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


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.

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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.

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Correspondence to Lígia O. Martins.

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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)

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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).

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