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New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications

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Abstract

This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4–5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10–30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.

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References

  • Ahmad M, Roberts JN, Hardiman EM, Singh R, Eltis LD, Bugg TD (2011) Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase. Biochemistry 50:5096–5107

    Article  CAS  PubMed  Google Scholar 

  • Ayala M (2010) Redox potential of peroxidases. Springer, Heidelberg

    Google Scholar 

  • Banci L, Bertini I, Turano P, Tien M, Kirk TK (1991) Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase. Proc Natl Acad Sci U S A 88:6956–6960

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Berry EA, Trumpower BL (1987) Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal Biochem 161:1–15

    Article  CAS  PubMed  Google Scholar 

  • Brown ME, Barros T, Chang MC (2012) Identification and characterization of a multifunctional dye peroxidase from a lignin-reactive bacterium. ACS Chem Biol 7:2074–2081

    Article  CAS  PubMed  Google Scholar 

  • Cao J, Woodhall MR, Alvarez J, Cartron ML, Andrews SC (2007) EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E. coli O157:H7. Mol Microbiol 65:857–875

    Article  CAS  PubMed  Google Scholar 

  • Chung N, Aust SD (1995) Veratryl alcohol-mediated indirect oxidation of phenol by lignin peroxidase. Arch Biochem Biophys 316:733–737

    Article  CAS  PubMed  Google Scholar 

  • Durão P, Bento I, Fernandes AT, Melo EP, Lindley PF, Martins LO (2006) Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies. J Biol Inorg Chem 11:514–526

    Article  PubMed  Google Scholar 

  • Fernandes AT, Martins LO, Melo EP (2009) The hyperthermophilic nature of the metallo-oxidase from Aquifex aeolicus. Biochim Biophys Acta 1794:75–83

    Article  CAS  PubMed  Google Scholar 

  • Hewson WD, Hager LP (1979) Oxidation of horseradish peroxidase compound II to compound I. J Biol Chem 254:3182–3186

    CAS  PubMed  Google Scholar 

  • Hiner AN, Hernandez-Ruiz J, Rodriguez-Lopez JN, Garcia-Canovas F, Brisset NC, Smith AT, Arnao MB, Acosta M (2002) Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation. J Biol Chem 277:26879–26885

    Article  CAS  PubMed  Google Scholar 

  • Hofrichter M, Ullrich R, Pecyna MJ, Liers C, Lundell T (2010) New and classic families of secreted fungal heme peroxidases. Appl Microbiol Biotechnol 87:871–897

    Article  CAS  PubMed  Google Scholar 

  • Jongbloed JD, Grieger U, Antelmann H, Hecker M, Nijland R, Bron S, van Dijl JM (2004) Two minimal Tat translocases in Bacillus. Mol Microbiol 54:1319–1325

    Article  CAS  PubMed  Google Scholar 

  • Kandelbauer A, Gübitz GM (2005) Bioremediation for the decolorization of textile dyes—a review. In: Lichtfouse E, Schwarzbauer J, Robert D (eds) Environmental chemistry. Springer, Berlin, pp 269–288

    Chapter  Google Scholar 

  • Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371

    Article  CAS  PubMed  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  PubMed Central  PubMed  Google Scholar 

  • Krishnappa L, Monteferrante CG, van Dijl JM (2012) Degradation of the twin-arginine translocation substrate YwbN by extracytoplasmic proteases of Bacillus subtilis. Appl Environ Microbiol 78:7801–7804

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kuan I-C, Johnson K, Tien M (1993) Kinetic analysis of manganese peroxidase. J Biol Chem 268:20064–20070

    CAS  PubMed  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  PubMed  Google Scholar 

  • Liers C, Ullrich R, Hofrichter M, Minibayeva FV, Beckett RP (2011) A heme peroxidase of the ascomyceteous lichen Leptogium saturninum oxidizes high-redox potential substrates. Fungal Genet Biol 48:1139–1145

    Article  CAS  PubMed  Google Scholar 

  • Liu X, Du Q, Wang Z, Zhu D, Huang Y, Li N, Wei T, Xu S, Gu L (2011) Crystal structure and biochemical features of EfeB/YcdB from Escherichia coli O157: ASP235 plays divergent roles in different enzyme-catalyzed processes. J Biol Chem 286:14922–14931

    Article  CAS  PubMed  Google Scholar 

  • Martinez AT, Ruiz-Duenas FJ, Martinez MJ, Del Rio JC, Gutierrez A (2009) Enzymatic delignification of plant cell wall: from nature to mill. Curr Opin Biotechnol 20:348–357

    Article  CAS  PubMed  Google Scholar 

  • Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, Henriques AO (2002) Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277:18849–18859

    Article  CAS  PubMed  Google Scholar 

  • Mendes S, Farinha A, Ramos CG, Leitao JH, Viegas CA, Martins LO (2011a) Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye-containing wastewaters. Bioresour Technol 102:9852–9859

    Article  CAS  PubMed  Google Scholar 

  • Mendes S, Pereira L, Batista C, Martins LO (2011b) Molecular determinants of azo reduction activity in the strain Pseudomonas putida MET94. Appl Microbiol Biotechnol 92:393–405

    Article  CAS  PubMed  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  PubMed Central  PubMed  Google Scholar 

  • Pereira L, Coelho A, Viegas AC, Ganachaud C, Lacazio G, Tron T, Robalo M, Martins LO (2009a) On the mechanism of biotransformation of the anthraquinonic dye Acid Blue 62 by laccases. Adv Synth Catal 351:1857–1865

    Article  CAS  Google Scholar 

  • Pereira L, Coelho AV, Viegas CA, Santos MM, Robalo MP, Martins LO (2009b) Enzymatic biotransformation of the azo dye sudan orange G with bacterial CotA-laccase. J Biotechnol 139:68–77

    Article  CAS  PubMed  Google Scholar 

  • Poulos TL, Kraut J (1980) The stereochemistry of peroxidase catalysis. J Biol Chem 255:8199–8205

    CAS  PubMed  Google Scholar 

  • Roberts JN, Singh R, Grigg JC, Murphy ME, Bugg TD, Eltis LD (2011) Characterization of dye-decolorizing peroxidases from Rhodococcus jostii RHA1. Biochemistry 50:5108–5119

    Article  CAS  PubMed  Google Scholar 

  • Rodriguez-Couto S (2009) Enzymatic biotransformation of synthetic dyes. Curr Drug Metab 10:1048–1054

    Article  CAS  PubMed  Google Scholar 

  • Ruiz-Duenas FJ, Martinez AT (2009) Microbial degradation of lignin: how a bulky recalcitrant polymer is efficiently recycled in nature and how we can take advantage of this. Microbiol Biotechnol 2:164–177

    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 β-carotene. Appl Microbiol Biotechnol 77:1241–1250

    Article  CAS  PubMed  Google Scholar 

  • Sezer S, Genebra T, Mendes S, Martins LO, Todorovic S (2012) A DyP-type peroxidase at a bio-compatible interface: structural and mechanistic insights. Soft Matter 8:10314–10321

    Article  CAS  Google Scholar 

  • Sezer S, Santos A, Kielb P, Pinto T, Martins LO, Todorovic S (2013) Distinct structural and redox properties of heme active in bacterial DyP-type peroxidases from two subfamilies: resonance Raman and electrochemistry study. Biochemistry 52:3074–3084

    Article  CAS  PubMed  Google Scholar 

  • Singh R, Grigg JC, Armstrong Z, Murphy ME, Eltis LD (2012) Distal heme pocket residues of B-type dye-decolorizing peroxidase: arginine but not aspartate is essential for peroxidase activity. J Biol Chem 287:10623–10630

    Article  CAS  PubMed  Google Scholar 

  • Strittmatter E, Liers C, Ullrich R, Wachter S, Hofrichter M, Plattner DA, Piontek K (2012) First crystal structure of a fungal high-redox potential dye-decolorizing peroxidase: substrate interaction sites and long-range electron transfer. J Biol Chem 288:4095–4102

    Article  PubMed  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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Sugano Y, Matsushima Y, Shoda M (2006) Complete decolorization of the anthraquinone dye reactive blue 5 by the concerted action of two peroxidases from Thanatephorus cucumeris Dec 1. Appl Microbiol Biotechnol 73:862–871

    Article  CAS  PubMed  Google Scholar 

  • Sugano Y, Muramatsu R, Ichiyanagi A, Sato T, Shoda M (2007) DyP, a unique dye-decolorizing peroxidase, represents a novel heme peroxidase family: ASP171 replaces the distal histidine of classical peroxidases. J Biol Chem 282:36652–36658

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed  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  PubMed  Google Scholar 

  • van Bloois E, Torres Pazmino DE, Winter RT, Fraaije MW (2010) A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily. Appl Microbiol Biotechnol 86:1419–1430

    Article  PubMed Central  PubMed  Google Scholar 

  • van der Ploeg R, Barnett JP, Vasisht N, Goosens VJ, Pother DC, Robinson C, van Dijl JM (2011) Salt sensitivity of minimal twin arginine translocases. J Biol Chem 286:43759–43770

    Article  PubMed  Google Scholar 

  • van der Ploeg R, Monteferrante CG, Piersma S, Barnett JP, Kouwen TR, Robinson C, van Dijl JM (2012) High-salinity growth conditions promote Tat-independent secretion of Tat substrates in Bacillus subtilis. Appl Environ Microbiol 78:7733–7744

    Article  PubMed Central  PubMed  Google Scholar 

  • Wong DW (2009) Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol 157:174–209

    Article  CAS  PubMed  Google Scholar 

  • Yoshida T, Tsuge H, Konno H, Hisabori T, Sugano Y (2011) The catalytic mechanism of dye-decolorizing peroxidase DyP may require the swinging movement of an aspartic acid residue. FEBS J 278:2387–2394

    Article  CAS  PubMed  Google Scholar 

  • Zubieta C, Joseph R, Krishna SS, McMullan D, Kapoor M, Axelrod HL, Miller MD, Abdubek P, Acosta C, Astakhova T, Carlton D, Chiu HJ, Clayton T, Deller MC, Duan L, Elias Y, Elsliger MA, Feuerhelm J, Grzechnik SK, Hale J, Han GW, Jaroszewski L, Jin KK, Klock HE, Knuth MW, Kozbial P, Kumar A, Marciano D, Morse AT, Murphy KD, Nigoghossian E, Okach L, Oommachen S, Reyes R, Rife CL, Schimmel P, Trout CV, van den Bedem H, Weekes D, White A, Xu Q, Hodgson KO, Wooley J, Deacon AM, Godzik A, Lesley SA, Wilson IA (2007a) Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA. Proteins 69:234–243

    Article  CAS  PubMed  Google Scholar 

  • 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 (2007b) Crystal structures of two novel dye-decolorizing peroxidases reveal a β-barrel fold with a conserved heme-binding motif. Proteins 69:223–233

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Rita Catarino for assistance in preliminary studies. Smilja Todorovic and Eduardo P. Melo are acknowledged for helpful discussions. This work was partially supported by the project grant BIORENEW-FP6-2004-NMP-NI-4/026456 from European Commission, PTDC/AGR-CFL/103840/2008 and PEst-OE/EQB/LA0004/2011 from Fundação para a Ciência e Tecnologia (FCT), Portugal.

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  1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av da República, 2780-157, Oeiras, Portugal

    Ana Santos, Sónia Mendes, Vânia Brissos & Lígia O. Martins

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  1. Ana Santos
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  2. Sónia Mendes
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  3. Vânia Brissos
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  4. Lígia O. Martins
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Correspondence to Lígia O. Martins.

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Ana Santos and Sónia Mendes contributed equally to this work.

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Santos, A., Mendes, S., Brissos, V. et al. New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications. Appl Microbiol Biotechnol 98, 2053–2065 (2014). https://doi.org/10.1007/s00253-013-5041-4

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