Electrochemistry and kinetics of fungal laccase mediators

Abstract

The screening of potential redox mediators for laccase was performed using homogeneous Trametes hirsuta laccase. Heterogeneous (electrochemical) and homogeneous (oxidation by laccase) reactions of the different types of the enhancers (mediators) of the enzyme were investigated. It was discovered that derivatives of phenyl-methyl-pyrazolones and benzoic acid, as well as N-hydroxynaphthalimide were efficient substrates for the laccase. The characterization of several representatives from each class was carried out using electrochemical and enzyme kinetics methods. The kinetic parameters for the oxidation of phenyl-methyl-pyrazolones and 3-(6-hylroxy)-aminobenzoic acid were comparable to those for 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by the laccase, whereas the rate of enzymatic oxidation of N-hydroxynaphthalimide was sufficiently lower. Electrochemical experiments demonstrated that only oxidation of phenyl-methyl-pyrazolones and N-hydroxynaphthalimide yielded several high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, whereas derivatives of benzoic acid showed low-potential intermediate, which was not able to oxidized lignin model compound. Phenyl-methyl-pyrazolones was about 50% as effective in degrading veratryl alcohol compared to ABTS as judged from HPLC kinetic studies, whereas N-hydroxynaphthalimide showed the same efficiency as ABTS. Phenyl-methyl-pyrazolones and hydroxynaphthalimides may be of commercial interest for oxidoreductase-catalyzed biodegradation of different xenobiotics.

Introduction

Laccase (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) catalyzes oxidation of ortho- and para-diphenols, aminophenols, polyphenols, polyamines, lignins and aryl diamine as well as some inorganic ions, coupled to the reduction of molecular dioxygen to water [1], [2]. The enzymes belong to a group of “blue” multicopper oxidases and they are classified into two groups in accordance with their source, i.e. plant and fungal. However, diphenol oxidases (laccases) have also been identified in eubacteria [3], [4], [5] and insects [6], [7]. Laccase contains four metal ions historically classified into three types, e.g. T1, T2, T3, according to their spectral characteristics [2].

The key characteristic of laccase is the standard redox potential of the T1 Cu site. The value of the redox potential of the T1 site has been determined using potentiometric titrations with redox mediators for a great number of different laccases and varies between 430 and 790 mV vs. NHE [8], [9], [10], [11], [12], [13], [14]. It has been shown for some laccases that the T1 site is the primary center accepting electrons from donor substrates [1], [2], [9] or electrodes [14], [15]. In addition, the catalytic efficiency (k_cat/K_M) for some donor substrates depends on the redox potential of the T1 site [9], [16]. This observation explains why laccases with high redox potentials of the T1 sites are of special interest in biotechnology, particularly for different bleaching [17], [18], [19] and bioremediation processes [20], [21].

Laccase is a mandatory enzyme for lignin conversion, since fungal laccase-deficient mutants completely loose the ability to degrade lignin [22], [23]. However, the mechanism of lignin transformation in nature is very complex, and the role of laccase in this process is not fully understood. Lignin, a highly branched, irregular three-dimensional organic polymer, is the most abundant biopolymer in Nature next to cellulose [24]. This natural polymer contains different structures including phenolic and non-phenolic compounds. Conventionally, the role of fungal laccases in lignin degradation was thought to be limited only to the oxidation of low-redox potential phenolic substructures of the polymer. This conclusion was based on the values of the redox potentials of T1 sites of the enzymes, which do not exceed 800 mV. When natural and artificial redox mediators (better referred as “enhancers”) of the enzyme were found, it was realized that laccase can play a broader role in lignin degradation [25], [26], [27]. Mediators or enhancers of laccase are enzyme substrates, which, being enzymatically oxidized, form highly reactive intermediates [28], [29], [30]. These intermediates can react with non-phenolic, strong lignin substructures, resulting in degradation of wood [26], [31], [32], [33]. The major difference between a mediator and an enhancer is the ability of the first one to cycle between its oxidized and reduced states for unlimited period of time. True redox mediators are not consumed during the redox transformation, whereas enhancers fall out from the catalytic cycle of the enzyme [34], [35], [36]. To a first approximation, only complexes of transition metals can be true mediators of laccase, whereas all organic substrates are essentially enzyme “enhancers” [35], [36]. However, the term “mediator” has been conventionally used for all enhancers of laccases. The confusion in the terminology is still unresolved. Therefore, although both terms are used in this work with respect to the studied substances, we actually mean the “enhancer” nature of the substrates.

Laccase-mediator (or laccase-enhancer) systems are employed in different biotechnological processes, such as green biodegradation of xenobiotics including pulp bleaching [17], [26], bioremediation [21], labeling in immunoassays [37], and green organic synthesis [38]. However, broader application of well-known mediators is restricted by their high price and limited effectiveness. One of the prospective trends in laccase research is screening and identification of new, low-cost redox mediators of laccases. The studies of new mediators are aimed at understanding the mechanism of their redox transformations, their efficiency in homogeneous reactions catalyzed by laccase, and, finally, their ability to degrade different kinds of xenobiotics.

Electrochemical studies of different laccase-mediator systems were performed previously [39], [40], [41], [42], [43]. We have shown in our recent publications that phenyl-methyl-pyrazolones are promising enhancers for laccase in the enzymatic degradation of lignin and its model compounds [36], [44]. Low-cost enhancers can broaden the application of laccase-mediator systems in large-scale biotechnological processes. In the present study, the effect of different substitutions into the structure of phenyl-3-methyl-pyrazolones on the efficiency of degradation of lignin model compounds has been investigated. The novel enhancers have been compared to the substances from other structural groups, namely, derivatives of benzoic acid and N-hydroxynaphthalimides, under the same experimental conditions.