Skip to content
2000
Volume 32, Issue 9
  • ISSN: 0929-8665
  • E-ISSN: 1875-5305

Abstract

Introduction

Superoxide Dismutases (SODs) are enzymes that catalyzes the conversion of toxic free radicals generated during stress conditions into nontoxic forms. Thus, the enzyme superoxide dismutase contributes to the adaptation and survival of microorganisms across a variety of environmental conditions, making it an indispensable enzyme during the response to stress. In this study, we embarked upon investigating and characterizing a Superoxide Dismutase (SOD) from DNA extracted directly from garden soil, where the average temperature ranges from 4°C- 45°C.

Materials and Methods

Metagenomic DNA was extracted by employing a kit. The gene was amplified using PCR. The amplified PCR product was gel eluted and ligated into the pGEMT-easy vector, followed by its subcloning in an expression vector. The protein was purified using Ni-NTA chromatography and characterized using biophysical, biochemical, and computational approaches.

Results

The recombinant SOD was expressed and purified; the purified protein exhibited activity and stability over a broad pH and temperature range, with optimal activity observed at 40°C and pH 8, respectively. The enzyme remains completely stable at 40°C for 3 h. However, in contrast, it loses 50% of its activity when incubated at 50°C and 60°C for 3 h. The biophysical investigation revealed stable conformation of the secondary structure of the protein, as evident from circular dichroism and intrinsic Tryptophan (Trp) fluorescence studies. sequence and structural analysis revealed a close similarity of the SOD reported in this study to the Mn SOD of multi- species. Molecular simulation dynamics experiments revealed the all-over conformational stability of protein structures at varying pH, indicating broad pH functioning of the enzyme.

Discussion

The study provides a comprehensive analysis of the structure and function of a superoxide dismutase enzyme derived from a soil metagenome. A Mn2+ binding site identified in the study offers an opportunity to further facilitate engineering and design of mutant SOD.

Conclusion

The enzyme exhibits distinct attributes that hold significant industrial relevance. Owing to the wide functionality of SOD at different pH and temperature, it can be tailored for its potential industrial applications, including therapeutic potential, thus opening new avenues for enhanced antioxidant therapies and novel biocatalyst designing.

© 2025 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/ppl/10.2174/0109298665415743250926072254
2025-10-03
2026-02-24

  • From This Site

    /content/journals/ppl/10.2174/0109298665415743250926072254
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. IghodaroO.M. AkinloyeO.A. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid.Alex. J. Med.201854428729310.1016/j.ajme.2017.09.001
     [Google Scholar]
  2. RajputV.D. Harish Singh R.K. Verma K.K. Sharma L. Quiroz-Figueroa F.R. Meena M. Gour V.S. Minkina T. Sushkova S. Mandzhieva S. Recent developments in enzymatic antioxidant defence mechanism in plants with special reference to abiotic stress.Biology202110426710.3390/biology10040267
     [Google Scholar]
  3. de Obeso Fernandez del ValleA. ScheckhuberC.Q. Superoxide dismutases in eukaryotic microorganisms: Four case studies.Antioxidants202211218810.3390/antiox11020188
     [Google Scholar]
  4. StephenieS. ChangY.P. GnanasekaranA. EsaN.M. GnanarajC. An insight on superoxide dismutase (SOD) from plants for mammalian health enhancement.J. Funct. Foods20206810391710.1016/j.jff.2020.103917
     [Google Scholar]
  5. SzőllősiR. Superoxide dismutase (SOD) and abiotic stress tolerance in plants: An overview.Oxidative Damage to Plants. AhmadP. Academic Press20148912910.1016/B978‑0‑12‑799963‑0.00003‑4
     [Google Scholar]
  6. ZhangJ. WangH. HuangQ. ZhangY. ZhaoL. LiuF. WangG. Four superoxide dismutases of Bacillus cereus 0-9 are non-redundant and perform different functions in diverse living conditions.World J. Microbiol. Biotechnol.20203611210.1007/s11274‑019‑2786‑7
     [Google Scholar]
  7. JomovaK. RaptovaR. AlomarS.Y. AlwaselS.H. NepovimovaE. KucaK. ValkoM. Reactive oxygen species, toxicity, oxidative stress, and antioxidants: Chronic diseases and aging.Arch. Toxicol.202397102499257410.1007/s00204‑023‑03562‑9
     [Google Scholar]
  8. HinsonJ.A. RobertsD.W. JamesL.P. Mechanisms of acetaminophen-induced liver necrosis.Adverse Drug Reactions. UetrechtJ. Heidelberg, BerlinSpringer-Verlag201036940510.1007/978‑3‑642‑00663‑0_12
     [Google Scholar]
  9. CarillonJ. RouanetJ.M. CristolJ.P. BrionR. Superoxide dismutase administration, a potential therapy against oxidative stress related diseases: Several routes of supplementation and proposal of an original mechanism of action.Pharm. Res.201330112718272810.1007/s11095‑013‑1113‑5
     [Google Scholar]
  10. KhanT.A. HassanI. AhmadA. PerveenA. AmanS. QuddusiS. AlhazzaI.M. AshrafG.M. AlievG. Recent updates on the dynamic association between oxidative stress and neurodegenerative disorders.CNS Neurol. Disord. Drug Targets201615331032010.2174/1871527315666160202124518 26831262
     [Google Scholar]
  11. ChaudharyN. RoyZ. BansalR. SiddiquiL. Understanding the role of free radicals and antioxidant enzymes in human diseases.Curr. Pharm. Biotechnol.202324101265127610.2174/1389201024666221121160822
     [Google Scholar]
  12. CheM. WangR. LiX. WangH.Y. ZhengX.F.S. Expanding roles of superoxide dismutases in cell regulation and cancer.Drug Discov. Today201621114314910.1016/j.drudis.2015.10.001
     [Google Scholar]
  13. ShengY. AbreuI.A. CabelliD.E. MaroneyM.J. MillerA.F. TeixeiraM. ValentineJ.S. Superoxide dismutases and superoxide reductases.Chem. Rev.201411473854391810.1021/cr4005296
     [Google Scholar]
  14. KarmakarA. DasA.K. GhoshN. SilP.C. Superoxide dismutase.Antioxidants Effects in Health. NabaviS.M. SilvaA.T.S. Elsevier202213916610.1016/B978‑0‑12‑819096‑8.00027‑6
     [Google Scholar]
  15. JiangM. LiL. ZhuD. ZhangH. ZhaoX. Oxygen reduction in the nanocage of metal–organic frameworks with an electron transfer mediator.J. Mater. Chem. A Mater. Energy Sustain.20142155323532910.1039/C3TA15319C
     [Google Scholar]
  16. SrivastavaA.K. SarojA. NayakA. NishadI. GautamK. Microbial life in stress of oxygen concentration: Physiochemical properties and applications.Microbial Versatility in Varied Environments.SingaporeSpringer202018119810.1007/978‑981‑15‑3028‑9_11
     [Google Scholar]
  17. PérezV. HengstM. KurteL. DoradorC. JeffreyW.H. WattiezR. MolinaV. Matallana-SurgetS. Bacterial survival under extreme UV radiation: A comparative proteomics study of Rhodobacter sp., isolated from high altitude wetlands in Chile.Front. Microbiol.20178117310.3389/fmicb.2017.01173
     [Google Scholar]
  18. PrabhakaranP. AshrafM.A. AqmaW.S. Microbial stress response to heavy metals in the environment.RSC Advances2016611110986210987710.1039/C6RA10966G
     [Google Scholar]
  19. CycońM. MrozikA. Piotrowska-SegetZ. Antibiotics in the soil environment—degradation and their impact on microbial activity and diversity.Front. Microbiol.20191033810.3389/fmicb.2019.00338
     [Google Scholar]
  20. WangY. BranickyR. NoëA. HekimiS. Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling.J. Cell Biol.201821761915192810.1083/jcb.201708007
     [Google Scholar]
  21. JenaA.B. SamalR.R. BholN.K. DuttaroyA.K. Cellular Red-Ox system in health and disease: The latest update.Biomed. Pharmacother.202316211460610.1016/j.biopha.2023.114606
     [Google Scholar]
  22. HuangX. HeD. PanZ. LuoG. DengJ. Reactive-oxygen-species-scavenging nanomaterials for resolving inflammation.Mater. Today Bio20211110012410.1016/j.mtbio.2021.100124
     [Google Scholar]
  23. LewandowskiŁ. KepinskaM. MilnerowiczH. The copper-zinc superoxide dismutase activity in selected diseases.Eur. J. Clin. Invest.2019491e1303610.1111/eci.13036 30316201
     [Google Scholar]
  24. LiuX. XuH. PengH. WanL. DiD. QinZ. HeL. LuJ. WangS. ZhaoQ. Advances in antioxidant nanozymes for biomedical applications.Coord. Chem. Rev.202450221561010.1016/j.ccr.2023.215610
     [Google Scholar]
  25. KurianG.A. RajagopalR. VedanthamS. RajeshM. The role of oxidative stress in myocardial ischemia and reperfusion injury and remodeling: Revisited.Oxid. Med. Cell. Longev.20162016165645010.1155/2016/1656450 27313825
     [Google Scholar]
  26. RaoM.K. SunkadG. Metaomics approaches to unravel the functioning of multispecies microbial communities.Microbiome Drivers of Ecosystem Function.Academic Press202439541610.1016/B978‑0‑443‑19121‑3.00009‑0
     [Google Scholar]
  27. NesmeJ. AchouakW. AgathosS.N. BaileyM. BaldrianP. BrunelD. FrostegårdÅ. HeulinT. JanssonJ.K. JurkevitchE. KruusK.L. KowalchukG.A. LagaresA. Lappin-ScottH.M. LemanceauP. Le PaslierD. Mandic-MulecI. MurrellJ.C. MyroldD.D. NalinR. NannipieriP. NeufeldJ.D. O’GaraF. ParnellJ.J. PühlerA. PylroV. RamosJ.L. RoeschL.F.W. SchloterM. SchleperC. SczyrbaA. SessitschA. SjölingS. SørensenJ. SørensenS.J. TebbeC.C. ToppE. TsiamisG. van ElsasJ.D. van KeulenG. WidmerF. WagnerM. ZhangT. ZhangX. ZhaoL. ZhuY-G. VogelT.M. SimonetP. Back to the future of soil metagenomics.Front Microbiol.201677310.3389/fmicb.2016.00073
     [Google Scholar]
  28. PeskinA.V. WinterbournC.C. A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1).Clin. Chim. Acta20002931-215716610.1016/S0009‑8981(99)00246‑6
     [Google Scholar]
  29. AshkenazyH. AbadiS. MartzE. ChayO. MayroseI. PupkoT. Ben-TalN. ConSurf 2016: An improved methodology to estimate and visualize evolutionary conservation in macromolecules.Nucleic Acids Res.201644W1W344W35010.1093/nar/gkw408
     [Google Scholar]
  30. JumperJ. EvansR. PritzelA. GreenT. FigurnovM. RonnebergerO. TunyasuvunakoolK. BatesR. ŽídekA. PotapenkoA. BridglandA. MeyerC. KohlS.A.A. BallardA.J. CowieA. Romera-ParedesB. NikolovS. JainR. AdlerJ. BackT. PetersenS. ReimanD. ClancyE. ZielinskiM. SteineggerM. PacholskaM. BerghammerT. BodensteinS. SilverD. VinyalsO. SeniorA.W. KavukcuogluK. KohliP. HassabisD. Highly accurate protein structure prediction with AlphaFold.Nature2021596787358358910.1038/s41586‑021‑03819‑2
     [Google Scholar]
  31. MaierJ.A. MartinezC. KasavajhalaK. WickstromL. HauserK.E. SimmerlingC. ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB.J. Chem. Theory Comput.20151183696371310.1021/acs.jctc.5b00255
     [Google Scholar]
  32. PettersenE.F. GoddardT.D. HuangC.C. CouchG.S. GreenblattD.M. MengE.C. FerrinT.E. Ucsf chimera - A visualization system for exploratory research and analysis.J. Comput. Chem.200425131605161210.1002/jcc.20084 15264254
     [Google Scholar]
  33. Salomon-FerrerR. CaseD.A. WalkerR.C. An overview of the amber biomolecular simulation package.Wiley Interdiscip. Rev. Comput. Mol. Sci.20133219821010.1002/wcms.1121
     [Google Scholar]
  34. PradeepN.V. AnupamaA.K. PoojaJ. Categorizing phenomenal features of α-amylase (Bacillus species) using bioinformatic tools.Adv. Lif Sci. Technol201242731
     [Google Scholar]
  35. EastmanP. SwailsJ. ChoderaJ.D. McGibbonR.T. ZhaoY. BeauchampK.A. WangL.P. SimmonettA.C. HarriganM.P. SternC.D. WiewioraR.P. BrooksB.R. PandeV.S. OpenMM 7: Rapid development of high performance algorithms for molecular dynamics.PLOS Comput. Biol.2017137e100565910.1371/journal.pcbi.1005659
     [Google Scholar]
  36. LemkulJ.A. Introductory tutorials for simulating protein dynamics with GROMACS.J. Phys. Chem. B2024128399418943510.1021/acs.jpcb.4c04901
     [Google Scholar]
  37. SinhaS. TamB. WangS.M. Applications of molecular dynamics simulation in protein study.Membranes202212984410.3390/membranes12090844
     [Google Scholar]
  38. HollingsworthS.A. DrorR.O. Molecular dynamics simulation for all.Neuron20189961129114310.1016/j.neuron.2018.08.011
     [Google Scholar]
  39. MoriT. JungJ. KobayashiC. DokainishH.M. ReS. SugitaY. Elucidation of interactions regulating conformational stability and dynamics of SARS-CoV-2 S-protein.Biophys. J.202112061060107110.1016/j.bpj.2021.01.012
     [Google Scholar]
  40. Martins de OliveiraV. LiuR. ShenJ. Constant pH molecular dynamics simulations: Current status and recent applications.Curr. Opin. Struct. Biol.20227710249810.1016/j.sbi.2022.102498
     [Google Scholar]
  41. KeQ. GongX. LiaoS. DuanC. LiL. Effects of thermostats/barostats on physical properties of liquids by molecular dynamics simulations.J. Mol. Liq.202236512011610.1016/j.molliq.2022.120116
     [Google Scholar]
  42. HessB. KutznerC. van der SpoelD. LindahlE. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation.J. Chem. Theory Comput.20084343544710.1021/ct700301q
     [Google Scholar]
  43. LüM. CaiR. WangS. LiuZ. JiaoY. FangY. ZhangX. Purification and characterization of iron-cofactored superoxide dismutase from Enteromorpha linza.Chin. J. Oceanology Limnol.20133161190119510.1007/s00343‑013‑3049‑3
     [Google Scholar]
  44. PadmapriyaV. AnandN. Purification and characterization of iron-containing superoxide dismutase from Anabaena variabilis Kutz ex Born. et Flah.Biomed. Pharmacol. J.20153349356
     [Google Scholar]
  45. PetkarM.B. PillaiM.M. KulkarniA.A. BondreS.H. RaoK. Purification and characterization of superoxide dismutase isolated from sewage isolated E. coli.J. Microb. Biochem. Technol.201305410.4172/1948‑5948.1000109
     [Google Scholar]
  46. KimS.W. LeeS.O. LeeT.H. Purification and characterization of superoxide dismutase from Aerobacter aerogenes.Agric. Biol. Chem.199155110110810.1080/00021369.1991.10870561
     [Google Scholar]
  47. Öztürk-ÜrekR. TarhanL. Purification and characterization of superoxide dismutase from chicken liver.Comp. Biochem. Physiol. B Biochem. Mol. Biol.2001128220521210.1016/S1096‑4959(00)00300‑6
     [Google Scholar]
  48. ReddyC.D. VenkaiahB. Purification and characterization of Cu-Zn superoxide dismutases from mungbean (Vigna radiata) seedlings.J. Biosci.19846111512310.1007/BF02702863
     [Google Scholar]
  49. HouZ. ZhaoL. WangY. LiaoX. Purification and characterization of superoxide dismutases from sea buckthorn and chestnut rose.J. Food Sci.201984474675310.1111/1750‑3841.14441 30861132
     [Google Scholar]
  50. FolgueiraI. LamasJ. de FelipeA.P. SueiroR.A. LeiroJ.M. Identification and molecular characterization of superoxide dismutases isolated from A scuticociliate parasite: Physiological role in oxidative stress.Sci. Rep.2019911332910.1038/s41598‑019‑49750‑5
     [Google Scholar]
  51. LiuQ. HangX. LiuX. TanJ. LiD. YangH. Cloning and heterologous expression of the manganese superoxide dismutase gene from Lactobacillus casei Lc18.Ann. Microbiol.201262112913710.1007/s13213‑011‑0237‑2
     [Google Scholar]
  52. PinmaneeP. SompinitK. JantimapornA. KhongkowM. HaltrichD. NimchuaT. SukyaiP. Purification and immobilization of superoxide dismutase obtained from Saccharomyces cerevisiae TBRC657 on bacterial cellulose and its protective effect against oxidative damage in fibroblasts.Biomolecules2023137115610.3390/biom13071156
     [Google Scholar]
  53. TidharA. RushingM.D. KimB. SlauchJ.M. Periplasmic superoxide dismutase SodCI of Salmonella binds peptidoglycan to remain tethered within the periplasm.Mol. Microbiol.201597583284310.1111/mmi.13067
     [Google Scholar]
  54. GhanmiF. Carré-MloukaA. ZaraiZ. MejdoubH. PeduzziJ. MaalejS. RebuffatS. The extremely halophilic archaeon Halobacterium salinarum ETD5 from the solar saltern of Sfax (Tunisia) produces multiple halocins.Res. Microbiol.20201712809010.1016/j.resmic.2019.09.003
     [Google Scholar]
  55. MarkillieL.M. VarnumS.M. HradeckyP. WongK.K. Targeted mutagenesis by duplication insertion in the radioresistant bacterium Deinococcus radiodurans: Radiation sensitivities of catalase (katA) and superoxide dismutase (sodA) mutants.J. Bacteriol.1999181266666910.1128/JB.181.2.666‑669.1999
     [Google Scholar]
  56. AssadyM. FarahnakA. GolestaniA. EsharghianM.R. Superoxide dismutase (SOD) enzyme activity assay in Fasciola spp. parasites and liver tissue extract.Iran. J. Parasitol.2011617
     [Google Scholar]
  57. RobinettN.G. PetersonR.L. CulottaV.C. Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains.J. Biol. Chem.2018293134636464310.1074/jbc.TM117.000182
     [Google Scholar]
  58. ChapT.Y. NathanV. BalasubramanyamT. Therapeutic potentials of superoxide dismutase: Current status and future prospects.Adv. Chem. Res.202272195216
     [Google Scholar]
  59. RobbinsD. ZhaoY. Manganese superoxide dismutase in cancer prevention.Antioxid. Redox Signal.201420101628164510.1089/ars.2013.5297
     [Google Scholar]
  60. TurnbaughP.J. LeyR.E. MahowaldM.A. MagriniV. MardisE.R. GordonJ.I. An obesity-associated gut microbiome with increased capacity for energy harvest.Nature200644471221027103110.1038/nature05414
     [Google Scholar]
  61. KaurR. KumarR. VermaS. KumarA. RajeshC. SharmaP.K. Structural and functional insights about unique extremophilic bacterial lipolytic enzyme from metagenome source.Int. J. Biol. Macromol.202015259360410.1016/j.ijbiomac.2020.02.210
     [Google Scholar]
  62. RosanoG.L. CeccarelliE.A. Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain.Microb. Cell Fact.2009814110.1186/1475‑2859‑8‑41
     [Google Scholar]
  63. OchnevaA. ZorkinaY. AbramovaO. PavlovaO. UshakovaV. MorozovaA. ZubkovE. PavlovK. GurinaO. ChekhoninV. Protein misfolding and aggregation in the brain: Common pathogenetic pathways in neurodegenerative and mental disorders.Int. J. Mol. Sci.202223221449810.3390/ijms232214498
     [Google Scholar]
  64. WangQ. YuanZ. WuH. LiuF. ZhaoJ. Molecular characterization of a manganese superoxide dismutase and copper/zinc superoxide dismutase from the mussel Mytilus galloprovincialis.Fish Shellfish Immunol.20133451345135110.1016/j.fsi.2013.01.011
     [Google Scholar]
  65. ThakurA. KumarP. LataJ. DeviN. ChandD. Thermostable Fe/Mn superoxide dismutase from Bacillus licheniformis SPB-13 from thermal springs of Himalayan region: Purification, characterization and antioxidative potential.Int. J. Biol. Macromol.20181151026103210.1016/j.ijbiomac.2018.04.155
     [Google Scholar]
  66. López-PeñaI. LeighB.S. SchlamadingerD.E. KimJ.E. Insights into protein structure and dynamics by ultraviolet and visible resonance raman spectroscopy.Biochemistry201554314770478310.1021/acs.biochem.5b00514
     [Google Scholar]
  67. PerryJ.J.P. ShinD.S. GetzoffE.D. TainerJ.A. The structural biochemistry of the superoxide dismutases.Biochim. Biophys. Acta. Proteins Proteomics20101804224526210.1016/j.bbapap.2009.11.004
     [Google Scholar]
  68. LiH. FengZ. SunY. NingS. ZhouW. LiuA. PanF. ZhaoX. ZhuH. LuJ.R. Engineering a thermostable iron superoxide dismutase based on manganese superoxide dismutase from Thermus thermophilus.Process Biochem.2016511394710.1016/j.procbio.2015.11.001
     [Google Scholar]
  69. BonettaV.R. The structure-function relationships and physiological roles of MnSOD mutants.Biosci. Rep.2022426BSR2022020210.1042/BSR20220202
     [Google Scholar]
  70. AreekitS. KanjanavasP. KhawsakP. PakpitchareonA. PotivejkulK. ChansiriG. ChansiriK. Cloning, expression, and characterization of thermotolerant manganese superoxide dismutase from Bacillus sp. MHS47.Int. J. Mol. Sci.201112184485610.3390/ijms12010844
     [Google Scholar]
/content/journals/ppl/10.2174/0109298665415743250926072254
Loading
Cloning, Expression, Purification, and Characterization of Superoxide Dismutase from the Soil Metagenome
PPL 32, 667 (2025); https://doi.org/10.2174/0109298665415743250926072254
/content/journals/ppl/10.2174/0109298665415743250926072254
/content/journals/ppl/10.2174/0109298665415743250926072254
Loading

Data & Media loading...


  • Article Type:     Research Article
Keyword(s): biocatalyst designing; metagenomics; molecular simulation; oxidative stress; reactive oxygen species; Superoxide dismutase

Most Cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test