Characteristics And Use Of Multicopper Oxidases Enzymes

Multicopper Oxidases, MCO are a family of enzymes that catalyse oxidation reactions of a substrate with simultaneous reduction of molecular oxygen to water. The universal catalytic centre is responsible for these reactions for all MCOs, composed of 4 or more copper atoms which form T1, T2 types and diatomic T3 types. Although the structure of the catalytic centre of most enzymes classified as MCO is similar, their biological functions and catalytic properties may be completely different.

So far (according to the UniProt database) circa 50 thousand various enzymes characterized by the presence of a catalytic centre typical of MCO and the ability to oxidize organic and inorganic compounds have been recognised. Many researchers have sought to analyse the characteristics on the basis of which it is possible to properly classify a newly identified enzyme which displays MCO properties. However, this problem still seems to be unresolved for most MCOs due to the highly similar properties of most substrates and considerable homology of amino acid sequences (especially within the catalytic centre). The most commonly described MCOs include laccases (especially from fungi belonging to the Basidiomycota division), and so-called Laccase-like Multicopper Oxidases (LMCO), ascorbate oxidase (mainly described in higher plants), bilirubin oxidase (e.g. Myrothecium verrucaria), some fungal pigments and ferroxidases (e.g. Fet3p of Saccharomyces cerevisiae yeasts) [12, 26, 44].

The very high oxidation-reducing potential of MCOs and the diversity of reactions they catalyse have caused these enzymes to become highly popular among researchers worldwide and offer many possibilities of application. MCOs are relatively stable enzymes, easy to separate from a culture and purify, which, combined with their low substrate specificity, makes them a valuable tool in drug production processes [5], elimination of phenolic compounds from alcohol products [13], dye synthesis [19], degradation and decolorization of wood pulp [93] or detoxification of xenobiotics [40]. It is mainly laccases and laccase-like enzymes that have attracted the attention of both the scientific community and industry [40, 93, 94]. Little attention has been devoted so far to other groups of MCO enzymes, which, although relatively less widespread in the natural environment, are an equally interesting object of research and offer a possibility of practical application.

A common feature of all enzymes included in the MCO family is the presence of a catalytic centre composed of at least four copper atoms divided – on account of their spectroscopic and magnetic properties – into three types: T1 and T2 types – containing one copper atom and the diatomic T3 type [67]. Type T1 gives the enzyme molecule a blue colouring and exhibits intense light absorption at a wavelength of 610 nm, resulting from a covalent copper-cysteine bond. In turn the T2 type is colourless and, similarly to the T1 type, detectable by means of electroparamagnetic resonance spectroscopy (EPR). The T3 type does not exhibit activity in EPR spectroscopy as a result of antiferromagnetic coupling of copper atoms. However, it is distinguished by a light absorption band at a wavelength of 330 nm [9] (Fig. 1).

Fig. 1.

In the T1 centre, where the substrate undergoes oxidation, the copper atom is bound to two histidine residues and one cysteine forming a distorted trigonal pyramid structure. The His-Cys-His sequence mentioned, which is characteristic of MCO, links T1 with T3. Sometimes the fourth amino acid residue, with weaker binding (most commonly methionine, leucine or phenylalanine), may occur in an axial position, which affects the oxidoreduction potential of the enzyme, stabilizes it and regulates its activity. The copper atom of T2 type and two atoms of T3 type, located in close proximity, are coordinated by the so-called interdomain copper binding sites, composed of 2 and 6 histidine residues respectively, and forming a triatomic copper cluster (known as Trinuclear Cu Cluster, TNC). It is a structure unique for the MCO family and is the place where binding and the four-electron reduction of molecular oxygen into water occurs [76].

The majority of MCOs contains about 500 amino acid residues and adopts the β-sheet layout in its secondary structure, shaped into the characteristic motif of the Greek Key [32, 43]. Typically, an MCO molecule consists of three domains formed in this manner. The T1 copper centre is located in domain 3 (blue copper-binding domain), and the T2/T3 triatomic copper cluster is located at the interface between domains 1 and 3, which is farther away from the protein surface compared to domain 3. However, apart from the three-domain MCOs, proteins possessing two or six domains have also been characterized [61].

The MCO catalytic mechanism includes (1) the reduction of the T1 Cu site by capturing an electron from the oxidized substrate, (2) transferring the electron from the T1 site to the TNC and (3) reduction of O₂ with formation of two water molecules (Fig. 2).

Fig. 2.

MCOs oxidize a wide spectrum of substrates, such as phenol, methoxyphenol, aromatic amines, multi-aromatic compounds, metal ions [39, 51]. MCO-catalysed reactions may occur directly (reactions of simple phenolic compound oxidation) or in the presence of a compound called a mediator, which mediates the transfer of electrons from the substrate to the active enzyme centre (Fig. 3). If direct oxidation of the phenolic substrate leads to the formation of its reactive and unstable radicals, these may, in the process of non-enzymatic, spontaneous coupling reactions combine to form dimers, oligomers or polymers [66].

Fig 3.

MCOs are a very diverse group of enzymes produced by both prokaryotic organisms and Eukaryota, and are characterized by various, not yet fully understood, biological functions. Although all MCOs exhibit the capability for oxidizing aromatic compounds, two functional classes can be distinguished among them [86]. The first one is the enzymes that oxidize organic substrates more readily than metal ions. The group consists mainly of laccases and laccase-like enzymes. The latter, in turn, oxidize metal ions, such as Fe (II), Cu (I) and/or Mn (II), with higher efficiency, compared to organic substrates. The latter enzymes are referred to as metal oxidases, and the most common ones among them are human ceruloplasmin (Cp) and yeast ferroxidase (Fet3p) [53]. The MCO division is not permanent and systematic, as there are no clear criteria for classification. For example, according to Hoegger et al. [26] multicopper oxidases form 10 enzyme groups: Basidiomycota laccases, Ascomycota laccases, insect laccases, MCO fungal pigments, fungal ferroxidases, plant and fungal ascorbate oxidases, plant-like enzymes like laccase, cooper resistance proteins (CopA), bilirubin oxidases and copper efflux proteins (CueO) (Table I). In turn, Sirim et al. [83] distinguished within the MCO family: laccases, ferroxidases, ascorbate oxidases (AO) and bilirubin oxidases (BOD). After integration of the sequence data and MCO structures, the Laccase Engineering Database (LccED) was constructed (https://lcced.biocatnet.de/). Currently, the database contains 16 MCO superfamilies containing over 14,000 amino acid sequences of 10,415 various proteins (Tab. I).

The identification of laccases from among multi-copper oxidases has remained ambiguous so far. Reiss et al. [71] proposed using the term “laccase” only for the enzyme isolated from the sap of Rhus vernicifera tree and introducing the term “laccase-like multi-copper oxidases” (LMCO) to account for the potential differences in their biological functions and biochemical features. In addition, Brander et al. [6] state that the term “laccases” was originally used in relation to plant-origin multicopper oxidases possessing three domains. Ihssen et al. [30] recommends classifying as laccases only those MCOs that have been isolated with urushiol – unsaturated alkyl catechol. The classification of enzymes in the MCO family is complex due to their broad substrate specificity, however, detailed biochemical characterization is necessary in order to organize the divisions proposed by the researchers. The division of MCOs is not permanent and systematic, as there are no clear criteria for classification. The division accepted by Hoegger et al. seems to be the most appropriate [26], however, due to slightly different biochemical properties and not fully specified biological functions, it seems reasonable to distinguish among the MCO also the group of laccase-like LMCO enzymes.

Until recently, the identification of enzymes included in the MCO was based mainly on the characteristics of their biochemical features and catalytic abilities. MCO interactions with substrates can be broadly divided into two categories and one can distinguish enzymes with low substrate specificity and enzymes with high specificity. The plant and fungal laccases belong to the first category and they can oxidize diphenols, aryl amines and aminophenols, and their Km values are generally within the range of 1–10 mM. The remaining MCOs have a significant degree of substrate specificity (Km < 1 mM) [85].

Some substances such as guaiacol, diammonium salt of 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP) and catechol have long been termed model laccase substrates [71]. However, it turned out that many of them are also oxidized by other enzymes from the MCO group, e.g. LMCO or bilirubin oxidases. Syringaldazine used to be considered to be a specific laccase substrate [64]. Syringaldazine and ABTS can be converted by MnP or LiP. However, the oxidation which depends on H₂O₂ allows for distinguishing these enzymes from MCO. MCO can also be distinguished from MnP using the leucoberbelin blue dye test. This compound reacts specifically with manganese ions released by MnP, resulting in the emergence of blue colour exhibiting the maximum absorption of light at the wavelength of 620 nm [18]. Figure 4 presents a scheme for the identification and differentiation of laccases from other ligninolitic enzymes proposed by Fernandes et al. [18].

Fig. 4.

While the ability to oxidize specific substrates allows one to quickly determine the activity of a given enzyme from the MCO group, in the era of the development of highly advanced molecular techniques, it should not be the only method of identification and characterization of the newly recognized protein. The development of omics techniques, such as genomics, transcriptomics and proteomics has contributed to determining the genes responsible for encoding enzymes, studying their expression at the level of the transcriptome and quantitative and qualitative analysis of the MCO against the background of other proteins in the body. Perry et al. [65] was the first to identify two genes encoding laccases Agaricus bisporus and found that these enzymes may exist as isoforms and be encoded by multigene gene families. Hence, hundreds of genes encoding MCO have been identified in both fungi and bacteria. The presence of many genes encoding enzyme isoforms in one strain may indicate the involvement of these enzymes in various physiological processes. The confirmation of this hypothesis may be the fact that individual enzyme isoforms often differ in substrate specificity and their activity may be different in different pH or temperature variants. Some of them have a constitutive character, and some may be induced, for example, by aromatic compounds or copper ions. Genomic and proteomic techniques have made it possible to identify consensus sequences for MCOs which distinguish them from other enzymes. These sequences contain four (L1, L2, L3, L4) contiguous fragments of copper-binding amino acid residues, whose degree of similarity, depending on the organism from which the enzyme is derived, can range from 75 to 85%. The L2 and L4 regions allow the enzyme to be classified as MCO, whereas the L1 and L3 sequences indicate the subgroup of the enzyme [23].

Among the microbial MCOs, laccases constitute the most studied and described group, being also the most numerous one. These are three-domain MCOs which were isolated for the first time by H. Yoshid in 1883 from resin from the Rhus vernicifera tree. Plant laccases, owing to the dehydrogenation mechanisms, play an important role in the polymerization of lignin-forming phenolic compounds, regeneration of damaged tissues and iron oxidation by converting Fe (II) to Fe (III) [15]. Although according to some researchers, the term laccase should be reserved exclusively for enzymes obtained from plants, also other three-domain MCOs, e.g. of microbial origin, are called laccase if only they exhibit the ability to oxidize aromatic compounds [61].

Laccases of fungal origin most often occur in the form of several monomers which oligomerize and then form multimeric complexes. The average molecular weight of the monomer ranges from about 50 to 110 kDa. An important feature of fungal laccases is a carbohydrate group with covalent binding, usually constituting 10 to 45% of the total enzyme molecule and consisting mainly of mannose, N-acetylglucosamine and galactose. All these features protect fungal laccases against proteolysis, high temperatures, extremely high or low pH values and other unfavourable factors [15, 74].

Among the fungi capable of the biosynthesis of laccases, the most numerous groups are those from the Basidiomycota division, e.g. Lentinus tigrinus, Agaricus bisporus, Trametes versicolor [82]. Amongst them, there occur quite often the so-called fungi of white wood rot, incl. strains of the species Phlebia radiate, Pleurotus ostreatus, Phanerochaete chrysosporium, Cerrena unicolor [87]. The synthesis of these enzymes has also been described in the fungi belonging to the Ascomycota division, e.g. Aspergillus niger, A. oryzae, Neurospora sp., Trichoderma atroviride and T. harzianum [87]. In fungi, laccases are involved in the processes of morphogenesis, lignin degradation and defence reactions to stress. These enzymes produced by saprophytic and mycorrhizal fungi are involved in the circulation of organic matter in the soil by degrading plant litter polymers or the formation of humic compounds [26]. Bacterial laccases have been identified in the cultures of strains including Azospirillum lipoferum, Escherichia coli, Bacillus subtilis and several species of Streptomycetes [7]. They were also described in Anabaena azollae cyanobacteria. Bacterial laccases are characterized by greater activity and stability than fungal enzymes at high temperatures, at alkaline pH and in the presence of high concentrations of chlorine and copper ions [15].

Both phenol and non-phenol substrates can undergo laccase-mediated catalytic reactions. In the case of molecules with high redox potential and with large size particles, which are not able to independently penetrate into the active enzyme centre, the action of the so called mediators is indispensable. They are organic compounds of low molecular weight which, when oxidized by laccase, form highly active cationic radicals capable of oxidising non-phenolic compounds. The most commonly used synthetic mediators are ABTS, hydroxyanthranilic acid (HAA), hydroxybenzotriazole (HBT) and hydroxyphthalimide (NPI). Natural mediators such as vanillin and syringaldehyde also have a similar effect [66].

Laccase-like multicopper oxidases (LMCO), similar to laccases, catalyse the oxidation of various substrates combined with the reduction of the O₂ molecule to two molecules of H₂O. Their biological functions are similar to the role of laccases, but not all of them have yet been recognized. LMCO have been described in many bacteria and fungi. The average molecular weight of LMCO is in the range of 51–66 kDa, while the number of amino acid sequences of enzymes is 470–600 aa. LMCO of Streptomyces bacterium have also been described, whose molecular mass is 32.6 kDa, and the amino acid sequence length is 297 aa. LMCOs of gram-negative bacteria differ from all other LMCOs through the presence of proline between the two histidines and the additional histidine in the second position after the HXH motif. LMCOs of bacterial origin are considered to be more effective in the decomposition of organic compounds than fungal LMCOs. On the other hand, fungal LMCOs have a wider substrate range than LMCOs from gram-negative bacteria [49].

The optimum pH value for LMCO activity is not the same because it depends on the substrate used for its measurement [49]. For example, three types of LMCO originating from Aspergillus niger: McoA, McoB and McoG were purified and characterized for their biocatalytic potential. All three enzymes were monomers with molecular weights in the range of 80 to 110 kDa. The highest McoA activity was observed in the pH 5.0 environment, while pH 6.0 was optimal for McoB and McoG. Additionally, McoA and McoB oxidized DMP-PDA (N, N-dimethyl-p-phenylenediamine) in a wider pH range than McoG [89]. The LMCO isolated from Myrothecium roridum showed activity of both MCO and bilirubin oxidase [35].

Bilirubin oxidase is a thermostable enzyme containing a disulphide bond. BOD catalyses the oxidation of tetrapyrroles, e.g. bilirubin to biliverdin as well as diphenols or aryl diamines with simultaneous reduction of four oxygen atoms to water [10]. This enzyme was discovered in the non-ligninolytic fungus Myrothecium verrucaria MT-1 in 1981 by Noriaki Tanaka and Sawao Murao. Unlike laccases, BODs are characterized by higher activity and stability at neutral pH and high temperature, however not higher than 60°C. They are also characterised by high tolerance to chloride anions and other chelators [52]. For example, bilirubin oxidase isolated from Myrothecium verrucaria (MvBOD) exhibited the highest catalytic activity in the temperature range of 30–60°C and pH from 7 to 8.5. However, in solutions with pH above 9, a decrease in BOD catalytic activity by as much as 50% has been observed [91].

Ascorbate oxidase catalyses the oxidation of ascorbate to dehydroascorbic acid with formation of H₂O in the presence of oxygen. It has been isolated from higher plants, in which it occurs in the largest amount in the cell wall and is involved in their growth [80, 85]. Ascorbate oxidase also participates in defence reactions by modifying the apoplastic space [26]. Its activity and expression are induced by auxin and light, which suggests that it is involved in signal transduction [95]. AO has also been described in microorganisms, including Myrothecium verrucaria, Aerobacter aerogenes, Acremonium sp. HI-25 [60, 80]. In contrast to laccases which act as monomers, it is necessary to create a homodimer structure for proper functioning of the AO. Such a protein structure also performs stabilizing functions [61].

The copper efflux oxidase (CueO), which is present in periplasm in E. coli, may oxidize p-phenylenediamine and 2,6-dimethoxyphenol. Like some other three-domain MCOs, it possesses ferroxidase activity. As a result, the enzyme not only protects the cell against the adverse effects of copper ions (through the oxidation of Cu⁺ to less harmful Cu²⁺), but also participates in iron homeostasis. CueO has been shown to oxidize the catechol groups of 2,3-dihydroxybenzoic acid, which is a precursor of enterobactin [24]. CueO has a structure similar to laccase and AO, but the conformation of the TNC makes it different from other MCOs. In addition, within the third domain, CueO contains methionine-rich regions which act as a copper ion sensor, in the presence of which the enzyme activity increases significantly [61, 72].

Another bacterial MCO is the copper resistance protein (CopA), consisting of three domains, described in Pseudomonas syringae or Xanthomonas campestris [26]. The activity of this enzyme was determined based on the ability to oxidize 2,6-dimethoxyphenol [84]. As reported by Nakamura and Go [61], in aerobic conditions CopA and CueO control copper metabolism by exporting the excess of Cu (I) from the cytoplasm and oxidation to Cu (II), which is less toxic.

The B. subtilis strain is capable of producing a thermostable protein – CotA, which coats endospores. CotA consists of more than 30 types of polypeptides and is resistant to both physical and chemical factors. It results from the function it performs, namely the production of melanin pigment, which protects against UV radiation and hydrogen peroxide [17, 29]. In addition, the protein is highly stable because the half-life of activity at 80°C was determined to be 2–4 hours [61]. According to Rajeswari [69], CotA laccase is similar to CueO in E. coli based on the construction of the catalytic centre, however, the cross-domain loop possesses sites at which allow for tighter packaging, which improves the stability of the entire structure and increases thermostability [61]. According to Enguit et al. [16], this segment contains only 4 of the 46 proline residues constituting the entire CotA. Therefore, it may suggest that the proline content both determines the thermostability of the protein and significantly increases it in combination with increased packaging [16].

MCO fungal pigments, found mainly in Ascomycota, including Aspergillus nidulans [90] are responsible for the oxidation of dihydroxyphenylalanine (DOPA) to dopaquinone along the melanin synthesis pathway. These enzymes differ significantly from other MCOs in the construction of the S2 region [44]. They oxidize typical laccase substrates, among others p-phenylenediamine, pyrogallol, gallic acid or ABTS [89].

MCO includes also ferroxidases, characterized by affinity to Fe (II), which is not shown by other multicopper oxidases. The most frequently described ferroxidases are the plasma membrane protein of Saccharomyces cerevisiae (Fet3p) and human ceruloplasmin (hCp) consisting of six domains. Based on X-ray examination of the crystal structure of ceruloplasmin, it was established that in the second, fourth and sixth domains there occurs a copper binding site, while the first and sixth domain divide the tri-nuclear, inter-domain copper binding site to form a pseudosymmetric C3 structure [61]. Cp exhibits the ability to oxidize aromatic diamines and other aromatic compounds [85]. Ferroxidases play an important role in iron homeostasis in yeasts and mammals [86]. Fet3p participates in the Fe (II) transport system with high affinity in yeast. Initially, Fe (III) is reduced to Fe (II) by ferroxidase, after which it is transported in the cell. Fet3p performs a protective role by suppressing the cytotoxic action of copper and iron [26, 85].

Another example of MCO is SLAC, a two-domain multicopper oxidase described in Streptomyces coelicolor, which exhibits the ability to oxidize aromatic and non-aromatic compounds containing amino and hydroxyl groups. Due to the similarity of the sequence to fungal laccases, but also smaller size, this enzyme has been defined as a small laccase. On the basis of the comparative analysis of protein sequences, the similarity of SLAC and other laccases in the position of metal ligands has been established. However, the occurrence of 24 histidine residues in the SLAC sequence has been established, which may indicate its role in binding the excess of intracellular copper ions in order to transfer them during export through the TAT secretory system [50]. SLAC is characterized by resistance to reducing compounds and thermal stability. In addition, this enzyme exhibits the highest activity in the environment with pH 9 [11, 50]. Owing to these features, SLAC have found their application in the pulp and paper industry for dye decolorization [11].

On account of the ability to oxidize many substrates, extracellular character and fairly high stability in a wide range of pH and temperatures, the MCO enzymes are characterized by a high application potential. So far enzymes from this group have found application in environmental protection, medicine, pharmaceutical industry, cosmetics and in the food industry. Laccases and laccase-like enzymes, which are the most common and characterized MCO group, are mainly used as biocatalysts in the synthesis reactions of new compounds, detection, biotransformation and biodegradation of toxic impurities (Table II).

MCO enzymes have been applied in many areas of the food industry, like baking, vegetable and fruit processing, winemaking and brewing. The bakery industry commonly uses laccases to improve bread structure, as well as the flavour and durability of pastries [73]. However, it has been proven that other MCOs, such as bilirubin oxidase, can be used to cross-link biopolymers by improving the physicochemical properties of food products [98]. The process increases the durability and stability of dough, at the same time reducing its viscosity. This effect was noted especially when using lower quality flour [51]. MCO is used, e.g., for cross-linking arabinoxylans so that the created network of transverse polymer bonds has a positive effect on crumb and crustiness of bread [40]. Laccases can be used instead of physical adsorbents like SO₂ to eliminate undesirable phenol derivatives, causing darkening and clouding of fruit juices, beers and wines [13, 40]. New reports have appeared lately, indicating the possible use of MCOs isolated from cultures of lactic acid fermentation bacteria, among others for removing biogenic amines from wine and some oriental cuisine products [2, 8, 25]. The use of MCO in the food industry is quite common. Preparations available on the market, such as Falouvorstar, Suberase or LACCASE Y120, which are based on laccase activity, are successfully used in brewing, production of corks for the wine industry and improvement of the colour values of food products [40].

Enzymes from the MCO group are used as biocatalysts in the reactions yielding many active substances which are components in the composition of household chemistry, body care products and medicines characterized by antimicrobial and antioxidant activity [79, 93]. Such activity is exhibited by molecular iodine (I₂), whose preparation through oxidising I^–has been described for MCOs isolated from the culture of Alphaproteobacterium sp. Q-1 and Roseovarius sp. A-2 [81, 88]. Strong antifungal activity has also been proven for iodinated phenolic compounds obtained in the reaction catalysed by laccase [31, 78]. Laccases are also successfully used as biocatalysts in the synthesis of drugs, among others β-lactam antibiotics and anti-cancer agents, e.g. vinblastines or mitomycin [45, 56]. Since 2006, when antiproliferative activity of laccase was demonstrated for the first time, intensive research has been conducted on the use of this enzyme as an anti-cancer agent [92]. The ability to inhibit cell division of breast, liver, colon and prostate cancer has been proven for laccases from various species of basidiomycetes [68, 70]. The application of laccase manufactured by Cerena unicolor in the treatment of blood and cervical cancer has been demonstrated and covered by patent protection [37, 54]. This enzyme, added in the right concentration, had a strong cytotoxic effect on cervical cancer cells of the SiHa and CaSki line and did not affect adversely the fibroblast cells constituting the reference system [59]. The same enzyme exerted pro-apoptotic action on blood cancer cells of Jurkat and RPMI 8226 lines [55]. MCOs may also have antiviral effects. Lentinus tigrinus, a fungal laccase, inhibits the activity of HIV-1 reverse transcriptase, without which the virus is unable to transcribe genetic material from RNA to DNA [96].

MCOs can be used for analytical purposes, including biological, enzymatic and immunochemical tests [94]. Laccase is used to detect the presence of morphine [5]. In turn, bilirubin oxidase-based (BOD) biosensors, designed in order to precisely determine the level of bilirubin, whose excessively high concentration in human serum is lethal, are characterized by high sensitivity and efficacy [28]. Furthermore, bilirubin and ascorbate oxidases have been used for clinical trials aimed at eliminating the effect of ascorbate, which adversely affected the liver [76]. The MCO catalytic activity, which is accompanied by the reduction of oxygen to water molecules, is used to obtain electrons, i.e. the driving force of biocells [52, 77].

In the cosmetics industry laccase is used for the production of dyes and can be used in non-toxic hair dyes instead of the oxidizing agent – hydrogen peroxide [4]. Such preparations are more convenient to use and less irritating to the organism due to the replacement of the oxidizing agent, which weakens hair and destroys their structure [13]. Moreover, laccases can be used in skin lightening preparations by reducing the content of melanin [40].

Biosynthesis of dyes using laccase is an environmentally friendly alternative to chemical synthesis of textile dyes and allows for reducing process costs. Substances obtained in this way are characterized by a wide range of colours and durability comparable to synthetic dyes [33]. On the other hand, these enzymes can also be used for decolorization of fabrics (e.g. jeans) or transforming dye precursors into their active forms, increasing the efficiency of the dyeing process [66]. This allows for limiting the application of chemical bleaches and is particularly useful for fabrics sensitive to chemical compounds [94].

Due to the ability to remove toxic phenols formed during the degradation of lignin, laccase participates in the reactions of its depolymerization [93]. In addition, these enzymes may be useful in the modification of cellulose fibres [40]. In order to improve the oxidation of non-phenolic compounds, laccase activity is supported by mediators, e.g. ABTS, TEMPO or HBT. However, the cost of synthetic mediators is an important limiting factor [94].

Mostly laccases of fungal origin have been widely applied to the process of bioremediation of contaminated areas [36]. These enzymes are used both in a free and immobilized form to eliminate a wide spectrum of toxic compounds, such as: phenolic compounds, chlorophenols, cyclic aromatic hydrocarbons or alkenes being components of, among others, pesticides. MCO enzymes can also be used for neutralization of compounds belonging to the group of the so-called hormone modulators (EDCs), i.e. compounds adversely affecting the function of the endocrine system of humans and animals [58]. The research carried out by Garcia-Morales et al. [22] demonstrated a high biocatalytic efficiency of the protein mixture (LacI and LacII) of the CS43 Pycnoporus sanguineus fungus, which was used in the biotransformation of EDCs such as bisphenol A, 4-nonylphenol, 17-α-ethinylestradiol and triclosan. The capabilities of different laccases, LMCO enzymes and bilirubin oxidase for decolorization of industrial dyes and their elimination from wastewater have also been widely explored [34, 94]. For example, a crude extract of the laccase derived from the fungus P. nebrodensis has shown effective decolorization (82.69%) of malachite green after just one hour of incubation [99].

MCO proteins are enzymes containing from one to six atoms of copper per molecule. Multicopper oxidases include laccases, ferroxidases, ascorbate oxidase, bilirubin oxidase, some fungal pigments with multicopper oxidase character and the so-called laccase-like enzymes. MCOs possess the ability to oxidize both organic and inorganic compounds. The reactions catalysed by MCO are accompanied by the reduction of molecular oxygen to water. These properties make them a valuable tool in bioremediation processes, medicine, pharmaceutical industry, cosmetics and food industry.