High-redox potential laccases

Directed Evolution of Ligninoiytic High-Redox Potential Laccases (HRPLs)... 

E., Ballesteros, A., Camarero, S., and Alcalde, M. (2010) Laboratory evolution of high redox potential laccases. Chem. Biol., 17, 1030-1041. 

Mate, D.M., Garcia-Ruiz, E., Camarero, S., Shubin, V.V., Falk, M., Shleev, S., Ballesteros, A.O., and Alcalde, M. (2013) Switching from blue to yellow altering the spectral properties of a high redox potential laccase by directed evolution. Biocatal. Biotransfor., 31, 8-21. 

Shleev S, Shumakovich G, Morozova O, Yaropolov A. Stable floating air diffusion biocathode based on direct electron transfer reactions between carbon particles and high redox potential laccase. Fuel Cells 2010 10 726-733. 

Gonzalez Arzola K, Gimeno Y, Arevalo MC, Falcon MA, Hernandez Creus A. Electrochemical and AFM characterization on gold and carbon electrodes of a high redox potential laccase from Fusarium proliferatum. Bioelectrochemistry 2010 79 17-24. 

Blue multicopper oxidases (BMCOs) such as laccase, ceruloplasmin, bilirubin oxidase (BOx), and ascorbate oxidase (AOx) have been extensively investigated as cathodic biocatalysts for DET-based biodevices [44]. BMCOs have a catalytic center consisting of four coppers a type 1 (Tl) Cu site, which accepts electrons from the substrate and from the electrode surface, and a type 2/type 3 (T2/T3) cluster, where O2 is reduced directly to water. High redox potential laccases and BOx, with redox potential up to 780 and 670 mV versus normal hydrogen electrode (NHE), respectively [44,45], can be used to create efficient biocathodes with current densities up to a few mA cm . In 2012, Shleev and coworkers used the DET ability of these enzymes to create several completely DET-based BFCs [42]. The enzymes have also been used in different MET-based approaches [46,47] specifically, Heller and coworkers... 

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]. 

The redox potential of blue copper oxidases varies from species to species. The high redox potential of around 700 mV in fungal laccase is primarily attributed to nonaxial methionine ligand, a geometry that stabilizes the reduced state. Other factors such as solvent accessibility, dipole orientation, and hydrogen bonding also play an important role. ... 

Visible MCD spectra of plastocyanin, azurin, Rhus vernicifera laccase, ascorbate oxidase and ceruloplasmin are similar on a per copper basis, but show differences from those of stellacyanin and fungal laccase. This is of interest in view of the absence of methionine from the coordination sphere of copper in stellacyanin, and the very high redox potential of fungal laccase.925... 

The redox properties can be divided among those involved with its reduction by potential substrates of the enzyme, its reoxidation by molecular oxygen, and by electron transfer to and from other redox centers within the molecule. Type 1 Cu + is reduced rapidly by a variety of both one-electron and potential two-electron substrates via a one electron process 63, 64). A likely reason for this ease of reduction of Type 1 Cu2+ ion lies with its exceptionally high redox potential (0.77 V at pH 6.2, Table 2). Introduction of molecular oxygen to reduced laccase results in a rapid reappearance of the blue color characteristic of Type 1 Cu2+. The question of intramolecular electron transfer occurring during reoxidation will be considered below. 

The Type 3 Coppers. There are two atoms of Cu bound to laccase which are not detected by EPR and which appear to be completely diamagnetic even at room temperature (57, 58). Three experimental observations which are thought to be relevant to these atoms are (a) The presence of a two-electron acceptor in laccase having a very high redox potential [61, 62) (b) The concomitant attenuation of 330 nm absorption associated with reduction of the two-electron acceptor [62) (c) all Cu in fungal laccase is in the Cu2+ form (55). 

The discovery of mediators (small molecules which when oxidised by peroxidases or laccases form highly reactive species), which have the abihty to oxidise high redox potential substrates difficult to be oxidised by the enzyme alone, has further expanded the applications of these enzymes in modifying inert polymers. Examples of widely investigated mediators are 1-hydroxybenzotriazole (HBT), violuric acid (VA), A-hydroxyacetanilide (NHA) and 2,2 -azinobis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS). Alternatively, active research is aimed at developing natural cost-effective lignin-derived mediators [10]. 

The first successful example of the directed evolution of fungal laccase involved the laccase from the thermophile ascomycete Myceliophihora thermophila laccase (MtL). This study led to subsequent directed evolution experiments in S. cerevisiae with several high-redox potential ligninolytic oxidoreductases (see below). MtL was subjected to 10 cycles of directed evolution to enhance its functional expression in S. cerevisiae [38]. The best performing variant of this process (the T2 mutant that harbored 14 mutations) exhibited a total improvement of 170-fold in activity its expression levels were enhanced 8-fold and the around 22-fold. The... 

Expression system of CotA-laccase for directed evolution and high-throughput screenings for the oxidation of high-redox potential dyes. Biotechnol. J., 4, 558-563. 

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