Advertisement

Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Molecular determinants of azo reduction activity in the strain Pseudomonas putida MET94

  • 604 Accesses

  • 22 Citations

Abstract

Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study, Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows a ping-pong bi–bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. Anjaneyulu Y, Chary N, Raj D (2005) Decolourization of industrial effluents—available methods and emerging technologies—a review. Rev Environ Sci Bio/Technol 4:245–273

  2. Bin Y, Jiti Z, Jing W, Cuihong D, Hongman H, Zhiyong S, Yongming B (2004) Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737. FEMS Microbiol Lett 236:129–136

  3. Blumel S, Stolz A (2003) Cloning and characterization of the gene coding for the aerobic azoreductase from Pigmentiphaga kullae K24. Appl Microbiol Biotechnol 62:186–190

  4. Blumel S, Knackmuss HJ, Stolz A (2002) Molecular cloning and characterization of the gene coding for the aerobic azoreductase from Xenophilus azovorans KF46F. Appl Environ Microbiol 68:3948–3955

  5. Chen H (2006) Recent advances in azo dye degrading enzyme research. Curr Protein Pept Sci 7:101–111

  6. Chen H, Wang RF, Cerniglia CE (2004) Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis. Protein Expr Purif 34:302–310

  7. Chen H, Hopper SL, Cerniglia CE (2005) Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein. Microbiology 151:1433–1441

  8. Chen H, Xu H, Kweon O, Chen S, Cerniglia CE (2008) Functional role of Trp-105 of Enterococcus faecalis azoreductase (AzoA) as resolved by structural and mutational analysis. Microbiology 154:2659–2667

  9. Chen H, Feng J, Kweon O, Xu H, Cerniglia CE (2010) Identification and molecular characterization of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24. BMC Biochem 11:13

  10. de Lorenzo V (2001) Cleaning up behind us. The potential of genetically modified bacteria to break down toxic pollutants in the environment. EMBO Rep 2:357–359

  11. Deller S, Macheroux P, Sollner S (2008) Flavin-dependent quinone reductases. Cell Mol Life Sci 65:141–160

  12. Hsueh CC, Chen BY (2007) Comparative study on reaction selectivity of azo dye decolorization by Pseudomonas luteola. J Hazard Mater 141:842–849

  13. Husain Q (2006) Potential applications of the oxidoreductive enzymes in the decolorization and detoxification of textile and other synthetic dyes from polluted water: a review. Crit Rev Biotechnol 26:201–221

  14. Ito K, Nakanishi M, Lee WC, Sasaki H, Zenno S, Saigo K, Kitade Y, Tanokura M (2006) Three-dimensional structure of AzoR from Escherichia coli. An oxidereductase conserved in microorganisms. J Biol Chem 281:20567–20576

  15. Kandelbauer A, Guebitz GM (2005) Bioremediation for the decolorization of textile dyes—a review. Env Chem 269–288

  16. Khersonsky O, Tawfik DS (2010) Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu Rev Biochem 79:471–505

  17. Leelakriangsak M, Huyen NT, Towe S, van Duy N, Becher D, Hecker M, Antelmann H, Zuber P (2008) Regulation of quinone detoxification by the thiol stress sensing DUF24/MarR-like repressor, YodB in Bacillus subtilis. Mol Microbiol 67:1108–1124

  18. Lewis JA, Escalante-Semerena JC (2006) The FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme of Salmonella enterica converts tricarballylate into cis-aconitate. J Bacteriol 188:5479–5486

  19. Liu G, Zhou J, Lv H, Xiang X, Wang J, Zhou M, Qu Y (2007a) Azoreductase from Rhodobacter sphaeroides AS1.1737 is a flavodoxin that also functions as nitroreductase and flavin mononucleotide reductase. Appl Microbiol Biotechnol 76:1271–1279

  20. Liu ZJ, Chen H, Shaw N, Hopper SL, Chen L, Chen S, Cerniglia CE, Wang BC (2007b) Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis. Arch Biochem Biophys 463:68–77

  21. Liu G, Zhou J, Wang J, Yan B, Li J, Lu H, Qu Y, Jin R (2008) Site-directed mutagenesis of substrate binding sites of azoreductase from Rhodobacter sphaeroides. Biotechnol Lett 30:869–875

  22. Liu G, Zhou J, Fu Q, Wang J (2009) The Escherichia coli AzoR is involved in resistance to thiol-specific stress caused by electrophilic quinones. J Bacteriol 191:6394–6400

  23. Loh KC, Cao B (2008) Paradigm in biodegradation using Pseudomonas putida—a review of proteomics studies. Enzym Microb Technol 42:1–12

  24. Maier J, Kandelbauer A, Erlacher A, Cavaco-Paulo A, Gubitz GM (2004) A new alkali-thermostable azoreductase from Bacillus sp. strain SF. Appl Environ Microbiol 70:837–844

  25. Mansour H, Corroler D, Barillier D, Ghedira K, Chekir L, Mosrati R (2007) Evaluation of genotoxicity and pro-oxidant effect of the azo dyes: acids yellow 17, violet 7, and orange 52, and of their degradation products by Pseudomonas putida mt-2. Food Chem Toxicol 45:1670–1677

  26. Mansour H, Mosrati R, Corroler D, Ghedira K, Barillier D, Chekir L (2009) In vitro mutagenicity of Acid Violet 7 and its degradation products by Pseudomonas putida mt-2: correlation with chemical structures. Environ Toxicol Pharmacol 27:231–236

  27. Matsumoto K, Mukai Y, Ogata D, Shozui F, Nduko J, Taguchi S, Ooi T (2010) Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus. Appl Microbiol Biotechnol 86:1431–1438

  28. Moutaouakkil A, Zeroual Y, Zohra Dzayri F, Talbi M, Lee K, Blaghen M (2003) Purification and partial characterization of azoreductase from Enterobacter agglomerans. Arch Biochem Biophys 413:139–146

  29. Nachyar C, Rajakumar G (2005) Purification and characterization of an oxygen insensitive azoreductase from Pseudomonas aeruginosa. Enz Microb Tecnhol 36:505–509

  30. Nakanishi M, Yatome C, Ishida N, Kitade Y (2001) Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase. J Biol Chem 276:46394–46399

  31. Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, Martins dos Santos VA, Fouts DE, Gill SR, Pop M, Holmes M, Brinkac L, Beanan M, DeBoy RT, Daugherty S, Kolonay J, Madupu R, Nelson W, White O, Peterson J, Khouri H, Hance I, Lee P, Holtzapple E, Scanlan D, Tran K, Moazzez A, Utterback T, Rizzo M, Lee K, Kosack D, Moestl D, Wedler H, Lauber J, Stjepandic D, Hoheisel J, Straetz M, Heim S, Kiewitz C, Eisen JA, Timmis KN, Dusterhoft A, Tummler B, Fraser CM (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4:799–808

  32. Pereira L, Coelho AV, Viegas CA, Ganachaud C, Lacazio G, Tron T, Robalo MP, Martins LO (2009a) On the mechanism of biotransformation of the anthraquinonic dye acid blue 62 by laccases. Adv Synth Catal 351:1857–1865

  33. 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

  34. Prigione V, Tigini V, Pezzella C, Anastasi A, Sannia G, Varese G (2008) Decolourisation and detoxification of textile effluents by fungal biosorption. Water Res 42:2911–2920

  35. Rai HS, Bhattacharyya MS, Singh J, Vats P, Banerjee UC (2005) Removal of dyes from the effluent of textile and dyestuff manufacturing industry: a review of emerging techniques with reference to biological treatment. Critical Rev Environ Sc Tech 35:219–238

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

  37. Ryan A, Laurieri N, Westwood I, Wang CJ, Lowe E, Sim E (2010) A novel mechanism for azoreduction. J Mol Biol 400:24–37

  38. Sollner S, Nebauer R, Ehammer H, Prem A, Deller S, Palfey BA, Daum G, Macheroux P (2007) Lot6p from Saccharomyces cerevisiae is a FMN-dependent reductase with a potential role in quinone detoxification. FEBS J 274:1328–1339

  39. Sollner S, Deller S, Macheroux P, Palfey BA (2009) Mechanism of flavin reduction and oxidation in the redox-sensing quinone reductase Lot6p from Saccharomyces cerevisiae. Biochemistry 48:8636–8643

  40. Suzuki Y, Yoda T, Ruhul A, Sugiura W (2001) Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp. OY1-2 isolated from soil. J Biol Chem 276:9059–9065

  41. Timmis KN, Pieper DH (1999) Bacteria designed for bioremediation. Trends Biotechnol 17:200–204

  42. Towe S, Leelakriangsak M, Kobayashi K, Van Duy N, Hecker M, Zuber P, Antelmann H (2007) The MarR-type repressor MhqR (YkvE) regulates multiple dioxygenases/glyoxalases and an azoreductase which confer resistance to 2- methylhydroquinone and catechol in Bacillus subtilis. Mol Microbiol 66:40–54

  43. van der Zee FP, Villaverde S (2005) Combined anaerobic–aerobic treatment of azo dyes—a short review of bioreactor studies. Water Res 39:1425–1440

  44. Wang CJ, Hagemeier C, Rahman N, Lowe E, Noble M, Coughtrie M, Sim E, Westwood I (2007) Molecular cloning, characterization and ligand-bound structure of an azoreductase from Pseudomonas aeruginosa. J Mol Biol 373:1213–1228

  45. Zimmermann T, Kulla HG, Leisinger T (1982) Properties of purified Orange II azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46. Eur J Biochem 129:197–203

Download references

Acknowledgements

We thank Diana Rocheta and Catarina Pimenta for their help in the preliminary studies. This work was supported by the FP6-NMP2-CT-2004-505899 (SOPHIED) and PTDC/BIO/72108/2006 project grants. L. Pereira holds a post-doctoral fellowship (SFRH/BPD/20744/2004) from Fundação para a Ciência e Tecnologia.

Author information

Correspondence to Lígia O. Martins.

Electronic supplementary materials

Below is the link to the electronic supplementary material.

Table 1S

Bacterial strains screened for decolourisation of synthetic dyes (DOC 78 kb)

Figure 1S

a pH profile of methyl red decolourisation by P. putida MET94 whole cells. b Dependence of decolourisation rates on the biomass concentration (DOC 41 kb)

Figure 2S

pH (a) and temperature (b) profile of PpAzoR. Reactions performed under anaerobic conditions using 2 mM of dye in the presence of 0.25 mM of NADPH (DOC 39 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mendes, S., Pereira, L., Batista, C. et al. Molecular determinants of azo reduction activity in the strain Pseudomonas putida MET94. Appl Microbiol Biotechnol 92, 393–405 (2011). https://doi.org/10.1007/s00253-011-3366-4

Download citation

Keywords

  • Pseudomonas putida
  • Azoreductase
  • Azo dyes
  • Decolourisation
  • Whole cell catalysis