Constraction Of HMn-SOD Recombinant Bacteria And The Preparation Of Selenium-enriched HMn-SOD

Superoxide dismutase(SOD)is one kind of metal enzymes that could catalyse the superoxide anion radical to molecular oxygen and hydrogen peroxide.SOD is critical for protecting the cell against the toxic products produced by aerobic respiration such as reactive free radicals.SOD has been widely used in health care,cosmetics and clinic because of its functions like antioxidant,enhancing immunity,radiation protection,and anti-aging etc.Selenium belongs to group VIA in the periodic table of elements and it was recognized as basic element of organisms in 1957.Selenium and selenoproteins play an important role in human health with their wide biological functions.In this paper,we firstly constructed three recombinant expression plasmids named hMn-sod-pET21a(+),hMn-sod-pET22b,and hMn-sod-pET28a(+).Then,these three plasmids were transformed into the expression host E.coli BL21(DE3)and hMn-sod-pET28a(+)renamed as 403 was selected as expression plasmid which had high expression level and enzyme activity.Secondly,hMn-sod-pET28a(+)was transformed into three expression hosts named E.coli Trx B,E.coli Rosseta,and E.coli BL21(DE3)respectively.E.coli BL21(DE3)was selected as a hMn-SOD recombinant which had high expression capability and was renamed as 403 BL.The 403BL recombinant was cultivated in 7L jar fermator and the final OD_600nmcould reached 118.The specific activity of recombinat hMn-SOD purified by Ni-chalting affinity chromatograph was 5-10 times higher than that of the broth,reaching 2710U/mg.Thirdly,selenium-enriched hMn-SOD was obtained under the condition of30?g/ml Na₂Se O₃during cultivation.The specific activity of selenium-enriched hMn-SOD containing 462.7?g/g selenium reached 3844U/mg which was 41%higher than that of the control.It was recognized that methionine and cysteine at the position24 and 141 rspectively in hMn-SOD were replaced by selenomethionine and selenocysteine by using protein MS analytic method.