Skip to main content
SpringerLink
Log in

Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace five Met residues in AnaPX with high redox residues Ile, Leu, or Phe was performed for the improvement of the enzyme stability toward H2O2. The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H2O2 stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, the M401F and M451I retained 16% and 5% activity at 100 mM H2O2, respectively, in addition to maintaining high dye-decolorization activity toward AQ and azo dyes at 5 mM H2O2 and showing a slower rate of heme degradation than the wildtype enzyme. The observed stabilization of AnaPX may be attributed to the replacement of potentially oxidizable Met residues either increasing the local stability of the heme pocket or limiting of the self-inactivation electron transfer pathways due to the above mutations. The increased stability of AnaPX variants coupled with the broad substrate specificity can be potentially useful for the further practical application of these enzymes especially in bioremediation of wastewater contaminated with recalcitrant AQ.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Cherry JR, Lamsa MH, Schneider P, Vind J, Svendsen A, Jones A, Pedersen AH (1999) Directed evolution of a fungal peroxidase. Nat Biotechnol 17:379–384

    Article  CAS  Google Scholar 

  • Christian VV, Shrivastava R, Novotny C, Vyas BR (2003) Decolorization of sulfonphthalein dyes by manganese peroxidase activity of the white-rot fungus Phanerochaete chrysosporium. Folia Microbiol (Praha) 48:771–774

    Article  CAS  Google Scholar 

  • Colonna S, Gaggero N, Richelmi C, Pasta P (1999) Recent biotechnological developments in the use of peroxidases. Trends Biotechnol 17(4):163–168

    Article  CAS  Google Scholar 

  • Dayhoff MO, Barker WC, McLaughlin PJ (1974) Inferences from protein and nucleic acid sequences: early molecular evolution, divergence of kingdoms and rates of change. Orig Life 5:311–330

    Article  CAS  Google Scholar 

  • Faraco V, Piscitelli A, Sannia G, Giardina P (2006) Identification of a new member of the dye-decolorizing peroxidase family from Pleurotus ostreatus. World J Microbiol Biotechnol 23:889–893

    Article  Google Scholar 

  • Hiner ANP, Ruiz JH, López JNRG, Cánovas FGA, Brisset NC, Smith AT, Arnao MB, Acosta M (2002) Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. J Biol Inorg Chem 277:26879–26885

    CAS  Google Scholar 

  • Johjima T, Ohkuma M, Kudo T (2003) Isolation and cDNA cloning of novel hydrogen peroxide-dependent phenol oxidase from the basidiomycete Termitomyces albuminosus. Appl Microbiol Biotechnol 61:220–225

    CAS  Google Scholar 

  • Kim K, Erman JE (1988) Methionine modification in cytochrome-c peroxidase. Biochim Biophys Acta 954:95–107

    CAS  Google Scholar 

  • Kim SJ, Shoda M (1999) Purification and characterization of a novel peroxidase from Geotrichum candidum Dec 1 involved in decolorization of dyes. Appl Environ Microbiol 65:1029–1035

    CAS  Google Scholar 

  • Kim YH, Berry AH, Spencer DS, Stites WE (2001) Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins. Protein Eng 14:343–347

    Article  CAS  Google Scholar 

  • Kitajima S (2008) Hydrogen peroxide-mediated inactivation of two chloroplastic peroxidases, ascorbate peroxidase and 2-cys peroxiredoxin. Photochem Photobiol 84:1404–1409

    Article  CAS  Google Scholar 

  • Kitajima S, Kitamura M, Koja N (2008) Triple mutation of Cys26, Trp35, and Cys126 in stromal ascorbate peroxidase confers H2O2 tolerance comparable to that of the cytosolic isoform. Biochem Biophys Res Commun 372:918–923

    Article  CAS  Google Scholar 

  • Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13:179–191

    Article  CAS  Google Scholar 

  • Liers C, Bobeth C, Pecyna M, Ullrich R, Hofrichter M (2010) DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes. Appl Microbiol Biotechnol 85:1869–1879

    Article  CAS  Google Scholar 

  • Miyazaki C, Takahashi H (2001) Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2. FEBS Lett 509:111–114

    Article  CAS  Google Scholar 

  • Morawski B, Quan S, Arnold FH (2001) Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnol Bioeng 76:99–107

    Article  CAS  Google Scholar 

  • Ogola HJ, Kamiike T, Hashimoto N, Ashida H, Ishikawa T, Shibata H, Sawa Y (2009) Molecular characterization of a novel peroxidase from the cyanobacterium Anabaena sp. strain PCC 7120. Appl Environ Microbiol 75:7509–7518

    Article  CAS  Google Scholar 

  • Ollikka P, Alhonmaki K, Leppanen VM, Glumoff T, Raijola T, Suominen I (1993) Decolorization of azo, triphenyl methane, heterocyclic, and polymeric dyes by lignin peroxidase isoenzymes from Phanerochaete chrysosporium. Appl Environ Microbiol 59:4010–4016

    CAS  Google Scholar 

  • Robinson T, McMullan G, Marchant R, Nigam P (2001) Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresour Technol 77:247–255

    Article  CAS  Google Scholar 

  • Ryan BJ, O'Fagain C (2007) Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide. Biochimie 89:1029–1032

    Article  CAS  Google Scholar 

  • Scheibner M, Hulsdau B, Zelena K, Nimtz M, de Boer L, Berger RG, Zorn H (2008) Novel peroxidases of Marasmius scorodonius degrade beta-carotene. Appl Microbiol Biotechnol 77:1241–1250

    Article  CAS  Google Scholar 

  • Shakeri M, Shoda M (2010) Efficient decolorization of an anthraquinone dye by recombinant dye-decolorizing peroxidase (rDyP) immobilized in silica-based mesocellular foam. J Mol Catal B Enzym 62:277–281

    Article  CAS  Google Scholar 

  • Stadtman ER, Moskovitz J, Levine RL (2003) Oxidation of methionine residues of proteins: biological consequences. Antioxid Redox Signal 5:577–582

    Article  CAS  Google Scholar 

  • Sturm A, Schierhorn A, Lindenstrauss U, Lilie H, Bruser T (2006) YcdB from Escherichia coli reveals a novel class of Tat-dependently translocated hemoproteins. J Biol Chem 281:13972–13978

    Article  CAS  Google Scholar 

  • Sugano Y (2009) DyP-type peroxidases comprise a novel heme peroxidase family. Cell Mol Life Sci 66:1387–1403

    Article  CAS  Google Scholar 

  • Valderrama B, Ayala M, Vazquez-Duhalt R (2002) Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes. Chem Biol 9:555–565

    Article  CAS  Google Scholar 

  • Valderrama B, Vazquez-Duhalt R (2005) Electron-balance during the oxidative self-inactivation of cytochrome c. J Mol Catal B Enzym 35:41–44

    Article  CAS  Google Scholar 

  • Valderrama B, Garcia-Arellano H, Giansanti S, Baratto MC, Pogni R, Vazquez-Duhalt R (2006) Oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering. FASEB J 20:1233–1235

    Article  CAS  Google Scholar 

  • van Bloois E, Torres Pazmino DE, Winter RT, Fraaije MW (2009) A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily. Appl Microbiol Biotechnol. doi:10.1007/s00253-009-2369-x

    Google Scholar 

  • Villegas JA, Mauk AG, Vazquez-Duhalt R (2000) A cytochrome c variant resistant to heme degradation by hydrogen peroxide. Chem Biol 7:237–244

    Article  CAS  Google Scholar 

  • Vogt W (1995) Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radic Biol Med 18:93–105

    Article  CAS  Google Scholar 

  • Welinder KG, Andersen MB (1993) Peroxidase variants with improved hydrogen peroxide stability. Patent WO 93/24618

  • Zamorano LS, Vilarmau SB, Arellano JB, Zhadan GG, Cuadrado NH, Bursakov SA, Roig MG, Shnyrov VL (2009) Thermal stability of peroxidase from Chamaerops excelsa palm tree at pH 3. Int J Biol Macromol 44:326–332

    Article  CAS  Google Scholar 

  • Zelena K, Hardebusch B, Hulsdau B, Berger RG, Zorn H (2009) Generation of norisoprenoid flavors from carotenoids by fungal peroxidases. J Agric Food Chem 57:9951–9955

    Article  CAS  Google Scholar 

  • Zorn H, Scheibner M, Hulsdau B, Berger RG, de Boer L, Meima RB (2008) Novel enzymes for use in enzymatic bleaching of food products. Patent Appl WO 2007006792

  • Zubieta C, Krishna SS, Kapoor M, Kozbial P, McMullan D, Axelrod HL, Miller MD, Abdubek P, Ambing E, Astakhova T, Carlton D, Chiu HJ, Clayton T, Deller MC, Duan L, Elsliger MA, Feuerhelm J, Grzechnik SK, Hale J, Hampton E, Han GW, Jaroszewski L, Jin KK, Klock HE, Knuth MW, Kumar A, Marciano D, Morse AT, Nigoghossian E, Okach L, Oommachen S, Reyes R, Rife CL, Schimmel P, van den Bedem H, Weekes D, White A, Xu Q, Hodgson KO, Wooley J, Deacon AM, Godzik A, Lesley SA, Wilson IA (2007) Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif. Proteins 69:223–233

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan

    Henry Joseph Oduor Ogola, Naoya Hashimoto, Suguru Miyabe, Takahiro Ishikawa, Hitoshi Shibata & Yoshihiro Sawa

  2. Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, Matsue, Shimane, Japan

    Hiroyuki Ashida

Authors
  1. Henry Joseph Oduor Ogola
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Naoya Hashimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suguru Miyabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroyuki Ashida
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Takahiro Ishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hitoshi Shibata
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yoshihiro Sawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yoshihiro Sawa.

Electronic supplementary materials

Below is the link to the electronic supplementary material.

S1

(DOC 506 kb)

S2

(DOC 203 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ogola, H.J.O., Hashimoto, N., Miyabe, S. et al. Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues. Appl Microbiol Biotechnol 87, 1727–1736 (2010). https://doi.org/10.1007/s00253-010-2603-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2603-6

Keywords

Search

Navigation