Enzymatic Degradation and Synthesis of Azo and Antraquinonic Dyes

Principal Investigator of the IST team: Cristina Anjinho Viegas

Principal investigator of gobal project: Lígia O  Martins (IBET, Oeiras)

Contract: PTDC/BIO/72108/2006   - Engenharia Bioquímica e Biotecnologia

Start date: 01/09/2007

Duration: 36 months


New legislations in conjunction with international trade pressures such as increasing competition and the introduction of ECOLABELS for textile, cosmetic, leather, paper products on the European and US markets, is threatening the very survival of the colour industry in many industrialized countries. About 15% of annual synthetic dye production is released in industrial effluents. These dyes are designed to resist fading upon exposure to sweat, light, water, oxidizing agents and microbial attack. Therefore they are hardly  removed from effluents by conventional wastewater treatment. There are now reasonable evidences that a significant proportion of existing dyes in waste water streams can be eliminated with the aid of enzymes under relatively gentle conditions. This proposal will focus primarily on research involving recombinant CotA-laccase from Bacillus subtilis in the degradation and synthesis of the most important groups of synthetic industrial colorants, namely the azo and anthraquinone dyes. Decoulorisation and dye removal using a biotechnological approach remains technically attractive and the development of a laccase based system is quite promising due to the high relative non-specific oxidation capacity of these enzymes. We want to get deep insight over the ability of CotA bacterial laccase to degrade azo and anthraquinonic dyes. The substrate specificity of CotA-laccase and the influence of chemical-physical parameters on the reaction towards structurally different dyes will be assessed. The integration of these results with the identification of degradation products and toxicological data obtained will allow the set-up of an effective enzymatic bioremediation process where the need of a highly pure enzyme preparation is probably useless and instead a cheaper crude extract could be used. In addition, the identification of degradation products will contribute to our knowledge on the catalytic mechanism employed by the enzyme. In fact, these data in combination with the existent X-ray structures for CotA-laccase  available could serve as basis for structure-function relationships studies by using site-directed mutagenesis under the frame of a financed FCT project underway and hopefully will allow the design of efficient laccase that would fit better in biotechnological applications. Attempts will also done towards the synthesis of dyes by using the laccase system. We will explore the laccase-mediated oxidative coupling reactions ability for the biological synthesis of industrial dye molecules with the view of replacing the chemical synthesis that operates at harsh conditions and high chemicals dosage. Any replacement technology is unlikely to provide cost reductions for industry but could have significant positive environmental impact. This approach is also an opportunity to find new dyes, less toxic and produced under safe conditions.

The IST team is assigned to the task of evaluating and comparing the potential toxicity of the industrial dyes under study with that of the respective degradation products. This will be based primarily on microplate susceptibility assays using the experimental eukaryotic model microorganism Saccharomyces cerevisiae. Results obtained will give indications on the potential modification of toxicity that may be associated with dye removal and decoulorisation.