Mediators laccase oxidation

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]. 

Desulfurization using purified enzymes Investigations into enzymatic desulfurization as an alternative to microbial desulfurization has revealed several enzymes capable of the initial oxidation of sulfur. A study reported use of laccase with azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a mediator for oxidation of DBT [181]. The rate of this reaction was compared to hydrogen peroxide-based phosphotungstic acid-catalyzed oxidation and the latter was found to be about two orders of magnitude higher. The authors also oxidized diesel oil sulfur to no detectable levels via extraction of the oxidized sulfur compounds from diesel. In Table 9, the enzymes used in oxidation of DBT to DBTO are reported. 

Mediator-assisted oxidation of fibre lignin Laccase Colloidal lignin [ox] Fibre lignin... 

The initial work on laccase oxidation of lignin in the presence of 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), ABTS, as mediator demonstrated that simple nonphenolic model compounds could be oxidized as shown in Figure 11.41 [184]. Thereby, the function of laccases was given a broader importance than just as oxidant of phenolic substrates. Subsequent work has demonstrated that in the... 

C Crestini, DS Argyropoulos. On the role of 1-hydroxybenzotriazole as mediator in laccase oxidation of residual kraft lignin. In DS Argyropoulos, ed. Oxidative Delignification Chemistry Fundamentals and Catalysis. ACS Symposium Series, 785, pp. 373-390, 2001. 

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. 

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. 

A Majcherczyk, C Johannes. Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase. Biochim. Biophys. Acta-General Subjects 1474(2) 157-162, 2000. 

In 1996 it was shown that by using ABTS as a mediator, laccase from Trametes versicolor was able to catalyze the aerobic oxidation of a series of benzylic alcohols to the corresponding benzaldehydes 46). Subsequently, Galli, and co-workers found that the stable N-oxy radical, 2,2,6,6-tetramethyl-piperidinyl-l-oxy (TEMPO, Fig. 4) in combination with laccase from Trametes villosa, catalyzes the aerobic oxidation of primary benzylic alcohols 47). The selective oxidation of the primary alcohol moiety in carbohydrates had been previously reported in two patents 48,49). 

Laccase/Mediator Cataeyzed Oxidation oe Benzyeic Aecohoes 47,50)... 

The oxidation of pollutants by laccase is normally limited to aromatic compounds with one phenolic group, but in the presence of low molecular weight compounds, known as mediators, it can also oxidize other compounds, such as dyes [58] or alkenes and polycyclic aromatic hydrocarbons [59]. 

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. 

The oxidized mediator form, produced in the course of the enzymatic reaction, can nonenzymatically oxidize compounds (including nonphenolic lignin structures) with ionization potentials exceeding the potentials of laccases (Morozova and others 2007). 

Polyphenol oxidase occurs within certain mammalian tissues as well as both lower (46,47) and higher (48-55) plants. In mammalian systems, the enzyme as tyrosinase (56) plays a significant role in melanin synthesis. The PPO complex of higher plants consists of a cresolase, a cate-cholase and a laccase. These copper metalloproteins catalyze the one and two electron oxidations of phenols to quinones at the expense of 02. Polyphenol oxidase also occurs in certain fungi where it is involved in the metabolism of certain tree-synthesized phenolic compounds that have been implicated in disease resistance, wound healing, and anti-nutrative modification of plant proteins to discourage herbivory (53,55). This protocol presents the Triton X-114-mediated solubilization of Vida faba chloroplast polyphenol oxidase as performed by Hutcheson and Buchanan (57). 

Figure 6.12 A mechanism for the generation of lignin phenoxy radicals due to direct oxidation by laccase and a mediation process involving colloidal lignin fragments, according to Felby etal. (1997b).

Scheme 7.8 Selective laccase/mediator oxidation of the natural glycoside thiocolchicoside...

---