Site-Directed Mutagenesis,Recombinant Expression And Application Research Of Human SOD1

Cells produce reactive oxygen species(ROS)during aerobic metabolism.ROS is one of the important causes of cell damage.Cellular oxidative damage further leads to disease and aging.Superoxide dismutase(SOD)is a metalloenzyme ubiquitous in various organisms,which can catalyze the conversion of superoxide anion into H2O2 with low activity.In addition to the special effects of anti-aging,SOD also has anti-inflammatory,anti-tumor and immune regulation functions.SOD is widely used in the medical,food and cosmetic industries.At present,the recombinant expression of SOD1 has received extensive attention.In this study,the physicochemical properties and secondary structures of the amino acid sequences of human SOD1 and bos grunniens SOD1 were predicted,and the homology modeling of human SOD1 and bos grunniens SOD1 was performed by swiss-model,and Py Mol was used to display and analyze them.Mt1 SOD1(E25G,P29 T,E101V,C112S),Mt2 SOD1(I18T,N20 H,K31V,C112S),and Mt1 SOD1(E25G,P29 T,E101V,C112S)and Mt2 SOD1(I18T,N20 H,K31V,C112S)were obtained through mutation of activity-related amino acid sites.The codonoptimized hSOD1,Mt1 SOD1,Mt2 SOD1,and bos grunniens SOD1 sequences were chemically synthesized and cloned into p ET-28a(+)to transform E.coli BL21(DE3).After induction with 1 m M IPTG,in LB medium containing 800 μM Cu2+ and 20 μM Zn2+ at 25 °C and 180 rpm for 16 h,all four SOD1 were highly expressed in soluble form,and nickel affinity chromatography can effectively purify the recombinant SOD1.The hSOD1 activity obtained by 100 ml LB shake flask culture was 71094 U/mg,and the yield was 4.57 mg.The Mt1 SOD1 activity was 128506 U/mg,and the yield was 3.13 mg.The Mt2 SOD1 activity was 58700.1U/mg,and the yield was 5.47 mg.The activity of yak SOD1 was 42969.5 U/mg,and the yield was 6.81 mg.The enzymatic properties of hSOD1 and Mt1 SOD1 showed that the enzymatic activity remained basically unchanged between 25 ℃ and 55 ℃,and the relative enzymatic activity of hSOD1 remained at about 50% at 75 ℃ for 30 min,while that of Mt1 SOD1 remained at30%..Under the condition of p H 3.6~10.4 for 30 min,bothSOD1 can maintain more than 70%of the enzyme activity.H2O2 sensitivity experiments showed that low concentrations of H2O2 could inactivate hSOD1 and Mt1 SOD1.The results of the effect of metal ions on the enzyme activity showed that the addition of Mn2+,Na+,Mg2+,K+,and Ba2+ all promoted the enzyme activity,among which Mn2+ had the most obvious promoting effect.At the same time,hSOD1 and Mt1 SOD1 have certain tolerance to ethanol,EDTA-2Na and SDS substances.The test results of enzymatic properties showed that hSOD1 and Mt1 SOD1 had high activity and high stability,which laid a foundation for the subsequent development and utilization of SOD1.Next,the effects of hSOD1 and Mt1 SOD1 on mouse melanoma B16 cells were determined.B16 cells were treated with 0 mg/ml,0.01 mg/ml,0.03 mg/ml,0.15 mg/ml,0.6mg/ml,1.2 mg/ml hSOD1,Mt1 SOD1 for 24 h,respectively.The results of the proliferation inhibition experiment showed that hSOD1 and Mt1 SOD1 could significantly inhibit the proliferation of B16 cells,and Mt1 SOD1 had a strong toxic effect on cells at a concentration of 1.2 mg/ml.The tyrosinase activity inhibition experiment showed that hSOD1 and Mt1SOD1 could significantly inhibit the tyrosinase activity in B16 cells in a concentrationdependent manner.The melanin synthesis inhibition experiment showed that hSOD1 and Mt1SOD1 could significantly inhibit the synthesis of melanin in B16 cells,and the inhibition rate could reach 50% at a concentration of 1.2 mg/ml.The results showed that hSOD1,Mt1 SOD1 had whitening function at the cellular level.Using SOD1 as the active ingredient,an anti-aging cosmetic product was developed,and the formula of oil phase components was screened and determined according to the principles of cosmetic formula design,which were 0.6% jojoba oil,0.1% white beeswax,1.2% vaseline,and No.26 white Mineral oil 1.4%,shea butter 0.8%,butanediol 0.5%,betaine 0.2%,trehalose0.3% and glycerol 0.5% were selected for the water phase components,and dd H2 O was added to 100%.The optimal ratio of emulsifier addition amount was determined by orthogonal experiment as 0.4% octadecanol,0.2% Tween-80 and 0.1% Span-60.Quality characterization of the obtained SOD1 anti-aging emulsion shows that it has fine texture,good stability and moisturizing properties.Based on the defects of recombinant SOD1 in the current state of the art,in this study,hSOD1 was used as a template,and the Mt1 SOD1 obtained by site-directed mutation had high activity and high stability,and solved the problem of heterogeneity in application.The application of cosmetics and other fields has laid the foundation.