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Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

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Abstract

A new gene encoding a superoxide dismutase (SOD) was identified from a thermophile Geobacillus sp. EPT3 isolated from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 437 amino acid residues. It was cloned, overexpressed in Escherichia coli (DE3), and the recombinant protein was purified to homogeneity. Geobacillus sp. EPT3 SOD was of the manganese-containing SOD type, as judged by the insensitivity of the recombinant enzyme to both KCN and H2O2, and the activity analysis of Fe or Mn reconstituted SODs by polyacrylamide gel electrophoresis. The recombinant SOD was determined to be a homodimer with monomeric molecular mass of 59.0 kDa. In comparison with other Mn–SODs, the manganese-binding sites are conserved in the sequence (His260, His308, Asp392, His396). The recombinant enzyme had high thermostability at 50 °C. It retained 57 % residual activity after incubation at 90 °C for 1 h, which indicated that this SOD was thermostable. The enzyme also showed striking stability over a wide range of pH 5.0–11.0. At tested conditions, the recombinant SOD from Geobacillus sp. EPT3 showed a relatively good tolerance to some inhibitors, detergents, and denaturants, such as β-mercaptoethanol, dithiothreitol, phenylmethylsulfonyl fluoride, Chaps, Triton X-100, urea, and guanidine hydrochloride.

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References

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein databases search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  Google Scholar 

  • Amo T, Atomi H, Imanaka T (2003) Biochemical properties and regulated gene expression of the superoxide dismutase from the facultatively aerobic hyperthermophile Pyrobaculum calidifontis. J Bacteriol 185:6340–6347

    Article  CAS  Google Scholar 

  • Angelova M, Dolashka-Angelova P, Ivanova E, Serkedjieva J, Slokoska L, Pashova S, Toshkova R, Vassilev S, Simeonov I, Hartmann HJ, Stoeva S, Weser U, Voelter W (2001) A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect. Microbiology 147:1641–1650

    CAS  Google Scholar 

  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201

    Article  CAS  Google Scholar 

  • Bagautdinov B, Yutani K (2011) Structure of indole-3-glycerol phosphate synthase from Thermus thermophilus HB8: implications for thermal stability. Acta Crystallogr D Biol Crystallogr 67:1054–1064

    Article  CAS  Google Scholar 

  • Barondeau DP, Kassmann CJ, Bruns CK, Tainer JA, Getzoff ED (2004) Nickel superoxide dismutase structure and mechanism. Biochemistry 43:8038–8045

    Article  CAS  Google Scholar 

  • Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287

    Article  CAS  Google Scholar 

  • Beyer WF Jr, Fridovich I (1991) In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli. J Biol Chem 266:303–308

    CAS  Google Scholar 

  • Bostwick DG, Alexander EE, Singh R, Shan A, Qian J, Santella RM, Oberley LW, Yan T, Zhong W, Jiang X, Oberley TD (2000) Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraepithelial neoplasia and cancer. Cancer 89:123–134

    Article  CAS  Google Scholar 

  • Boyadzhieva IP, Atanasova M, Emanuilova E (2010) A novel, thermostable manganese-containing superoxide dismutase from Bacillus licheniformis. Biotechnol Lett 32:1893–1896

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Cullen JJ, Weydert C, Hinkhouse MM, Ritchie J, Domann FE, Spitz D, Oberley LW (2003) The role of manganese superoxide dismutase in the growth of pancreatic adenocarcinoma. Cancer Res 63:1297–1303

    CAS  Google Scholar 

  • De Vendittis A, Marco S, Di Maro A, Chambery A, Albino A, Masullo M, Michniewicz A, Parlato G, De Angelis A, De Vendittis E, Rullo R (2012) Properties of a putative cambialistic superoxide dismutase from the aerotolerant bacterium Streptococcus thermophilus strain LMG 18311. Protein Pept Lett 19:333–344

    Article  Google Scholar 

  • Delboni LF, Mande SC, Rentier-Delrue F, Mainfroid V, Turley S, Vellieux FM, Martial JA, Hol WG (1995) Crystal structure of recombinant triosephosphate isomerase from Bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three-dimensional structures points to the importance of hydrophobic interactions. Protein Sci 4:2594–2604

    Article  CAS  Google Scholar 

  • Dello Russo A, Rullo R, Nitti G, Masullo M, Bocchini V (1997) Iron superoxide dismutase from the archaeon Sulfolobus solfataricus: average hydrophobicity and amino acid weight are involved in the adaptation of proteins to extreme environments. Biochim Biophys Acta 1343:23–30

    Article  CAS  Google Scholar 

  • Emerit J, Samuel D, Pavio N (2006) Cu–Zn super oxide dismutase as a potential antifibrotic drug for hepatitis C related fibrosis. Biomed Pharmacother 60:1–4

    Article  CAS  Google Scholar 

  • Fridovich I (1975) Superoxide dismutases. Annu Rev Biochem 44:147–159

    Article  CAS  Google Scholar 

  • Guo FX, SJ E, Liu SA, Chen J, Li DC (2008) Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum. Mycologia 100:375–380

    Article  CAS  Google Scholar 

  • He YZ, Fan KQ, Jia CJ, Wang ZJ, Pan WB, Huang L, Yang KQ, Dong ZY (2007) Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring. Appl Microbiol Biotechnol 75:367–376

    Article  CAS  Google Scholar 

  • Hennig M, Darimont B, Sterner R, Kirschner K, Jansonius JN (1995) 2.0 Å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability. Structure 3:1295–1306

    Article  CAS  Google Scholar 

  • Jackson SM, Cooper JB (1998) An analysis of structural similarity in the iron and manganese superoxide dismutases based on known structures and sequences. Biometals 11:159–173

    Article  CAS  Google Scholar 

  • Kardinahl S, Anemüller S, Schäfer G (2000) The hyper-thermostable Fe-superoxide dismutase from the archaeon Acidianus ambivalens: characterization, recombinant expression, crystallization and effects of metal exchange. Biol Chem 381:1089–1101

    Article  CAS  Google Scholar 

  • Klenk HP, Schleper C, Schwass V, Brudler R (1993) Nucleotide sequence, transcription and phylogeny of the gene encoding the superoxide dismutase of Sulfolobus acidocaldarius. Biochim Biophys Acta 1174:95–98

    Article  CAS  Google Scholar 

  • Knapp S, Kardinahl S, Hellgren N, Tibbelin G, Schäfer G, Ladenstein R (1999) Refined crystal structure of a superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius at 2.2 Å resolution. J Mol Biol 285:689–702

    Article  CAS  Google Scholar 

  • Kreil DP, Ouzounis CA (2001) Identification of thermophilic species by the amino acid compositions deduced from their genomes. Nucleic Acids Res 29:1608–1615

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Lancaster VL, LoBrutto R, Selvaraj FM, Blankenship RE (2004) A cambialistic superoxide dismutase in the thermophilic photosynthetic bacterium Chloroflexus aurantiacus. J Bacteriol 186:3408–3414

    Article  CAS  Google Scholar 

  • Lee HJ, Kwon HW, Koh JU, Lee DK, Moon JY, Kong KH (2010) An efficient method for the expression and reconstitution of thermostable Mn/Fe superoxide dismutase from Aeropyrum pernix K1. J Microbiol Biotechnol 20:727–731

    CAS  Google Scholar 

  • Li DC, Gao J, Li YL, Lu J (2005) A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus. Extremophiles 9:1–6

    Article  Google Scholar 

  • Lim JH, Yu YG, Choi IG, Ryu JR, Ahn BY, Kim SH, Han YS (1997) Cloning and expression of superoxide dismutase from Aquifex pyrophilus, a hyperthermophilic bacterium. FEBS Lett 406:142–146

    Article  CAS  Google Scholar 

  • Liu B, Wang YQ, Zhang XB (2006) Characterization of a recombinant maltogenic amylase from deep sea thermophilic Bacillus sp. WPD616. Enzyme Microb Tech 39:805–810

    Article  CAS  Google Scholar 

  • Liu JG, Yin MM, Zhu H, Lu JR, Cui ZF (2011) Purification and characterization of a hyperthermostable Mn-superoxide dismutase from Thermus thermophilus HB27. Extremophiles 15:221–226

    Article  CAS  Google Scholar 

  • Luisa Corvo M, Jorge JC, van’t Hof R, Cruz ME, Crommelin DJ, Storm G (2002) Superoxide dismutase entrapped in long-circulating liposomes: formulation design and therapeutic activity in rat adjuvant arthritis. Biochim Biophys Acta 1564:227–236

    Article  CAS  Google Scholar 

  • McCord JM (1976) Iron- and manganese-containing superoxide dismutases: structure, distribution, and evolutionary relationships. Adv Exp Med Biol 74:540–550

    Article  CAS  Google Scholar 

  • McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049–6055

    CAS  Google Scholar 

  • McCord JM, Fridovich I (1988) Superoxide dismutase: the first twenty years (1968–1988). Free Radic Biol Med 5:363–369

    Article  CAS  Google Scholar 

  • Meier B, Parak F, Desideri A, Rotilio G (1997) Comparative stability studies on the iron and manganese forms of the cambialistic superoxide dismutase from Propionibacterium shermanii. FEBS Lett 414:122–124

    Article  CAS  Google Scholar 

  • Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, Clayton PE, Wallace DC, Malfroy B, Doctrow SR, Lithgow GJ (2000) Extension of life-span with superoxide dismutase/catalase mimetics. Science 289:1567–1569

    Article  CAS  Google Scholar 

  • Miroshnichenko ML, Bonch-Osmolovskaya EA (2006) Recent developments in the thermophilic microbiology of deep-sea hydrothermal vents. Extremophiles 10:85–96

    Article  Google Scholar 

  • Moreno C, Romero J, Espejo RT (2002) Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio. Microbiology 148:1233–1239

    CAS  Google Scholar 

  • Oppenheimer CH, ZoBell CE (1952) The growth and viability of sixty-three species of marine bacteria as influenced by hydrostatic pressure. J Mar Res 11:10–18

    Google Scholar 

  • Parker MW, Blake CC (1988) Iron- and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett 229:377–382

    Article  CAS  Google Scholar 

  • Raimondi S, Uccelletti D, Matteuzzi D, Pagnoni UM, Rossi M, Palleschi C (2008) Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity. Appl Microbiol Biotechnol 77:1269–1277

    Article  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  Google Scholar 

  • Sato S, Harris JI (1977) Superoxide dismutase from Thermus aquaticus. Isolation and characterisation of manganese and apo enzymes. Eur J Biochem 73:373–381

    Article  CAS  Google Scholar 

  • Song NN, Zheng Y, E SJ, Li DC (2009) Cloning, expression, and characterization of thermostable manganese superoxide dismutase from Thermoascus aurantiacus var. levisporus. J Microbiol 47:123–130

    Article  CAS  Google Scholar 

  • Song CF, Sheng LQ, Zhang XB (2012) Preparation and characterization of a thermostable enzyme (Mn–SOD) immobilized on supermagnetic nanoparticles. Appl Microbiol Biotechnol 96:123–132

    Article  CAS  Google Scholar 

  • Stewart RRC, Bewley JD (1980) Lipid peroxidation associated with accelerated aging of soybean axes. Plant Physiol 65:245–248

    Article  CAS  Google Scholar 

  • Ursby T, Adinolfi BS, Al-Karadaghi S, De Vendittis E, Bocchini V (1999) Iron superoxide dismutase from the archaeon Sulfolobus solfataricus: analysis of structure and thermostability. J Mol Biol 286:189–205

    Article  CAS  Google Scholar 

  • Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43

    Article  CAS  Google Scholar 

  • Vorauer-Uhl K, Fürnschlief E, Wagner A, Ferko B, Katinger H (2001) Topically applied liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation. Eur J Pharm Sci 14:63–67

    Article  CAS  Google Scholar 

  • Wang X, Yang H, Ruan L, Liu X, Li F, Xu X (2008) Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10. J Ind Microbiol Biotechnol 35:133–139

    Article  CAS  Google Scholar 

  • Weisiger RA, Fridovich I (1973) Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization. J Biol Chem 248:4793–4796

    CAS  Google Scholar 

  • Whittaker MM, Whittaker JW (1999) Thermally triggered metal binding by recombinant Thermus thermophilus manganese superoxide dismutase, expressed as the apo-enzyme. J Biol Chem 274:34751–34757

    Article  CAS  Google Scholar 

  • Whittaker MM, Whittaker JW (2000) Recombinant superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilium. J Biol Inorg Chem 5:402–408

    CAS  Google Scholar 

  • Wu SJ, Liu B, Zhang XB (2006) Characterization of a recombinant thermostable xylanase from deep-sea thermophilic Geobacillus sp. MT-1 in East Pacific. Appl Microbiol Biotechnol 72:1210–1216

    Article  CAS  Google Scholar 

  • Yamano S, Maruyama T (1999) An azide-insensitive superoxide dismutase from a hyperthermophilic archaeon, Sulfolobus solfataricus. J Biochem 125:186–193

    Article  CAS  Google Scholar 

  • Yamano S, Sako Y, Nomura N, Maruyama T (1999) A cambialistic SOD in a strictly aerobic hyperthermophilic archaeon, Aeropyrum pernix. J Biochem 126:218–225

    Article  CAS  Google Scholar 

  • Yoo HY, Kim SS, Rho HM (1999) Overexpression and simple purification of human superoxide dismutase (SOD1) in yeast and its resistance to oxidative stress. J Biotechnol 68:29–35

    Article  CAS  Google Scholar 

  • Yost FJ Jr, Fridovich I (1973) An iron-containing superoxide dismutase from Escherichia coli. J Biol Chem 248:4905–4908

    CAS  Google Scholar 

  • Youn HD, Kim EJ, Roe JH, Hah YC, Kang SO (1996) A novel nickel-containing superoxide dismutase from Streptomyces spp. Biochem J 318:889–896

    CAS  Google Scholar 

  • Yu P (2007) A new approach to the production of the recombinant SOD protein by methylotrophic Pichia pastoris. Appl Microbiol Biotechnol 74:93–98

    Article  CAS  Google Scholar 

  • Yu J, Yu XM, Liu JH (2004) A thermostable manganese-containing superoxide dismutase from pathogen Chlamydia pneumoniae. FEBS Lett 562:22–26

    Article  CAS  Google Scholar 

  • Yunoki M, Kawauchi M, Ukita N, Sugiura T, Ohmoto T (2003) Effects of lecithinized superoxide dismutase on neuronal cell loss in CA3 hippocampus after traumatic brain injury in rats. Surg Neurol 59:156–160

    Article  Google Scholar 

  • Zhang LQ, Guo FX, Xian HQ, Wang XJ, Li AN, Li DC (2011) Expression of a novel thermostable Cu, Zn-superoxide dismutase from Chaetomium thermophilum in Pichia pastoris and its antioxidant properties. Biotechnol Lett 33:1127–1132

    Article  CAS  Google Scholar 

  • Zou Y, Yang L, Liu G, Li Y, Zhu Y, Zhang Z (2011) Identification of three superoxide dismutase genes from a Geobacillus sp. Protein J 30:66–71

    Article  CAS  Google Scholar 

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Acknowledgments

This work is financially supported by the Foundation for Innovative Research Team of Jimei University, China (No. 2010A006), National Natural Science Foundation of China (No. 31271914), and Science and Technology Program of Xiamen, China (No. 201303120001).

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  1. School of Biotechnology Engineering, Jimei University, 43 Yindou Rd., Xiamen, 361021, People’s Republic of China

    Yanbing Zhu, Hui Ni, Anfeng Xiao & Huinong Cai

  2. Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, 361021, People’s Republic of China

    Yanbing Zhu, Hui Ni, Anfeng Xiao & Huinong Cai

  3. Research Center of Food Microbiology and Enzyme Engineering Technology, Fujian Province University, Xiamen, 361021, People’s Republic of China

    Yanbing Zhu, Hui Ni, Anfeng Xiao & Huinong Cai

  4. Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, 453003, People’s Republic of China

    Guohong Wang

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Zhu, Y., Wang, G., Ni, H. et al. Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3. World J Microbiol Biotechnol 30, 1347–1357 (2014). https://doi.org/10.1007/s11274-013-1536-5

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