Cloning,Expression And Characterization Of Superoxide Dismutase From Acidothermus Cellulolyticus 11B

Superoxide dismutase?SOD EC 1.15.1.1?is an antioxidant metalloenzyme that catalyzes the disproportionation reaction of superoxide anions to produce hydrogen peroxide and oxygen,effectively protecting against oxidative stress and oxidative damage.In this paper,we extracted and purified the superoxide dismutase?AcSOD?from Acidothermus cellulolyticus 11B.The AcSOD gene was cloned using molecular biology techniques,different expression vectors were constructed,the recombinant protein was expressed and purified,the enzymatic properties were characterized,and the catalytic type of the enzyme was discussed.Multiple sequence alignment analysis revealed that AcSOD had a high degree of sequence conservation with known Ni-SOD.The nine residues of Ni-hook?His-Cys-X-X-X-Pro-Cys-Gly-X-Tyr?might be the key judgment of nickel-type superoxide dismutase.The homology modeling results showed that the similarity between AcSOD and Ni-SOD?PDB:3G50?reached 68.38%.It is suggested that this enzyme may be a new type of Ni-SOD.To elucidate the catalytic mechanism of this enzyme,we purified the enzyme 331 times from the original strain by ammonium sulfate precipitation and anion exchange chromatography,and the specific activity reached 2450 U/mg.However,the purity of the enzyme still did not meet the requirements for further analysis.The complete open reading frame of the AcSOD gene was cloned using molecular biology methods,and three expression vectors?pET28a-SOD1.0,pET28a-SOD2.0,and pET20b-SOD3.0?were further constructed to verify the effect of Ni-SOD leader sequence effective cleavage on the formation of enzyme activity.The results showed that only after the leader peptide was excised could the enzyme exhibit its high activity.In this paper,the enzymatic properties of the recombinant enzymes?AcSOD2.0,expressed from pET28a-2.0?were characterized.The specific activity of AcSOD2.0was 2125 U/mg.Inhibitor analysis showed that the enzyme was sensitive to hydrogen peroxide and was weakly inhibited by azide,and the inhibition result was consistent with the Ni-SOD inhibition mode.AcSOD2.0 showed high thermal stability,and retained 80%of the enzyme activity at 50?for 2 h,and showed thermal activation at 60?.The relative enzyme activity of AcSOD2.0 in the pH range of 4-7 was over95%;Metal ions Fe²⁺and Ni²⁺could inhibit its activity,while Fe³⁺,Mg²⁺,Zn²⁺,Ca²⁺,Mn²⁺,Cu²⁺would enhance its activity.DTT caused the inactivation of this enzyme and other organic compounds such as ethanol,?-mercaptoethanol,Tween-80,SDS,urea,Triton X-100,EDTA had inhibitory effects on its activity.In summary,an AcSOD was purified from the thermophilic bacteria A.cellulolyticus 11B,related genes were cloned,different expression vectors were constructed,and recombinant enzymes were expressed and obtained.Sequence analysis and inhibition mode determination indicated that the enzyme might be a new type of Ni-SOD.The characterization of enzymatic properties showed that AcSOD2.0had a high thermal stability,a wide pH catalytic range,and a variety of metal ion tolerance or activation characteristics.This paper lays the foundation for explaining the catalytic mechanism of AcSOD,and the enzyme also shows good application prospects.