Biocatalyst Application

We have focused on the biodegradation of the most important groups of synthetic industrial colorants, namely the azo and anthraquinonic dyes. Around 106 tons of synthetic dyes are produced annually, of which 1–1.5 x 105 tons are released to the environment in wastewaters. In general, dyes are stable xenobiotic organic pollutants persisting in the environment and concerns have been raised that such artificial compounds may be carcinogenic. We showed that CotA-laccase is able to decolourise, at neutral or alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes (Pereira et al. 2009a). The enzymatic biotransformation of the azo dye Sudan Orange G and the anthraquinonic dye Acid Blue 62 have been addressed in more detail through a multidisciplinary approach that combined enzymology, electrochemistry, mass spectrometry, NMR and toxicology (Pereira et al. 2009 a, b). This had provided mechanistic insight over the enzymatic biotransformation process and will guide us to develop useful enzymatic technology tools. Additionaly, we have constructed an E. coli strain co-expressing the genes coding for the azoreductase of P. putida MET94 and CotA-laccase (Mendes et al. 2011b). Whole cell assays of the recombinant strain were performed in a sequential anaerobic vs aerobic process leading to high levels of decolourisation as well as detoxification of model dye-containing wastewaters.

We have recently investigated the enzymatic bioconversion of lignin model units (Rosado et al. 2012). Lignin is a heterogeneous aromatic polymer, highly recalcitrant towards degradation, however its chemical nature makes this polymer an interesting source of aromatic chemicals. Laccases are naturally involved in both lignin biosynthesis and biodegradation. Therefore, they have the highest potential for modification of lignocellulosic materials and isolated lignins. We provide insight into the mechanism of laccase mediator reactions using as mediators “natural” phenolic compounds. The efficiency of these mediators on the conversion of non-phenolic lignin units was tested at different pH values and increasing mediator/non-phenolic ratios using CotA-laccase and a fungal high redox laccase. The intermediates and products of reactions were identified by LC-MS and 1H NMR. These approaches allow concluding on the 1) mechanism involved in the oxidation of phenolics by laccases, 2) importance of the chemical nature and properties of phenolic mediators, 3) apparent independence of the enzyme’s properties on the yields of non-phenolics conversion, 4) competitive routes involved in the catalytic cycle of the laccase-mediator system with several new C-O coupling type products.

Coloured disubstituted benzoquinonimine trimeric structures were obtained as main reaction products of the oxidation of p-electron donor primary aromatic amines using two different laccases, CotA-laccase from Baccilus subtilus and TvL from Trametes versicolor (Sousa et al 2013). Our results also show that the occurrence and/or rates of oxidation of aromatic amines are strongly dependent on the presence of p-electron releasing substituents in the aromatic ring and are independent on the properties of the enzyme used. This work contribute for 1) understanding key features of laccase reactivity with p-substituted aromatic amines and 2) establishing enzymatic processes that lead to the synthesis of coloured bio-products in mild conditions with potential impact in the cosmetic and dye industries.


MET | Microbial & Enzyme Technology 2013