducing by this 3 genes had the highest similarity with the Geobacillus kaustophilus HAT426's SOD. We cloned these genes to expression vector pPIC9 .The recombinant plasmids were transformed into yeasts GS115.Results showed that only one express interesting protein not obviously. Also we cloned the gene Mn-sod to another vector pET-28a to express in E coli.BL21,and got a high expression.After purification,the activity of the MnSOD protein was 2726.7U/ mg.As an enzyme,it showed great stabilization at high temperatures:it was stable at 60℃.It had 70% activities at 80℃after 2.5 hours ,and it also had 30% activities left after 2.5 hours even at 90℃.We found Mn2+ is very important for the activity.Through the results of native PAGE and SDS-PAGE,we confered that this enzyme was a homotetramer.With the method of random cloning of the genome of Pseudelatia separata granulovirus sequence, we got a fragment encoding Superoxide dismutase(PsGV-sod). This sequence has a 462bp ORF, encoding 153 amino acids. By comparing the sequence identities of DNA and amino acids, we found that it had high homologies with other granulovirus sod genes . The results showed that it encoded Cu,Zn-SOD by analyzing the conserved sequences .