Laccases redox

Figure 17.3 Anatomy of a redox enzyme representation of the X-ray crystallographic structure of Trametes versicolor laccase III (PDB file IKYA) [Bertrand et al., 2002]. The protein is represented in green lines and the Cu atoms are shown as gold spheres. Sugar moieties attached to the surface of the protein are shown in red. A molecule of 2,5-xyhdine that co-crystallized with the protein (shown in stick form in elemental colors) is thought to occupy the broad-specificity hydrophobic binding pocket where organic substrates ate oxidized by the enzyme. Electrons from substrate oxidation are passed to the mononuclear blue Cu center and then to the trinuclear Cu active site where O2 is reduced to H2O. (See color insert.)...

Figure 17.6 Redox hydrogel approach to immobilizing multiple layers of a redox enzyme on an electrode, (a) Structure of the polymer, (b) Voltammograms for electrocatalytic O2 reduction by a carbon fiber electrode modified with laccase in the redox hydrogel shown in (a) (long tether) or a version with no spacer atoms in the tether between the backbone and the Os center (short tether). Reprinted with permission fi om Soukharev et al., 2004. Copyright (2004) American Chemical Society.

Mano N, Soukharev V, Heller A. 2006. A laccase-wiring redox hydrogel for efficient catalysis of O2 electroreduction. J Phys Chem B 110 11180-11187. 

The low specificity of electron-donating substrates is remarkable for laccases. These enzymes have high redox potential, making them able to oxidize a broad range of aromatic compounds (e.g. phenols, polyphenols, methoxy-substituted phenols, aromatic amines, benzenethiols) through the use of oxygen as electron acceptor. Other enzymatic reactions they catalyze include decarboxylations and demethylations [66]. 

Reported redox potentials of laccases are lower than those of non-phenolic compounds, and therefore these enzymes cannot oxidize such substances [7]. However, it has been shown that in the presence of small molecules capable to act as electron transfer mediators, laccases are also able to oxidize non-phenolic structures [68, 69]. As part of their metabolism, WRF can produce several metabolites that play this role of laccase mediators. They include compounds such as /V-hvdi oxvacetan i I ide (NHA), /V-(4-cyanophenyl)acetohydroxamic acid (NCPA), 3-hydroxyanthranilate, syringaldehyde, 2,2 -azino-bis(3-ethylben-zothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP), violuric acid, 1-hydroxybenzotriazole (HBT), 2,2,6,6-tetramethylpipperidin-iV-oxide radical and acetovanillone, and by expanding the range of compounds that can be oxidized, their presence enhances the degradation of pollutants [3]. 

Some proteins may have redox centers with ° s significantly higher than +0.4 V (e.g., the multicopper protein laccase). No general approach is presently available to deal with the high end of the biological redox window, and, in fact, the reduction potentials of these systems (again, e.g., laccases) are frequently poorly defined. 

The laccases, classed as polyphenol oxidases, catalyze the oxidation of diphenols, polyamines, as well as some inorganic ions, coupled to the four-electron reduction of oxygen to water see Fig. 12.4 for the proposed catalytic cycle. Due to this broad specificity, and the recognition that this specificity can be extended by the use of redox mediators [27], laccases show promise in a range of applications [28], from biosensors [29-32], biobleaching [27, 33-35] or biodegradation [36], to biocatalytic fuel cells [1-3, 18, 26, 37-42]. 

Co-immobilization of this redox polymer with a fungal laccase from Trametes versicolor, possessing a Tl copper site reduction potential of +0.57 V vs Ag/AgCl ( +0.77 vs NHE), was achieved using a diepoxide cross-linker, in an approach... 

An alternative strategy for co-immobilization of mediator and GOx is based on adsorption of enzyme, cross-linked, as was described for the laccase-based biocatalytic cathodes [30, 37 42], to an osmium-based redox polymer film, on carbon electrodes [1-3, 54],... 

The laccase molecule is a dimeric or tetrameric glycoprotein, which contains four copper atoms per monomer, distributed in three redox sites. More than 100 types of laccase have been characterized. These enzymes are glycoproteins with molecular weights of 50-130 kDa. Approximately 45% of the molecular weight of this enzyme in plants are carbohydrate portions, whereas fungal laccases contain less of a carbohydrate portion (10-30%). Some studies have suggested that the carbohydrate portion of the molecule ensures the conformational stability of the globule and protects it from proteolysis and inactivation by radicals (Morozova and others 2007). 

Although several works suggested that these enzymes cannot oxidize nonphenolic compounds because the redox potentials of laccases are lower than those of the... 

The discovery of ABTS as a laccase substrate mediating or enhancing the enzyme action was essential to increase the range of molecules that can be converted by laccases (Fig. 4.5). Such a mediator requires several conditions (1) it must be a good laccase substrate (2) its oxidized and reduced forms must be stable (3) it must not inhibit the enzymatic reaction and (4) its redox conversion must be cyclic. 

Claus H, Faber G, Koenig H (2002) Redox-mediated decolorization of synthetic dyes by fungal laccases. Appl Microbiol Biotechnol 59 672-678... 

Dye decolorizing potential of the WRF Ganoderma lucidum KMK2 was demonstrated for recalcitrant textile dyes. G. lucidum produced laccase as the dominant lignolytic enzyme during SSF of wheat bran, a natural lignocellulosic substrate. Crude enzyme shows excellent decolorization activity to anthraquinone dye Rema-zol Brilliant Blue R without redox mediator, whereas diazo dye Remazol Black-5 (RB-5) requires a redox mediator [43]. 

Both purified laccase as well as the crude enzyme from the WRF Cerrena unicolor were used to convert the dyes in aqueous solution. Biotransformation of the dyes was followed spectrophotometrically and confirmed by high performance liquid chromatography. The results indicate that the decolorization mechanism follows MichaeliseMenten kinetic and that the initial rate of decolorization depends both on the structure of the dye and on the concentration of the dye. Surprisingly, one recalcitrant azo dye (AR 27) was decolorized merely by purified laccase in the absence of any redox mediator [46],... 

In Phanerochaete flavido-alba, an induction of ligninolytic activities that was ascribed to phenolic compounds was evidenced [69]. Phenols have also been shown to have an important role as redox mediators for dye degradation with laccases from Pycnoporus cinnabarinus and Trametes villosa, and they resulted to be necessary to degrade a strongly recalcitrant azo dye, the Reactive Black 5 [70]. 

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