Laccases redox mediators

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]. 

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]. 

The fuel cell described above exhibited three key flaws. First, the anode redox mediator operates at a redox potential well above that of glucose oxidase, raising the operating potential of the anode and lowering the achievable cell potential. Second, the cell operates at pH 5, near-optimal for the laccase electrode but suboptimal for the current-limiting glucose... 

The redox potential of the Tl Cu-site has been determined using potentiometric titrations with redox mediators for a large number of different laccases and varies between 410 mV vs. NHE for Rhus vernicifera [67] and 790 mV for laccases from Polyporus versicolor and Coriolus hirsutus [244,251]. The T2 and T3 sites have higher potentials [251]. 

The results of a simultaneous spectrophotometric titration at 330 and 610 nm are depicted in a double Nemst plot in Figure 5, giving an E o,sso value for the type 3 center that is nearly identical to E o.eio of the type 1 copper. However, at lower temperatures (e.g., 10°C) a value of 30 10 mV was measured for the diflFerence E 0,330 E o,qio- A similar redox situation is found for tree laccase (E o for the type 1 copper, 394 mV E o for the type 3 copper, 434 mV). However, at 25°C, where the difference E 0,330 E o.eio is considerably diminished in ascorbate oxidase (in the presence of the redox mediator ferricyanide) thermodynamic control for the occupancy of the individual copper sites is less pronounced. 

To better understand the mechanisms for the oxidation of lignin by a laccase mediator system, a laccase from Polyporous sp, kindly provided by Novozymes, was used in combination with 1-HBT. The redox mediator was found to be partly regenerated during the oxidation of lignin dimer 1 in the presence of laccase. A free radical of 1-HBT generated by laccase was probably responsible for the oxidation of I [146]. The free radical of 1-HBT was, however, transformed to benzotriazole, which could not mediate the oxidation of I. A proposed mechanism for the laccase mediator oxidation of nonphenolic lignins is given in Scheme 14.1. 

To investigate the importance, not only of laccase mediators, but also of lacca-ses per se, several laccases were studied for the oxidation of the nonphenolic lignin dimer I. In the presence of the redox mediators 1-HBT or violuric acid, it was found that the oxidation rates of dimer I by the laccases differed considerably. In oxidation of dimer I, both 1-HBT and violuric acid were to some extent, consumed. The consumption rate followed the same order of laccases as the oxidation rates of dimer I. The oxidation rate of dimer I was found to be dependent on both k, jt and the stability of the laccase in question. Both 1-HBT and violuric acid inactivated the laccases— violuric acid to a greater extent then 1-HBT. The presence of dimer I in the reaction mixture slowed down this inactivation. Inactivation seems to be mainly due to the reaction of the redox mediator free-radical with the laccases. No relationship between the carbohydrate content of the laccases and their inactivation was found. When the redox potential of the laccases is in the range of 750-800 mV, i.e., about that of the redox mediator, a further increase in redox potential does not affect k(,jt and the oxidation rate of dimer I [147]. 

Li, K.C., E. Xu, and K.E.L. Eriksson. Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound. Appl Environ Microbiol 65(6) 2654-2660, 1999. 

Fig. (10). The three model compounds, used for assessing the redox mediating role of 3-hydroxyanthranilic acid in delignification by laccase...

Interestingly, ABTS was much less effective as a redox mediator than HA when P. cinnabarinus laccase acted on III. Moreover, veratryl alcohol (which is a non-substrate for laccase alone) was efficiently converted to veratraldehyde by laccase when ABTS was added to the reaction mixture, whereas less than 10% was oxidised to the aldehyde when the mediator was HA. The reason of this mechanistic difference in regioselectivity of... 

The vast majority of enzyme biofuel cells is based on the electroenzymatic oxidation of glucose by glucose oxidase (GOX) and oxygen reduction by laccase, rarely, bilirubin oxidase, or even ascorbate oxidase. Usually two couples of redox mediators are involved in the functioning of the enzymatic biofuel cell. One is required to establish an electrical connection between the electrode surface and the reduced form of flavin adenine dinucleotide, the prosthetic center of GOX. The second couple, located at the cathode, allows the electron transfer from the electrode siuface to the copper center of laccase where the oxygen reduction takes place (Fig. 3.2). 

Covalent functionlization of CNTs was employed in a flexible way to modify single-walled carbon nanotubes (SWCNTs) by the corresponding redox mediator prior to their deposition on electrode. R. Bilewicz et al. reported the covalent functionalization of SWCNTs with ferrocene and ABTS [6]. Immobilized ferrocene acts as a redox bridge for the electrical wiring of GOX while at the anode and ABTS-modified SWCNTs serve for the electrical connection of laccase at the cathode. 

First examples of partially mediatorless-based glucose biofuel cells (GBFCs) showed DET at the laccase-modified cathode while a redox mediator still had to be used to connect an enzyme at the anode. 

Treatment of lignins or aromatics by laccases/peroxidases in the presence of different redox mediators has been shown to produce different effects as summarised by Sena-Martins et al. [82], Stewart [85], Kobayashia and Higashimura [44]. Several authors have observed a decrease in phenolic and carboxylic groups, and an increase in new C-C, aryl-aryl and aryl-alkyl bonds [28, 65, 78, 84]. 

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