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- Comparison of Laccases and Hemeproteins Systems in Bioremediation of Organic PollutantsPublication . Lopes, João M.; Marques-da-Silva, Dorinda; Videira, Paula Q.; Lagoa, Ricardo L.Aim: Laccases and peroxidases have attracted great interest for industrial and environmental applications. These enzymes have a broad substrate range and a robust oxidizing ability. Moreover, using mediators or co-oxidants makes it possible to increase their catalytic activity and extend their substrate scope to more resistant chemical structures. Background: Fungal laccases and ligninolytic peroxidases, mainly lignin and manganese peroxidases, are the privileged oxidoreductases for bioremediation processes. Nonetheless, an increasing diversity of laccases and peroxidase-type enzymes has been proposed for environmental technologies. Objective: This article aims to provide an overview of these enzymes and compare their applicability in the degradation of organic pollutants. Methods: Fundamental properties of the proteins are covered and applications towards polycyclic aromatic hydrocarbons (PAHs) and pesticides are specially focused. Results: Laccases are multicopper oxidases initially studied for applications in the pulp and paper in-dustry but able to oxidize a variety of environmentally concerning compounds. Relying on O2, laccases do not require peroxides nor auxiliary agents, like Mn2+, although suitable redox mediators are needed to attack the more recalcitrant pollutants (e.g., PAHs). True and pseudo-peroxidases use a stronger oxi-dant (H2O2) and the redox chemistry at the heme site generates high potential species that allow the oxidation of dyes and some pesticides. Conclusion: Lately, research efforts have been directed to enzyme discovery, testing with micropollu-tants, and improving biocatalysts’ stability by immobilization and protein engineering. Further under-standing of the effects of natural media components and solvents on the enzymes might lead to compet-itive enzymatic treatments of highly toxic media.
- Use of Lipid Vesicles for Revealing the Potential Contribution of Cytochrome C in the Metabolism of Environmental ToxicantsPublication . Lopes, João M.; Marques-da-Silva, Dorinda; Videira, Paula A.; Lagoa, RicardoCytochrome c (Cc) is well-known as an electron carrier at mitochondria but can also catalyze peroxidase-like reactions. Although the reported catalytic abilities are low, the role of Cc in transforming environmental toxicants requires additional studies. We employed cardiolipin vesicles to stimulate the enzymatic activity of Cc and tested the catalytic system towards the dye methyl orange and the polycyclic aromatic hydrocarbons (PAHs) benzo[b]fluoranthene and benzo[a]pyrene. The results showed that cardiolipin-containing vesicles promoted dye decolorization and the oxidation of both PAHs by Cc, indicating that lipid membranes can be useful to further investigate Cc–mediated metabolism of toxicants.
- Comparison of cytochrome c with conventional biocatalysts in the degradation of environmental toxicantsPublication . Lagoa, Ricardo; Lopes, João M.; Rodrigues, J. Rui; Marques-da-Silva, DorindaPeroxidases and laccases are amongst the most attractive enzymes for the degradation of concerning pollutants like polycyclic aromatic hydrocarbons (PAHs) and organic dyes. In spite of the availability of high activity enzymes, their applicability is hindered by specificity and stability limitations in real environmental conditions, so more suitable biocatalysts are demanded. Cytochrome c (Cc) is best known as a protein electron carrier at mitochondria, but it also displays (pseudo-)peroxidase activity. In this work, we aimed to evaluate the potential of Cc as biocatalyst of PAHs’ and azo dyes’ degradation and compare it with two more conventional enzymes - plant peroxidases and fungi laccases. The studies were carried out with Cc from horse heart, horseradish peroxidase (HRP) and laccase from Trametes versicolor. The enzymes were tested with two major PAHs, anthracene and benzo[a]pyrene (BaP), and with methyl orange (MO) as a model azo dye. The enzyme-catalyzed oxidation of PAHs was determined by HPLC, and MO decolorization was followed in spectrophotometric kinetic assays. Several degradation studies were performed to assess the catalytic capacity at different pH and the effect of the redox mediator ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)). Peroxidase reactions of HRP and Cc were initiated with 100 μM H2O2 (in media containing 100 μM DTPA as metal chelator). In pH 5 acetate buffer, the specific ABTS-oxidizing activity of Cc was much lower than that of laccase and HRP. However, these two enzymes lost almost all activity at pH 7, while Cc exhibited catalytic activity even at pH 8 (phosphate buffer). Decolorization assays showed laccase alone to be a weak catalyst of MO degradation, although addition of ABTS to the reaction media greatly accelerated the transformation. On the contrary, both HRP and Cc directly catalyzed MO decolorization, including at neutral pH, and the redox mediator offered no advantage. As for the PAHs, HRP and laccase catalysis benefited from the presence of ABTS in the media, but again Cc oxidized anthracene and BaP directly. Indeed, 24h incubation of BaP (1 mg/L) with Cc (0.1 mg/mL) resulted in the transformation of 70±4% of the PAH and generation of major reaction products different of the BaP quinones produced by laccase- and HRP-ABTS systems. This work disclosed catalytic properties of Cc different from laccase and HRP that are relevant for the design of new enzymatic remediation processes.
- Removal of Hydrophobic Organic Pollutants and Copper by Alginate-Based and Polycaprolactone MaterialsPublication . Marques-da-Silva, Dorinda; Lopes, João M.; Correia, Iris; Silva, João S.; Lagoa, RicardoOrganic pollutants (OPs) and heavy metals are environmental toxicants associated with great concerns. Decontamination processes are urgent for both, and the possibility to achieve their simultaneous removal from polluted waters is highly interesting. Additionally, in many cases, the effect of organic matter in the removal process is overlooked and must be considered. This work aimed to study the potential of alginate-based and polycaprolactone (PCL) materials to remove OPs and copper ions in the absence and presence of organic matter. The OPs investigated were the polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, and the pesticide chlorpyrifos, both hydrophobic compounds. Copper (II) ions were used as a model of heavy metals. Alginate-based spheres were prepared by gelation, and PCL microparticles were obtained by oil/water emulsion solvent evaporation. The materials with the highest efficiencies for OP removal from aqueous solutions were those with activated carbon and PCL. Furthermore, the spheres with activated carbon could remove anthracene and copper simultaneously, even in the presence of humic acid. This work points to activated carbon–alginate spheres as a multifunctional adsorbent able to remove different pollutants and to PCL for potential applications in OP decontamination processes.