Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) belong to the multicopper oxidase family, along with such different proteins as plant ascorbic oxidase, mammalian ceruloplasmin or Fet3p ferroxidase from Saccharomyces cerevisiae, among others [1]. These copper-containing enzymes catalyze the oxidation of various substrates with the simultaneous reduction of molecular oxygen to water [2]. Yoshida first discovered laccases in 1883 after observing that latex from the Japanese lacquer tree (Rhus vernicifera) hardened in the presence of air [3,4]. This makes laccase as one of the oldest enzymes ever described. Since then, laccase activity has been found in plants, some insects [5,6], and few bacteria [7]. However, most biotechnologically useful laccases (i.e. those with high redox potentials) are of fungi origin. Over 60 fungal strains belonging to Ascomycetes, Deuteromycetes and especially Basidiomycetes show laccase activities. Among the latter group, white-rot fungi are the highest producers of laccases but also litter-decomposing and ectomycorrhizal fungi secret laccases [8].

Laccases are typically monomeric extracellular enzymes containing four copper atoms bound to 3 redox sites (T1, T2 and T3). The termed "blue copper" at the T1 site-because of its greenish-blue colour in its oxidized resting state-is responsible of the oxidation of the reducing substrate. The trinuclear cluster (containing one Cu T2 and two Cu T3) is located approx. 12 Å away from the T1 site, and it is the place where molecular oxygen is reduced to water [1]. Laccases catalyze one-electron substrate oxidation coupled to the four-electron reduction of O₂. It is assumed that laccases operate as a battery, storing electrons from the four individual oxidation reactions of four molecules of substrate, in order to reduce molecular oxygen to two molecules of water.

Fungal laccases often occur as multiple isoenzymes expressed under different cultivation conditions (e.g. inducible or constitutive isoforms). Most are monomeric proteins, although laccases formed by several units have been also described [9,10]. They are glycoproteins with average molecular mass of 60–70 kDa, and carbohydrate contents of 10–20% which may contribute to the high stability of laccases. The covalently linked carbohydrate moiety of the enzyme is typically formed by mannose, N-acetylglucosamine and galactose. The amino acid chain contains about 520–550 amino acids including a N-terminal secretion peptide [4].

Biological functions attributed to laccases include spore resistance and pigmentation [11,12], lignification of plant cell walls [13], lignin biodegradation, humus turnover and detoxification processes [8], virulence factors [12], and copper and iron homeostasis [14].

Laccases exhibit an extraordinary natural substrate range (phenols, polyphenols, anilines, aryl diamines, methoxy-substituted phenols, hydroxyindols, benzenethiols, inorganic/organic metal compounds and many others) which is the major reason for their attractiveness for dozens of biotechnological applications [15-17]. Moreover, in the presence of small molecules, known as redox mediators, laccases enhance their substrate specificity. Indeed, laccase oxidizes the mediator and the generated radical oxidizes the substrate by mechanisms different from the enzymatic one, enabling the oxidative transformation of substrates with high redox potentials-otherwise not oxidized by the enzyme-, Figure 1A. The industrial applicability of laccase may therefore be extended by the use of a laccase-mediator system (LMS). Thus, laccase and LMS find potential application in delignification and biobleaching of pulp [18-21]; treatment of wastewater from industrial plants [22,23]; enzymatic modification of fibers and dye-bleaching in the textile and dye industries [24,25]; enzymatic crosslinking of lignin-based materials to produce medium density fiberboards [26]; detoxification of pollutants and bioremediation [27-31]; detoxification of lignocellulose hydrolysates for ethanol production by yeast [32,33]; enzymatic removal of phenolic compounds in beverages-wine and beer stabilization, fruit juice processing [34-36]-; and construction of biosensors and biofuel cells [37].

In organic synthesis, laccases have been employed for the oxidation of functional groups [38-42], the coupling of phenols and steroids [43-45], the construction of carbon-nitrogen bonds [46] and in the synthesis of complex natural products [47] and more.

As mentioned above, many of these applications require the use of redox mediators opening a big window for new biotransformations of non-natural substrates towards which laccase alone hardly shows activity. On the other hand, in most of the cases large quantities of enzymes are required, which makes the efficient expression of laccase in heterologous systems an important issue. Moreover, the protein engineering of fungal laccases with the aim of improving several enzymatic features (such as activity towards new substrates, stability under harsh operating conditions -e.g. presence of organic cosolvents, extreme pH values-, thermostability, and others) is a critical point in the successful application of this remarkable biocatalyst. All these issues are addressed in the following lines, paying special attention to their application in organic synthesis.

Enzymatic polymerization using laccases has drawn considerable attention recently since laccase or LMS are capable of generating straightforwardly polymers that are impossible to produce through conventional chemical synthesis [127].

For example, the polymerization ability of laccase has been applied to catechol monomers for the production of polycatechol [127]. Polycatechol is considered a valuable redox polymer; among its applications are included chromatographic resins and the formation of thin films for biosensors. Former methods for the production of polycatechol used soybean peroxidase or horseradish peroxidase (HRP), which suffer from the common "suicide H₂O₂ inactivation". The main limitation of all heme-containing peroxidases is their low operational stability, mostly due to their rapid deactivation by H₂O₂-with half-lifes in the order of minutes in the presence of 1 mM H₂O₂ [127,137].

Inert phenolic polymers, for example poly(1-napthol), may also be produced by laccase-catalyzed reactions [125,138-140]. These polymers have application in wood composites, fiber bonding, laminates, foundry resins, abrasives, friction and molding materials, coatings and adhesives [125,141].

The enzymatic preparation of polymeric polyphenols by the action of laccases has been investigated extensively in the past decades as a viable and non-toxic alternative to the usual formaldehyde-based chemical production of these compounds [142-144]. Poly(2,6-dimethyl-1,4-oxyphenylene)-"poly(phenylene oxide)", PPO-, is widely used as high-performance engineering plastic, since the polymer has excellent chemical and physico-mechanical properties. PPO was first prepared from 2,6-dimethylphenol monomer using a copper/amine catalyst system. 2,6-Dimethylphenol was also polymerized through HRP catalysis to give a polymer consisting of exclusively 1,4-oxyphenylene units [145]. On the other hand, a small amount of Mannich-base and 3,5,3'5'-tetramethyl-4,4'-diphenoquinone units are contained in the commercially available PPO. The polymerization also proceeded under air in the presence of laccase derived from Pycnoporus coccineus without the addition of H₂O₂ [123,146].

It has been also reported that laccase induced a new type of oxidative polymerization of 4-hydroxybenzoic acid derivatives, 3,5-dimethoxy-4-hydroxybenzoic acid (syringic acid) and 3,5-dimethyl-4-hydroxybenzoic acid. The polymerization involved elimination of CO₂ and H₂ from the monomer to give PPO derivatives with molecular weight up to 1.8 × 10⁴ (Figure 5A) [145,147].

A novel system of enzymatic polymerization, i.e. a laccase-catalyzed cross-linking reaction of new "urushiol analogues" for the preparation of "artificial urushi" polymeric films (Japanese traditional coating) has been demonstrated (Figure 5B) [148-151]. Flavonoids have been also polymerized by polyphenol oxidase and laccase. The flavonoid-containing polymers showed good antioxidant properties and enzyme inhibitory effect [152].

It has been reported that laccase induced radical polymerization of acrylamide with or without mediator [146]. Laccase has been also used for the chemo-enzymatic synthesis of lignin graft-copolymers [153]. Along these lines, the potential of this enzyme for crosslinking and functionalizing lignocellulose compounds is also reported [154]. Laccases can be used in the enzymatic adhesion of fibers in the manufacturing of lignocellulose-based composite materials, such as fiber boards. In particular, laccase has been proposed to activate the fiberbound lignin during manufacturing of the composites, and boards with good mechanical properties without toxic synthetic adhesives have been obtained by using laccases [155,156]. Another possibility is to functionalize lignocellulosic fibers by laccases in order to improve the chemical or physical properties of the fiber products. Preliminary results have shown that laccases are able to graft various phenolic acid derivatives onto kraft pulp fibers [157,158]. This ability could be used in the future to attach chemically versatile compounds to the fiber surfaces, possibly resulting in fiber materials with completely novel properties, such as hydrophobicity or charge.

Finally, laccase-TEMPO mediated system has been also used to catalyze the regioselective oxidation of the primary hydroxyl groups of sugar derivatives or even starch, pullulan and cellulose allowing the polymer functionalization [132,159]. The efficiency of this system was initially tested with mono- and disaccharides (i.e., phenyl β-D-glucopyranoside), and the corresponding glucopyranosiduronates were isolated and characterized (Figure 6A). Subsequently, this chemo-enzymatic approach has been exploited to achieve the partial oxidation of a water soluble cellulose sample. Also, the same approach has been applied for the mild oxidation of the glycosylated saponin, asiaticoside [160] (Figure 6B), and a series of natural glycosides [133].

Laccases have been used to synthesize products of pharmaceutical importance. The first chemical that comes to mind is actinocin, synthesized via a laccase-catalyzed reaction from 4-methyl-3-hydroxyanthranilic acid as shown in Figure 7A. This pharmaceutical product has proven effective in the fight against cancer as it blocks transcription of tumor cell DNA [161,162].

Other examples of the potential application of laccases for organic syntheses include the oxidative coupling of katarantine and vindoline to yield vinblastine. Vinblastine is an important anti-cancer drug, especially useful in the treatment of leukemia. Vinblastine is a natural product that may be extracted from the plant Catharanthus roseus. The compound is however only produced in small quantity in the plant, whereas the precursors-namely katarantine and vindoline- are at much higher concentrations, and thus are relatively inexpensive to obtain and purify. A method of synthesis has been developed through the use of laccase with preliminary results reaching 40% conversion of the precursors to vinblastine [2]. Laccase coupling has also resulted in the production of several other novel compounds that exhibit beneficial properties, e.g. antibiotic properties [163].

The study of new synthetic routes to aminoquinones is of great interest because a number of antineoplast drugs in use, like mitomycin, or under development, like nakijiquinone-derivatives [164] or herbamycin-derivatives [165], contain an aminoquinone moiety. Several simple aminoquinones possess activity against a number of cancer cell-lines [166-168] as well as antiallergic or 5-lipoxygenase inhibiting activity [168,169].

Laccases have also been employed to synthesize new cyclosporin derivatives [170]. Cyclosporin A was converted to cyclosporin A Methyl vinyl ketone [R¹ = (E)-2-butenyl to R¹ = (E)-3-oxo-1-butenyl] by HBT-mediated laccase oxidation [170], (Figure 7B).

Laccases are also able to oxidize catechins. These molecules are the condensed structural units of tannins, which are considered important antioxidants found in herbs, vegetables and teas. Catechins ability to scavenge free radicals makes them important in preventing cancer, inflammatory and cardiovascular diseases. Oxidation of catechin by laccase has yielded products (Figure 7C) with enhanced antioxidant capability [136,171].

Last but not least, laccase finds applications in the synthesis of hormone derivatives (generating dimers or oligomers by the coupling of the reactive radical intermediates). Intra et al. [172] and Nicotra et al. [44] have recently exploited the laccase capabilities to isolate new dimeric derivatives of the hormone β-estradiol (Figure 8A) and of the phytoalexin resveratrol (Figure 8B), respectively. Similarly, laccase oxidation of totarol, and of isoeugenol or coniferyl alcohol gave novel dimeric derivatives [134] and a mixture of dimeric and tetrameric derivatives [173] respectively, whereas an even more complex mixture of products was observed in the oxidation of substituted imidazole (Figure 9A) [126]. These novel substituted imidazoles or oligomerization products (2–4) are applicable for pharmacological purposes. In another study, derivatization of the natural compound 3-(3,4-dihydroxyphenyl)-propionic acid can be achieved by laccase-catalyzed N-coupling of aromatic and aliphatic amines (Figure 9B). The derivatives of this antiviral natural compound 3-(3,4-dihydroxyphenyl)-propionic acid may have interesting pharmaceutical uses. More recently, nuclear amination of p-hydroquinones with primary aromatic amines catalyzed by laccases in the presence of O₂resulted in the formation of the corresponding monoaminated or diaminated quinones [174,175], (Figure 9C).