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

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.

Bio-removal of Azo Dyes: A Review

“…This fact reveals that dye reducing activity is not dependent on the intracellular uptake of the dye (Pearce et al, 2003). On the basis of reducing equivalent used to reduce azo linkages, enzymes have been identified as FMN-dependent reductases (Burger and Stolz, 2010), FMN-independent reductases (Burger and Stolz, 2010), NADH-dependent reductases (Misal et al, 2011), NADPH-dependent reductases (Mendes et al, 2011) and NADH-DCIP reductases (Phugare et al, 2010). NADH-DCIP reductases are believed to be marker enzymes of bacterial and fungal mixed function oxidase systems, and mediate the detoxification of xenobiotic compounds (Bhosale et al, 2006).…”

Improving Kinetic or Thermodynamic Stability of an Azoreductase by Directed Evolution

“…These enzymes require two cycles of NADPH-dependent reduction of FMN to FMNH 2 for reducing the azo substrate to two amines and the quinone substrate to a hydroquinone. Pseudomonas putida MET94 is a bacteria that degrades a wide range of structurally distinct azo dyes with high efficiency and the azoreductase PpAzoR was shown to play a key role in this process [29]. Its broad substrate specificity makes it attractive for bioremediation processes [30] but its low kinetic stability impairs exploitation of its full potential for environmentally related applications.…”

Aromatic Amines Sources, Environmental Impact and Remediation

“…Monitorization of aromatic amines degradation by HPLC has been done by many authors (Carvalho et al 2010;Khalid et al 2009). Aromatic amines resulted from azo dye reduction have also been commonly identified by HPLC, using standard compounds for comparison (Carvalho et al 2008;Mendes et al 2011;Ramalho et al 2004) (Fig. 7.9).…”

“…. Products of the reaction were identified, in comparison to the standards: 2-aminobenzoic acid (1), N,N′-dimethyl-p-phenylenediamine (2), 4-aminoresorcinol (3) and aniline (4) (Mendes et al 2011) …”