Skip to content


  • Review
  • Open Access

Engineering and Applications of fungal laccases for organic synthesis

  • 1,
  • 1,
  • 1,
  • 1,
  • 1 and
  • 1Email author
Microbial Cell Factories20087:32

  • Received: 26 August 2008
  • Accepted: 20 November 2008
  • Published:


Laccases are multi-copper containing oxidases (EC, widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.

Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed.


  • Redox Mediator
  • Medium Density Fiberboard
  • Fungal Laccases
  • Vindoline
  • Asiaticoside

Laccases: general features


Laccases (benzenediol:oxygen oxidoreductase, EC 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].

Biochemical features

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 and industrial applications

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 [1517]. 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 [1821]; 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 [2731]; 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 [3436]-; and construction of biosensors and biofuel cells [37].
Figure 1
Figure 1

Expanded role of laccase oxidizing non-usual substrates by the action of redox mediators (A); and redox potentials of the oxidation reactions of ABTS and HBT by laccase (B).

In organic synthesis, laccases have been employed for the oxidation of functional groups [3842], the coupling of phenols and steroids [4345], 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.

Laccase-mediator system (LMS)

The combination of the laccase with low molecular weight molecules such as 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) not only lead to higher rates and yields in the transformation of laccase substrates but also add new oxidative reactions to the laccase repertory towards substrates in which the enzyme alone had no or only marginal activity, Figure 1A, B. Thus, LMS enlarges substrate range being able to oxidize compounds with redox potential (E°) higher than that of laccase (typically, laccase E° at the T1 site is in the range +475 to +790 mV but the LMS allows to oxidize molecules with E° above +1100 mV) [48, 49]. Besides, the mediator acts as a diffusible electron carrier enabling the oxidation of high molecular weight biopolymers such as lignin, cellulose or starch [1]. Hence, the steric issues that hinder the direct interaction between enzyme and polymer are overcome by the action of the redox mediator.

LMS has resulted highly efficient in many biotechnological and environmental applications as regards the numerous research articles and invention patents published [50, 51]. Many artificial mediators have been widely studied, from ABTS the first described laccase mediator [52], to the use of synthetic mediators of the type -NOH- (such as HBT, violuric acid (VIO), N-hydroxyphtalimide (HPI) and N-hydroxyacetanilide (NHA), the stable 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO), or the use of phenothiazines and other heterocycles (e.g. promazine or 1-nitroso-naphthol-3,6-disulfonic acid), Figure 2[18, 38, 53]. More recently, complexes of transition elements (polyoxometalates) have been also demonstrated to mediate lignin degradation catalyzed by laccase [54, 55].
Figure 2
Figure 2

Chemical structures of some representative artificial (ABTS, HBT, violuric acid -VIO-, TEMPO, promazine -PZ- and 1-nitroso-naphthol-3,6-disulfonic acid -NNDS-) and lignin-derived natural mediators (acetosyringone, syringaldehyde, vanillin, acetovanillone, p -coumaric acid, ferulic acid and sinapic acid).

The choice of a proper mediator (over 100 redox mediators have been described [56]) represents a key consideration for a given biotransformation. The use of different mediators may yield different final products when using the same precursors. This is basically due to the fact that substrate oxidation in laccase-mediator reactions occurs via different mechanisms. The mediator radicals preferentially perform a specific oxidation reaction based on its chemical structure and effective redox potential (or dissociation bond energy) [43, 38, 53, 57]. For example, ABTS and HBT follow two different radical pathways: i) electron transfer (ET) in the case of ABTS radicals (ABTS•+or ABTS2+) and ii) hydrogen atom transfer (HAT) for nitroxyl radicals (N-O) of HBT, Figure 3. On the contrary, the stable radical TEMPO follows an ionic oxidation mechanism [38, 39], Figure 4.
Figure 3
Figure 3

Diagram showing the differences between the oxidation mechanisms followed by ABTS radicals (Electron Transfer route, ET) and HBT radicals (Hydrogen Atom Transfer route, HAT) in LMS for oxidation of non-phenolic substrates (according to Galli and Gentili[52]).

Figure 4
Figure 4

Mechanisms of the laccase-TEMPO oxidation of hydroxymethyl groups to aldehyde groups by TEMPO according to d'Acunzo et al. [43].

Despite all the associated advantages of LMS, there are two major drawbacks hindering the use of mediators: they are expensive and they can generate toxic derivatives. Moreover, in some cases, while oxidizing the mediator, laccase is inactivated by the mediator radicals, or the latter can be transformed into inactive compounds with no more mediating capability (e.g. generation of benzotriazol from HBT by losing the hydroxyl group). Last trends are focusing in the use of low-cost and eco-friendly alternative mediators; in this sense, several naturally occurring mediators produced by fungi (phenol, aniline, 4-hydroxybenzoic acid and 4-hydroxybenzyl alcohol) have been identified [49]. More recently, phenolic compounds derived from lignin degradation (such as acetosyringone, syringaldehyde, vanillin, acetovanillone, ferulic acid or p-coumaric acid) have been demonstrated to be highly-efficient laccase mediators of natural origin (even better than the powerful artificial ones) for dye decolorization, removal of polycyclic aromatic hydrocarbons, pulp bleaching and pitch removal [5861], Figure 2. These natural compounds can be obtained at low cost due to their abundance in nature and also in industrial paper pulp wastes, smoothing the progress to a more environmental-friendly and sustainable white biotechnology processes.

Heterologous expression of fungal laccases

Biotechnological and environmental applications require large amounts of enzymes. Laccases secreted from wild-type fungal organisms may not be suitable for commercial purposes mainly because the low yields and undesirable preparation procedures (such as presence of toxic inducers) are not economically advantageous; however recent advances in bioreactor design and culture conditions have significantly increased the production yields [62].

Heterologous expression should be better suited for large-scale production, because of the potential of expressing different laccases in one selected optimised host. Laccases, like other oxidative enzymes, are difficult to express in non-fungal systems. The heterologous expression of active laccases has been reported mainly in filamentous fungi (Aspergillus oryzae, Aspergillus niger, Aspergillus sojae and Trichoderma reseei) and yeasts (Saccharomyces cerevisiae, Pichia pastoris, Pichia methalonica, Yarrowia lipolytica and Kluyveromyces lactis), Table 1. There is one remarkable exception of homologous expression, in which the basidiomycete fungus Pycnoporus cinabarinus was used as host to overexpress the active laccase (up to 1.2 g l-1) [63]. Unfortunately, the functional expression of fungal laccases in bacteria (Escherichia coli) has not been yet accomplished (perhaps due to the requirement of glycosylation, missing chaperones, and different codon usage, among other shortcomings).
Table 1

List of heterologously expressed laccases







Coriolus hirsutus

Saccharomyces cerevisiae

Active laccase secreted in the medium.

Kojima et al. [64]



Active laccase secreted in the medium.

Kojima et al. [64]


Phlebia radiata

Trichoderma reesei

Laccase secreted activity of 7.7 nkat ml-1 (ABTS). The enzyme was purified and partially characterized.

Saloheimo and Niku-Paavola [65]


Rhizoctonia solani

Aspergillus oryzae

Laccase activity secreted in the medium. The enzyme was purified and partially characterized.

Wahleithner et al. [66]



Active laccase secreted in the medium.

Wahleithner et al. [66]


Trametes villosa

Aspergillus oryzae

Active laccase secreted in the medium. The enzyme was purified and partially characterized.

Yaver et al. [9]


Myceliophtora thermophila

Aspergillus oryzae

Laccase secreted activity of 0.85 U ml-1 (SGZ). The enzyme was purified and partially characterized.

Berka et al. [67]


Saccharomyces cerevisiae

Laccase secreted activity of 0.6 U l-1 (ABTS). Total activity was enhanced 170-fold by directed evolution (18 mg l-1).

Bulter et al. [68]


Trametes versicolor

Pichia pastoris

Active laccase secreted in the medium. Production yield was further optimised.

Jönsson et al. [69]; O'Callaghan et al. [70]; Hong et al. [71]



Saccharomyces cerevisiae

Undetectable laccase activity in the medium.

Cassland and Jönsson [72]



Saccharomyces cerevisiae

Active laccase secreted in the medium. Production of ethanol from raw materials (0.12 U l-1).

Cassland and Jönsson [72] Larsson et al. [73]



Pichia pastoris

Active laccase secreted in the medium. The enzyme and a truncated version (LCCIa) were purified and partially characterized.

Gelo-Pujic et al. [74]



Pichia pastoris

Laccase secreted activity of 0.15 U ml-1 (ABTS). The enzyme was purified and partially characterized.

Brown et al. [75]



Zea mays L

Laccase activity was found in the seed, and variability in the amount was seen. The highest level was 0.55% TSP (respect to total soluble protein).

Hood et al. [76]



Pichia methalonica

9.79 U ml-1 of laccase acivity in recombinant with the α-factor signal peptide.

Guo et al. [77]



Yarrowia lipolytica

2.5 mg l-1 (0.23 U ml-1) of active enzyme with limited excess of glycosylation.

Jolivalt et al. [78]



Saccharomyces cerevisiae

0.035 U l-1 of laccase activity produced by S. cerevisiae.

Necochea et al. [79]



Pichia pastoris Aspergillus niger

2.8 U l-1 of laccase activity produced by P. pastoris and up to 2700 U l-1 by A. niger.

Bohlin et al. [80]

Gene IV


Aspergillus niger

592 U l-1 of enzyme activity in solid-state fermentation produced by A. niger.

Téllez-Jurado et al. [81]


Schizophyllum commune

Aspergillus sojae

Laccase secreted activity of 774 U ml-1 (Gallic acid).

Hatamoto et al. [82]


Coprinus cinereus

Aspergillus oryzae

Transformants secreted from 8.0 to 135 mg of active laccase per liter. The enzyme was purified and partially characterized.

Yaver et al. [83]


Coprinopsis cinerea

Coprinopsis cinerea

Maximal activity (3 U ml-1) reached with the gpdII promoter and 0. 1 μM CuSO4 (homologous expression).

Kilaru et al. [84]


Liriodendron tulipifera

Tobacco cells

Protoplasts retained laccase activity which could be measured once the protoplasts were lysed.

LaFayette et al. [85]


Pycnoporus cinnabarinus

Pichia pastoris

Transformants secreted 8.0 mg l-1 of hyperglycosylated active laccase.

Otterbein et al. [86]



Aspergillus niger

70 mgl-1 of active laccase using the A. niger signal peptide which represent a 77-fold increased activity (7000 U ml-1) (ABTS). The enzyme was purified and partially characterized.

Record et al. [87]



Aspergillus oryzae

80 mgl-1 of active laccase.

Sigoillot et al. [88]



Pycnoporus cinnabarinus

Laccase secreted activity of 1200 mg l-1 (homologous expression)

Alves et al. [63]



Yarrowia lipolytica

20 mg l-1 of active enzyme in bioreactor.

Madzak et al. [89]


Loblolly pine (Pinus taeda)

Saccharomyces cerevisiae

Yeast cells accumulated the expected fusion protein in insoluble fractions without degradation of products, but no laccase activity was detected.

Sato et al. [90]


Marinomonas mediterranea

Escherichia coli

Production of recombinant protein, with the most of activity, located in the membrane fraction rather than in the soluble one.

Sanchez-Amat et al. [91]


Pleurous sajor-caju

Pichia pastoris

Transformants produced 4.85 mg l-1 of active laccase. The enzyme was purified and partially characterized.

Soden et al. [92]


Solanum tuberosum L.

Lycopersicon esculentum

Active laccases secreted in the medium conferring resistance to pathogen Pseudomonas syringae pv tomato.

Li and Steffens [93]


Melanocarpus albomyces

Trichoderma reesei

920 mg L l-1 of active laccase

Kiiskinen et al. [94]



Saccharomyces cerevisiae

168 U l-1 of laccase activity produced (around 3 mg l-1)

Kiiskinen et al. [94]


Trametes sp. strain C30

Saccharomyces cerevisiae

2 mg l-1 of rLAC3 produced in bioreactor.

Klonowska et al. [95]


Pleurotus ostreatus

Kluyveromyces lactis Saccharomyces cerevisiae

K. lactis was more effective host (1.1 of POXA1b and 1.4 mg l-1 of POXC laccase) than S. cerevisiae.

Piscitelli et al. [96]

3M7C mutant


Saccharomyces cerevisiae

~30 mU OD600 l-1 after 6 days of incubation in shaken flask.

Festa et al. [97]



Kluyveromyces lactis

80 U l-1 after 10 days of incubation.

Faraco et al. [98]


Pycnoporus coccineus

Aspergillus oryzae Saccahromyces cerevisiae

High copper concentrations are required for the production of active laccase.

Hoshida et al. [99]


Coprinopsis cinerea

Coprinopsis cinerea

Maximal activity (3 U ml-1) reached with the gpdII promoter and 0. 1 μM CuSO4

Kilaru et al. [84]


Tametes trogii

Pichia pastoris

17 mg l-1 of active enzyme, reaching up to 2520 U l-1 in fed-batch culture.

Colao et al. [100]



Kluyveromyces lactis

6.6 U l-1 of bioactive molecule produced by K. lactis.

Camattari et al. [101]


Trametes sp.

Pichia pastoris

Overexpression (1.01 U/mg) of active laccase (32000 U ml-1).

Li et al. [102]


Trametes sp 420

Pichia pastoris

8.3 × 104 U l-1+of active laccase.

Hong et al. [103]


Pleurotus eryngii

Aspergillus niger

Partially characterization of recombinant laccase.

Rodríguez et al. [104]



Saccharomyces cerevisiae

139 mU ml-1 of laccase in alginate immobilized cells and 18°C.

Bleve et al. [105]


Fome lignosus

Pichia pastoris

3.7-fold expression improvement (up to 144 mg l-1) with EMS random mutagenesis.

Hu et al. [106]

Laccase engineering

Crystallographic structure determination is an essential tool for structure-function relationships studies (i.e. rational design). However, since the crystallization of the first (but inactive) laccase from Coprinus cinereus in 1998 by Ducros et al.[107], few crystal structures of active laccases have been published: one from the ascomycete Melanocarpus albomyces [108], two from basidomycetes Trametes versicolor [109] and Rigidosporus lignosun [110] and another from Bacillus subtilis [111]. Based on these laccase structures, over the last decade several residues in the neighbourhood of the catalytic copper ions have been subjected to site-directed mutagenesis to determine the parameters that define the catalytic activity and the E° of fungal laccases [112, 113]. One consequence of these comprehensive structure-function studies has been the generation of a collection of mutants with structural perturbations at the T1 copper center.

To overcome many of the limitations of the rational design, and in the absence of enough structural information, directed molecular evolution represents a promising alternative. This methodology recreates in the laboratory the key events of natural evolution (mutation, recombination and selection) doing in such a manner those more efficient enzymes-even with novel functions-can be tailored. Diversity is mimicked by inducing mutations and/or recombination in the gene encoding a specific protein. Afterwards, the best performers in each generation are selected and further used as the parental types for a new round of evolution. The process is repeated as many times as necessary enhancing exponentially the targeted features, until a biocatalyst with the desired traits is obtained: stability at high temperature or in organic solvents; improved catalytic activities; higher specificity; etc.

A thorough understanding of efficient and reliable high-throughput screening methodologies is a prerequisite for the design and validation of this type of experiments [114]. A key query result of smart laboratory evolution is the improvement of several enzymatic properties at the same time (e.g. stability and activity). The first successful example of directed laccase evolution reported came from Arnold group [68]. They carried out the functional expression of a thermophilic laccase in S. cerevisiae by directed evolution: after ten rounds of laboratory evolution and screening, the total enzymatic activity was improved 170-fold along with better performances at high temperatures.

It is well known that most of the laccase catalysed transformations for organic syntheses (from the oxidation of steroid hormones to the enzymatic polymerisation required for the synthesis of phenolic-based resins such as poly-α-naphtol, poly-pyrogallol and poly-catechol [1, 115]., as well as conductive water-soluble polymers [116]) must be carried out in the presence of organic solvents. However, at high concentrations of organic co-solvents laccases undergo unfolding, therefore losing their catalytic activity. Recently, our group generated a thermostable laccase-the genetic product of five rounds of directed evolution expressed in S. cerevisiae [117, 118]-that tolerates high concentrations of co-solvents. This evolved laccase mutant is capable of resisting a wide array of biotechnologically relevant miscible co-solvents at concentrations as high as 50% (v/v). Indeed, in 40% (v/v) ethanol or in 30% (v/v) acetonitrile the performance of the laccase mutant was comparable to that of the parental enzyme in aqueous solution, a capacity that has not been acquired in nature. Intrinsic electrochemical laccase features such as the redox potential at the T1 and T2/T3 sites and the geometry and electronic structure of the catalytic coppers varied slightly during the course of the in vitro evolution. Indeed, some mutations at the protein surface stabilized the evolved laccase by allowing additional electrostatic and hydrogen-bonding to occur [117]. Additionally, the protein folding in the post-translational maturation steps seemed to be modified by mutations in processing regions [119].

Besides methods that involve iterative steps of random mutagenesis and/or DNA recombination, semi-rational studies-which take advantage from both protein structure and combinatorial libraries constructed by saturation mutagenesis- are being employed successfully. This approach involves the mutation of any single amino acid codon to all the other codons that will generate the 20 naturally occurring amino acids coupled to screen for the desire function. This technique is commonly employed to improve the characteristics of enzymes at "hot-spot" residues already identified by conventional random mutagenesis. In addition, it can be employed to simultaneously mutate several codons (combinatorial saturation mutagenesis), which will enable all possible combinations of interesting residues to be evaluated in order to identify their optimal interactions and synergies.

In a recent study [120] of the evolved Myceliophthora thermophila laccase variant T2 (MtLT2) expressed in S. cerevisiae [68], we applied combinatorial saturation mutagenesis to residues L513 (the axial non-coordinating ligand supposedly essential for the E° at the T1 site) and S510 (belonging to the tripeptide 509VSG511 that is common to the low-medium E° laccases). A mutant with 3-fold higher turnover rates than the parent type, contained one beneficial mutation (TCGS510GGGG) that could not be achieved by conventional error-prone PCR techniques, since it was dependent on the two consecutive nucleotide changes. In a more exhaustive study [119], several regions of the same variant were investigated by combinatorial saturation mutagenesis. After exploring over 180,000 clones, the S510G mutant revealed a direct interaction between the conserved 509VSG511 tripeptide located in the neighbourhood of the T1 site and the C-terminal plug.

Applications of laccases in organic synthesis

Organic synthesis of chemicals suffers from several drawbacks, including the high cost of chemicals, cumbersome multi-step reactions and toxicity of reagents [2, 17]. Laccases might prove to be very useful in synthetic chemistry, where they have been proposed to be applicable for production of complex polymers and medical agents [16, 121]. Indeed, the application of laccase in organic synthesis has arisen due to its broad substrate range, and the conversion of substrates to unstable free (cation) radicals that may undergo further non-enzymatic reactions such as polymerization or hydration. The list of laccases used for organic synthesis is presented in Table 2.
Table 2

List of laccases used for organic synthesis

Laccase source



Coriolus hirsutus

Synthesis of an indamine dye

Baker et al. [122]


Synthesis of conducting polyaniline

Karamyshev et al. [116]

Pycnoporus cinnabarinus

Synthesis of 3-(3,4-dihydroxyphenyl)-propionic acid derivatives

Mikolasch et al. [45]

Pycnoporus coccineus

Polymerization to functional polymers

Uyama and Kobayashi [123]

Pyricularia oryzae

Oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone and methoxyphenols

Setti et al. [124]

Trametes versicolor

Synthesis of aromatic aldehydes

Fritz-Langhals and Kunath [40]


Polymerization of 1-napthol

Akta et al. [125]


Synthesis of substituted imidazoles and dimerization products

Schäfer et al. [126]


Polymerization of catechol

Akta and Tanyolaç [127]


Cross-linking of a protein

Boumans et al. [128]


Synthesis of 3,4-dihydro-7,8-dihydroxy-2H-dibenzofuran-1-ones

Hajdok et al. [129]

Trametes villosa

Polymerization of bisphenol A

Uchida et al. [130]

Trametes hirsuta

Oligomerization of protein

Mattinen et al. [131]

Trametes pubescens

Oxidation of sugars derivatives

Marzorati et al. [132]


Oxidation of natural glycosides

Baratto et al. [133]


Synthesis of totarol

Ncanana et al. [134]

Pyricularia oryzae

Crosslinking of recombinant proteins

Suderman et al. [135]

Agaricus bisporus

Synthesis of 3,4-dihydro-7,8-dihydroxy-2H-dibenzofuran-1-ones

Hajdok et al. [129]


Synthesis of poly(catechin)

Kurisawa et al. [136]

Laccases for enzymatic polymerization and polymer functionalization

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, 138140]. 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 [142144]. 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].
Figure 5
Figure 5

(A) PPO derivatives obtained from 4-hydroxybenzoic acid derivatives by laccase catalysis, and (B) "Artificial Urushi" prepared from new "urushiol analogues" by a laccase-catalyzed cross-linking reaction.

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) [148151]. 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].
Figure 6
Figure 6

(A) Products obtained by the oxidation of sugars using laccase and TEMPO, and (B) enzymatic modification of the natural glycoside asiaticoside.

Oxidative transformation of organic compounds by laccase

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].
Figure 7
Figure 7

(A) Synthesis of actinocin via a laccase-catalyzed reaction, (B) Synthesis of novel cyclosporin reaction product obtained from cyclosporin A by HBT-mediated laccase oxidation, (C) Products obtained by the laccase/hydroquinone-mediated oxidation of (+)-catechin.

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 [166168] 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).
Figure 8
Figure 8

(A, ii-v) Dimeric products obtained by the oxidation of β-estradiol, (B) Dimeric product obtained by the oxidation of the phytoalexin resveratrol.

Figure 9
Figure 9

(A) N -[2-alkylamino-4-phenylimidazol-1-yl]-acetamide (substrate 1) and products 2–4 formed during incubation with T. versicolor laccase, (B) The natural compound 3-(3,4-dihydroxyphenyl)-propionic acid derivative can be synthesized by laccase-catalyzed N-coupling of aromatic and aliphatic amines, and (C) the coupling of p -hydroquinones with primary aromatic amines by laccases.


The use of laccases in organic synthesis does show as a promising green alternative to the classical chemical oxidation with a wide range of substrates. In the near future, the practical use of fungal laccases for troublesome transformations (digestion of lignocellulose to use as a carbon source; modifications of lignosulfonates for production of emulsifiers, surfactants and adhesives; synthesis of polymers with properties as redox films for bioelectronic devices; synthesis of antibiotics and much more) will expand the need for this biocatalyst. Meanwhile, the development of more robust fungal laccases tailored by protein engineering and the search for environment-friendly mediators along with further research on heterologous expression are significant hurdles that must be overcome.



This material is based upon work funded by the Spanish Ministry of Education and Science (projects VEM2004-08559, CTQ2005-08925-C02-02/PPQ, PIE200880I033); EU project NMP2-CT-2006-026456; CSIC project 200580M121 and Ramon y Cajal Programme. Dr. A. Kunamneni is the recipient of a Marie Curie fellowship (MIF1-CT-2006-040163) of EU's FP6.

Authors’ Affiliations

Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049 Madrid, Spain


  1. Alcalde M: Laccase: biological functions, molecular structure and industrial applications. Industrial Enzymes: structure, function and applications. Edited by: J Polaina J, MacCabe AP. 2007, 459-474. New York, SpringerGoogle Scholar
  2. Yaropolov AI, Skorobogat'ko OV, Vartanov SS, Varfolomeyev SD: Laccase: Properties, catalytic mechanism, and applicability. Appl Biochem Biotechnol. 1994, 49: 257-280.Google Scholar
  3. Call HP, Mücke I: History, overview and applications of mediated ligninolytic systems, especially laccase-mediator-systems (Lignozyme®-process). J Biotechnol. 1997, 53: 163-202.Google Scholar
  4. Gianfreda L, Xu F, Bollag JM: Laccases: a useful group of oxidoreductive enzymes. Bioremed J. 1999, 3: 1-25.Google Scholar
  5. Dittmer NT, Suderman RJ, Jiang H, Zhu YC, Gorman MJ, Kramer KJ, Kanost MR: Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae. Insect Biochem Mol Biol. 2004, 34: 29-41.Google Scholar
  6. Kramer KJ, Kanost MR, Hopkins TL, Jing H, Zhu YC, Xhu R, Kerwin JL, Turecek F: Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron. 2001, 57: 385-392.Google Scholar
  7. Claus H: Laccases and their occurrence in prokaryotes. Arch Microbiol. 2003, 179: 145-150.Google Scholar
  8. Baldrian P: Fungal laccases – occurrence and properties. FEMS Microbiol Rev. 2006, 30: 215-242.Google Scholar
  9. Yaver DS, Xu F, Golightly EJ, Brown KM, Brown SH, Rey MW, Schneider P, Halkier T, Mondorf K, Dalboge H: Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa. Appl Environ Microbiol. 1996, 62: 834-841.Google Scholar
  10. Marques De Souza CG, Peralta RM: Purification and characterization of the main laccase produced by the white-rot fungus Pleurotus pulmonarius on wheat bran solid state medium. J Basic Microbiol. 2003, 43 (4): 278-286.Google Scholar
  11. Aramayo R, Timberlake WE: The Aspergillus nidulans yA gene is regulated by abaA. EMBO J. 1993, 12: 2039-2048.Google Scholar
  12. Williamson PR, Wakamatsu K, Ito S: Melanin biosynthesis in Cryptococcus neoformans. J Bacteriol. 1998, 180: 1570-1572.Google Scholar
  13. O' Malley DM, Whetten R, Bao W, Chen CL, Seedorf RR: The role of laccase in lignification. Plant J. 1993, 4: 751-757.Google Scholar
  14. Stoj C, Kosman DJ: Cuprous oxidase activity of yeast Fet3p and human ceruloplasmin: implication for function. FEBS Lett. 2003, 554: 422-426.Google Scholar
  15. Nyanhongo GS, Gomes J, Gubitz GM, Zvauya R, Read JS, Steiner W: Decolorization of textile dyes by laccases from a newly isolated strain of Trametes modesta. Water Res. 2002, 36: 1449-1456.Google Scholar
  16. Xu F: Applications of oxidoreductases: recent progress. Industrial Biotechnol. 2005, 1: 38-50.Google Scholar
  17. Riva S: Laccases: blue enzyme for green chemistry. Trends Biotechnol. 2006, 24: 219-226.Google Scholar
  18. Bourbonnais R, Paice MG, Freiermuth B, Bodie E, Borneman S: Reactivities of various mediators and laccases with kraft pulp and lignin model compounds. Appl Environ Microbiol. 1997, 63 (12): 4627-4632.Google Scholar
  19. Smith M, Thurston CF, Wood DA: Fungal laccases: role in delignification and possible industrial applications. Multi-copper oxidases. Edited by: Messerschmidt A. 1997, 201-224. Singapore, World ScientificGoogle Scholar
  20. Camarero S, García O, Vidal T, Colom J, del Río JC, Gutiérrez A, Gras JM, Monje R, Martínez MJ, Martínez AT: Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system. Enzyme Microb Technol. 2004, 35: 113-120.Google Scholar
  21. Ibarra D, Camarero S, Romero J, Martínez MJ, Martínez AT: Integrating laccase-mediator treatment into an industrial-type sequence for totally chlorine free bleaching eucalypt kraft pulp. J Chem Technol Biotechnol. 2006, 81: 1159-1165.Google Scholar
  22. Bergbauer M, Eggert C, Kraepelin G: Degradation of chlorinated lignin compounds in a bleach plant effluent by the white-rot fungus Trametes versicolor. Appl Microbiol Biotechnol. 1991, 35: 105-109.Google Scholar
  23. Berrio J, Plou FJ, Ballesteros A, Martinez AT, Martinez MJ: Immobilization of Pycnoporus coccineus laccase on Eupergit C: Stabilization and treatment of olive oil mill wastewaters. Biocatal Biotransform. 2007, 25: 130-134.Google Scholar
  24. Abadulla E, Tzanov T, Costa S, Robra KH, Cavaco-Paulo A, Gübitz GM: Decolourisation and detoxification of textile dyes with laccase from Trametes hirsuta. Appl Environ Microbiol. 2000, 66: 3357-3362.Google Scholar
  25. Kunamneni A, Ghazi I, Camarero S, Ballesteros A, Plou FJ, Alcalde M: Decolorization of synthetic dyes by laccase immobilized on epoxy-activated carriers. Process Biochem. 2008, 43: 169-178.Google Scholar
  26. Widsten P, Tuominen S, Qvintus-Leino P, Laine JE: The influence of high defibration temperature on the properties of medium-density fiberboard (MDF) made from laccase-treated softwood fibers. Wood Sci Technol. 2004, 38: 521-528.Google Scholar
  27. Keum YS, Li QX: Reduction of nitroaromatic pesticides with zero-valent iron. Chemosphere. 2004, 54 (3): 255-263.Google Scholar
  28. Bollag JM, Chu HL, Rao MA, Gianfreda L: Enzymatic oxidative transformation of chlorophenol mixtures. J Environ Qual. 2003, 32: 63-69.Google Scholar
  29. Gianfreda L, Rao MA: Potential of extracellular enzymes in remediation of polluted soils: a review. Enzyme Microb Technol. 2004, 35: 339-354.Google Scholar
  30. Alcalde M, Ferrer M, Plou FJ, Ballesteros A: Environmental biocatalysis: from remediation with enzymes to novel green processes. Trends Biotechnol. 2006, 24 (6): 281-287.Google Scholar
  31. Zumarraga M, Plou FJ, Garcia-Arellano H, Ballesteros A, Alcalde M: Bioremediation of polycyclic aromatic hydrocarbons by fungal laccases engineered by directed evolution. Biocatal Biotrans. 2007, 25: 219-228.Google Scholar
  32. Jonsson LJ, Palmqvist E, Nilvebrant NO, Hahn Hagerdal B: Detoxification of wood hydrolysates with laccase and peroxidase from the White-rot fungus Trametes versicolor. Appl Microbiol Biotechnol. 1998, 49: 691-697.Google Scholar
  33. Larsson S, Reimann A, Nilvebrant NO, Jonsson LJ: Comparison of different methods for the detoxification of lignocellulose hydrolysates of spruce. Appl Biochem Biotechnol. 1999, 77–79: 91-103.Google Scholar
  34. Cantarelli C, Brenna O, Giovanelli G, Rossi M: Beverage stabilization through enzymatic removal of phenolics. Food Biotechnol. 1989, 3: 203-214.Google Scholar
  35. Servili M, De Stefano G, Piacquadio P, Sciancalepore V: A novel method for removing phenols from grape must. Am J Enol Viticul. 2000, 51: 357-361.Google Scholar
  36. Minussi RC, Pastore GM, Duran N: Potential applications of laccase in the food industry. Trends Food Sci Technol. 2002, 13: 205-216.Google Scholar
  37. Ghindilis A: Direct electron transfer catalysed by enzymes: application for biosensor development. Biochem Soc Trans. 2000, 28: 84-89.Google Scholar
  38. Baiocco P, Barreca AN, Fabbrini M, Galli C, Gentili P: Promoting laccase activity towards non-phenolic substrates: A mechanistic investigation with some laccase-mediator systems. Org Biomol Chem. 2003, 1: 191-197.Google Scholar
  39. Fabbrini M, Galli C, Gentili P: Comparing the efficiency of some mediators of laccase. J Mol Catal B Enzym. 2002, 16: 231-240.Google Scholar
  40. Fritz-Langhals E, Kunath B: Synthesis of aromatic aldehydes by laccase mediator assisted oxidation. Tetrahedron Lett. 1998, 39: 5955-5956.Google Scholar
  41. Potthast A, Rosenau T, Chen CL, Gratzl JS: A novel method for the conversion of benzyl alcohols to benzaldehydes by laccase-catalyzed oxidation. J Mol Catal A Chem. 1996, 108: 5-9.Google Scholar
  42. Fabbrini M, Galli C, Gentili P, Macchitella D: An oxidation of alcohols by oxygen with the enzyme laccase, and mediation by TEMPO. Tetrahedron Lett. 2001, 42: 7551-7553.Google Scholar
  43. d'Acunzo F, Galli C, Masci B: Oxidation of phenols by laccase and laccase-mediator systems: Solubility and steric issues. Eur J Biochem. 2002, 269: 5330-5335.Google Scholar
  44. Nicotra S, Cramarossa MR, Mucci A, Pagnoni UM, Riva S, Forti L: Biotransformation of resveratrol: synthesis of trans-dehydrodimers catalyzed by laccases from Myceliophtora thermophyla and from Trametes pubescens. Tetrahedron. 2004, 60: 595-600.Google Scholar
  45. Ponzoni C, Beneventi E, Cramarossa MR, Raimondi S, Trevisi G, Pagnoni UM, Riva S, Forti L: Laccase-catalyzed dimerization of hydroxystilbenes. Adv Synth Catal. 2007, 349: 1497-1506.Google Scholar
  46. Mikolasch A, Hammer E, Jonas U, Popowski K, Stielow A, Schauer F: Synthesis of 3-(3, 4-dihydroxyphenyl)-propionic acid derivatives by N-coupling of amines using laccase. Tetrahedron. 2002, 58: 7589-7593.Google Scholar
  47. Barilli A, Belinghieri F, Passarella D, Lesma G, Riva S, Silvani A, Danieli B: Enzyme assisted enantioselective synthesis of the alkaloid (+)-aloperine. Tetrahedron Asym. 2004, 15: 2921-2925.Google Scholar
  48. Fernández-Sánchez C, Tzanov T, Gübitz GM, Cavaco-Paulo A: Voltametric monitoring of laccase-catalysed mediated reactions. Bioelectrochemistry. 2002, 58 (2): 149-156.Google Scholar
  49. Johannes C, Majcherczyk A: Laccase activity tests and laccase inhibitors. J Biotechnol. 2000, 78: 193-199.Google Scholar
  50. Morozova OV, Shumakovich GP, Shleev SV, Iaropolov YI: Laccase-mediator systems and their applications: A review. Prikl Biokhim Mikrobiol. 2007, 43 (5): 583-597.Google Scholar
  51. Kunamneni A, Plou FJ, Alcalde M, Ballesteros A: Laccases and their applications: A patent review. Recent Patent Biotechnol. 2008, 2: 10-24.Google Scholar
  52. Bourbonnais R, Paice MG: Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS Lett. 1990, 267: 99-102.Google Scholar
  53. Galli C, Gentili P: Chemical messengers: mediated oxidations with the enzyme laccase. J Phys Org Chem. 2004, 17: 973-977.Google Scholar
  54. Gamelas JAF, Tavares APM, Evtuguin DV, Xavier AMB: Oxygen bleaching of kraft pulp with polyoxometalates and laccase applying a novel multi-stage process. J Mol Catal B: Enzym. 2005, 33: 57-64.Google Scholar
  55. Gamelas JAF, Pontes ASN, Evtuguin DV, Xavier AMRB, Esculcas AP: New polyoxometalate-laccase integrated system for kraft pulp delignification. Biochem Eng J. 2007, 33: 141-147.Google Scholar
  56. Call HP: Mehrkomponentensystem zum Verändern, Abbau oder Bleichen von Lignin, ligninhaltigen Materialien oder ähnlichen Stoffen sowie Verfahren zu seiner Anwendung. EP 0717143A1. 1996Google Scholar
  57. d'Acunzo F, Galli C, Gentili P, Sergi F: Mechanistic and steric issues in the oxidation of phenolic and non-phenolic compounds by laccase or laccase-mediator systems. The case of bifunctional substrates. New J Chem. 2006, 30: 583-591.Google Scholar
  58. Camarero S, Ibarra D, Martínez MJ, Martínez AT: Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl Environ Microbiol. 2005, 71: 1775-1784.Google Scholar
  59. Cañas A, Alcalde M, Plou FJ, Martínez MJ, Martínez AT, Camarero S: Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil. Environ Sci Technol. 2007, 41: 2964-2971.Google Scholar
  60. Camarero S, Ibarra D, Martínez AT, Romero J, Gutiérrez A, del Río JC: Paper pulp delignification using laccase and natural mediators. Enzyme Microb Technol. 2007, 40: 1264-1271.Google Scholar
  61. Gutiérrez A, Rencores J, Ibarra D, Molina S, Camarero S, Romero J, del Río JC, Martínez AT: Removal of lipophilic extractives from paper pulp by laccase and lignin-derived phenols as natural mediators. Environ Sci Technol. 2007, 41: 4124-4129.Google Scholar
  62. Couto SR, Toca-Herrera JL: Laccase production at reactor scale by filamentous fungi. Biotechnol Adv. 2007, 25: 558-569.Google Scholar
  63. Alves AMCR, Record E, Lomascolo A, Scholtmeijer K, Asther M, Wessels JGH, Wosten HAB: Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus. Appl Environ Microbiol. 2004, 70: 6379-6384.Google Scholar
  64. Kojima Y, Tsukuda Y, Hawai Y, Tsukamoto A, Sugiura J, Sakaino M, Kita Y: Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus. J Biol Chem. 1990, 265: 15224-15230.Google Scholar
  65. Saloheimo M, Niku-Paavola ML: Heterologous production of a ligninolytic enzyme: expression of the Phlebia radiata laccase gene in Trichoderma reesei. Biotechnol. 1991, 9: 987-990.Google Scholar
  66. Wahleithner JA, Xu F, Brown SH, Golightly EJ, Halkier T, Kauppinen S, Pederson A, Schneider P: The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani. Curr Genet. 1996, 29: 395-403.Google Scholar
  67. Berka RMP, Schneider EJ, Golightly SH, Brown M, Madden KM, Brown T, Halkier K, Mondorf , Xu F: Characterization of the gene encoding an extracellular laccase of Myceliophtora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae. Appl Environ Microbiol. 1997, 63: 3151-3157.Google Scholar
  68. Bulter T, Alcalde M, Sieber V, Meinhold P, Schlachtbauer C, Arnold FH: Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl Environ Microbiol. 2003, 69: 987-995.Google Scholar
  69. Jönsson LJ, Saloheimo M, Penttila M: Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of lcc1 and expression in Pichia pastoris. Curr Genet. 1997, 32: 425-430.Google Scholar
  70. O'Callaghan J, O'Brien MM, McClean K, Dobson ADW: Optimisation of the expression of Trametes versicolor laccase gene in Pichia pastoris. J Ind Microbiol Biotechnol. 2002, 29: 55-59.Google Scholar
  71. Hong F, Meinander QM, Jönsson LF: Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. Biotechnol Bioeng. 2002, 79 (4): 438-449.Google Scholar
  72. Cassland P, Jönsson LJ: Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature. Appl Microbiol Biotechnol. 1999, 52: 393-400.Google Scholar
  73. Larsson S, Cassland P, Jönsson LJ: Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase. Appl Environ Microbiol. 2001, 67: 1163-1170.Google Scholar
  74. Gelo-Pujic M, Kim HH, Butlin NG, Palmore GT: Electrochemical studies of a truncated laccase produced in Pichia pastoris. Appl Environ Microbiol. 1999, 65: 5515-5521.Google Scholar
  75. Brown MA, Zhao Z, Mauk AG: Expression and characterization of a recombinant multi-copper oxidase: laccase IV from Trametes versicolor. Inorg Chim Acta. 2002, 331: 232-238.Google Scholar
  76. Hood EE, Bailey MR, Beifuss K, Magallanes-Lundback M, Horn ME, Callaway E, Drees C, Delaney DE, Clough R, Howard JA: Criteria for high-level expression of a fungal laccase gene in transgenic maize. Plant Biotechnol J. 2003, 1: 129-140.Google Scholar
  77. Guo M, Lu FP, Du LX, Pu J, Bai DQ: Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica. Appl Microbiol Biotechnol. 2006, 71: 848-852.Google Scholar
  78. Jolivalt C, Madzak C, Brault A, Caminade E, Malosse C, Mougin C: Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications. Appl Microbiol Biotechnol. 2005, 66: 450-456.Google Scholar
  79. Necochea R, Valderrama B, Diaz-Sandoval S, Folch-Mallol JL, Vazquez-Duhalt R, Iturriaga G: Phylogenetic and biochemical characterisation of a recombinant laccase from Trametes versicolor. FEMS Microbiol Lett. 2005, 244: 235-241.Google Scholar
  80. Bohlin C, Jönsson LJ, Roth R, vanZyl WH: Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger. Appl Biochem Biotechnol. 2006, 129: 195-214.Google Scholar
  81. Téllez-Jurado A, Arana-Cuenca A, González Becerra AE, Viniegra-González G, Loera O: Expression of a heterologous laccase by Aspergillus niger cultured by solid-state and submerged fermentations. Enzyme Microb Technol. 2006, 38: 665-669.Google Scholar
  82. Hatamoto O, Sekine H, Nakano E, Abe K: Cloning and expression of a cDNA encoding the laccase from Schizophyllum commune. Biosci Biotechnol Biochem. 1999, 63: 58-64.Google Scholar
  83. Yaver DS, Overjero MD, Xu F, Nelson BA, Brown KM, Halkier T, Bernauer S, Brown SH, Kauppinen S: Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc 1. Appl Environ Microbiol. 1999, 65: 4943-4948.Google Scholar
  84. Kilaru S, Hoegger PJ, Majcherczyk A, Burns C, Shishido K, Bailey A, Foster GD, Kues U: Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters. Appl Microbiol Biotechnol. 2006, 71 (2): 200-210.Google Scholar
  85. LaFayette PR, Eriksson KE, Dean JF: Characterization and heterologous expression of laccase cDNAs from xylem tissues of yellow-poplar (Liriodendron tulipifera). Plant Mol Biol. 1999, 40: 23-35.Google Scholar
  86. Otterbein L, Record E, Longhi S, Asther M, Moukha S: Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris. Eur J Biochem. 2000, 267: 1619-1625.Google Scholar
  87. Record E, Punt PJ, Chamkha M, Labat M, Hondel van Den CAMJJ, Esther M: Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme. Eur J Biochem. 2002, 269: 602-609.Google Scholar
  88. Sigoillot C, Record E, Belle V, Robert JL, Levasseur A, Punt PJ, Hondel Van Den CA, Fournel A, Sigoillot JC, Asther M: Natural and recombinant fungal laccases for paper pulp bleaching. Appl Microbiol Biotechnol. 2004, 64: 346-352.Google Scholar
  89. Madzak C, Otterbein L, Chamkha M, Moukha S, Asther M, Gaillardin C, Beckerich JM: Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the dimorphic yeast Yarrowia lipolytica. FEMS Yeast Res. 2005, 5: 635-646.Google Scholar
  90. Sato Y, Wuli B, Sederoff R, Whetten R: Molecular cloning and expression of eight cDNAs in loblolly pine (Pinus taeda). J Plant Res. 2001, 114: 147-155.Google Scholar
  91. Sanchez-Amat A, Lucas-Elio P, Fernandez E, Garcia-Borron JC, Solano F: Molecular cloning and functional characterisation of a unique multipotent polyphenol oxidase from Marinomonas mediterranea. Biochim Biophys Acta. 2001, 1547: 104-116.Google Scholar
  92. Soden DM, O'Callaghan J, Dobson AD: Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology. 2002, 148 (Pt 12): 4003-4014.Google Scholar
  93. Li L, Steffens JC: Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta. 2002, 215: 239-247.Google Scholar
  94. Kiiskinen LL, Kruus K, Bailey M, Ylosmaki E, Siika-Aho M, Saloheimo M: Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterisation of the purified. Microbiol. 2004, 150: 3065-3074.Google Scholar
  95. Klonowska A, Gaudin C, Asso M, Fournel A, Reglier M, Tron T: LAC3, a new low redox potential laccase from Trametes sp. strain C30 obtained as a recombinant protein in yeast. Enzyme Microb Technol. 2005, 36: 34-41.Google Scholar
  96. Piscitelli A, Giardina P, Mazzoni C, Sannia G: Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2005, 69: 428-439.Google Scholar
  97. Festa G, Autore F, Fraternali F, Giardina P, Sannia G: Development of new laccases by directed evolution: Functional and computational analyses. Proteins. 2008, 72 (1): 25-34.Google Scholar
  98. Faraco V, Ercole C, Festa G, Piscitelli A, Sannia G: Heterologous expression of heterodimeric laccase from Pleurotus ostreatus in Kluyveromyces lactis. Appl Microbiol Biotechnol. 2008, 77: 1329-1335.Google Scholar
  99. Hoshida H, Fujita T, Murata K, Kubo K, Akada R: Copper-dependent production of a Pycnoporus coccineus extracellular laccase in Aspergillus oryzae and Saccharomyces cerevisiae. Biosci Biotechnol Biochem. 2005, 69: 1090-1097.Google Scholar
  100. Colao MC, Lupino S, Garzillo AM, Buonocore V, Ruzzi M: Heterologous expression of lccl gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme. Microb Cell Fact. 2006, 5: doiGoogle Scholar
  101. Camattari A, Bianchi MM, Branduardi P, Porro D, Brambilla L: Induction by hypoxia of heterologous-protein production with the Kl PDC1 promoter in yeasts. Appl Environ Microbiol. 2007, 73: 922-929.Google Scholar
  102. Li F, Hong YZ, Xiao YZ, Xu YH, Fang W: High production of laccase B from Trametes sp. in Pichia pastoris. World J Microbiol Biotechnol. 2007, 23: 741-745.Google Scholar
  103. Hong YZ, Zhou HM, Tu XM, Li JF, Xiao YZ: Cloning of a laccase gene from a novel basidiomycete Trametes sp 420 and its heterologous expression in Pichia pastoris. Curr Microbiol. 2007, 54: 260-265.Google Scholar
  104. Rodríguez E, Ruiz-Dueñas FJ, Kooistra R, Ram A, Martínez AT, Martínez MJ: Isolation of two laccase genes from the white-rot fungus Pleurotus eryngii and heterologous expression of the pel 3 encoded protein. J Biotechnol. 2008, 134: 9-19.Google Scholar
  105. Bleve G, Lezzi C, Mita G, Rampino P, Perrotta C, Villanova L, Grieco F: Molecular cloning and heterologous expression of a laccase gene from Pleurotus eryngii in free and immobilized Saccharomyces cerevisiae cells. Appl Microbiol Biotechnol. 2008, 79: 731-741.Google Scholar
  106. Hu MR, Chao YP, Zhang GQ, Yang XQ, Xue ZQ, Qian SJ: Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro. Biomol Eng. 2007, 24: 619-624.Google Scholar
  107. Ducros V, Brzozowski AM, Wilson KS, Brown SH, Ostergaard P, Schneider P, Yaver DS, Pedersen AH, Davies GJ: Crystal structure of the type-2 Cu depleted laccase from Coprinus cinereus at 2.2 angstrom resolution. Nat Struct Biol. 1998, 5: 310-316.Google Scholar
  108. Bertrand TC, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, Mougin C: Crystal structure of four-copper laccase complexed with an arylamin: insights into substrate recognition and correlation with kinetics. Biochemistry. 2002, 41 (23): 7325-7333.Google Scholar
  109. Piontek K, Antorini M, Choinowski T: Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-Å resolution containing a full complement of coppers. J Biol Chem. 2002, 277: 37663-37669.Google Scholar
  110. Garavaglia S, Cambria MT, Miglio M, Ragusa S, Lacobazzi V, Palmieri F, D'Ambrosio C, Scaloni A, Rizzi M: The structure of Rigidoporus lignosus laccase containing a full complement of copper ions, reveals an asymmetrical arrangement for the T3 copper pair. J Mol Biol. 2004, 342: 1519-1531.Google Scholar
  111. Enguita FJ, Martins LO, Henriques AO, Larrondo MA: Crystal structure of a bacterial endospore coat component: A laccase with enhanced thermostability properties. J Biol Chem. 2003, 278: 19416-19425.Google Scholar
  112. Xu F, Berka RM, Wahleithner JA, Nelson BA, Shuster JR, Brown SH, Palmer AE, Solomon EI: Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile. Biochem J. 1998, 334: 63-70.Google Scholar
  113. Palmer AE, Szilagyi RK, Cherry JR, Jones A, Xu F, Solomon EI: Spectroscopic characterization of the Leu513His variant of fungal laccase: Effect of increased axial ligand interaction on the geometric and electronic structure of the type 1 Cu site. Inorg Chem. 2003, 42: 4006-4017.Google Scholar
  114. Alcalde M, Bulter T, Arnold FH: Colorimetric assays for biodegradation of polycyclic aromatic hydrocarbons by fungal laccases. J Biomol Screen. 2002, 7 (6): 547-553.Google Scholar
  115. Torres E, Bustos-Jaimes I, Le Borgne S: Potential use of oxidative enzymes for the detoxification of organic pollutants. Appl Catal B-Environ. 2003, 46: 1-15.Google Scholar
  116. Karamyshev AV, Shleev SC, Koroleva OV, Yaropolov AI, Sakharov IY: Laccase-catalyzed synthesis of conducting polyaniline. Enzyme Microb Tech. 2003, 33: 556-564.Google Scholar
  117. Zumarraga M, Bulter T, Shleev S, Polaina J, Plou FJ, Ballesteros A, Alcalde M: In vitro evolution of a fungal laccase in high concentrations of organic cosolvents. Chem Biol. 2007, 14: 1052-1064.Google Scholar
  118. Zumarraga M, Camarero S, Martinez-Arias A, Ballesteros A, Plou FJ, Alcalde M: Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra. Proteins. 2008, 71 (1): 250-260.Google Scholar
  119. Zumarraga M, Vaz Domínguez C, Camarero S, Shleev S, Polaina J, Martínez-Arias A, Ferrer M, de Lacey A, Fernández V, Ballesteros A, Plou FJ, Alcalde M: Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved 509VSG511tripeptide. Comb Chem High-Throughput Scr. 2008Google Scholar
  120. Alcalde M, Zumarraga M, Polaina J, Ballesteros A, Plou FJ: Combinatorial saturation mutagenesis by in vivo overlap extension for the engineering of fungal laccases. Comb Chem High Throughput Screen. 2006, 9 (10): 719-727.Google Scholar
  121. Xu F: Recent progress in laccase study: properties, enzymology, production, and applications. The encyclopedia of bioprocessing technology: fermentation, biocatalysis and bioseparation. Edited by: Flickinger MC, Drew SW. 1999, 1545-1554. NY, John Wiley & SonsGoogle Scholar
  122. Baker WL, Sabapathy K, Vibat M, Lonergan G: Laccase catalyzes formation of an indamine dye between 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Enzyme Microb Technol. 1996, 18: 90-94.Google Scholar
  123. Uyama H, Kobayashi S: Enzyme-catalyzed polymerization to functional polymers. J Mol Catal B: Enzym. 2002, 19–20: 117-127.Google Scholar
  124. Setti L, Giuliani S, Spinozzi G, Pifferi PG: Laccase catalyzed oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone and methoxyphenols. Enzyme Microb Technol. 1999, 25: 285-289.Google Scholar
  125. Aktaş N, Çiçek H, Ünal AT, Kibarer G, Kolankaya N, Tanyolaç A: Reaction kinetics for laccase-catalyzed polymerization of 1-napthol. Bioresour Technol. 2001, 80: 29-36.Google Scholar
  126. Schäfer A, Specht M, Hetzheim A, Francke W, Schauer F: Synthesis of substituted imidazoles and dimerization products using cells and laccase from Trametes versicolor. Tetrahedron. 2001, 57: 7693-7699.Google Scholar
  127. Aktas N, Tanyolac A: Reaction conditions for laccase catalyzed polymerization of catechol. Bioresour Technol. 2003, 87: 209-214.Google Scholar
  128. Boumans JWL, Nagtegaal RMA, Dunnewind A, Happe RP, Bos MA, Faegemand M, Degn P: A method for enzymatic cross-linking of a protein, cross-linked protein thus obtained and use thereof. WO Patent 2006/016809. 2006Google Scholar
  129. Hajdok S, Leutbecher H, Greiner G, Conrad J, Beifuss U: Laccase initiated oxidative domino reactions for the efficient synthesis of 3, 4-dihydro-7, 8-dihydroxy-2 H-dibenzofuran-1-ones. Tetrahedr Lett. 2007, 48: 5073-5076.Google Scholar
  130. Uchida H, Fukuda T, Miyamoto H, Kawabata T, Suzuki M, Uwajima T: Polymerization of bisphenol A by purified laccase from Trametes villosa. Biochem Biophys Res Commun. 2001, 287: 355-358.Google Scholar
  131. Mattinen ML, Hellman M, Permi P, Autio K, Kalkkinen N, Buchert J: Effect of protein structure on laccase-catalyzed protein oligomerization. J Agric Food Chem. 2006, 54: 8883-8890.Google Scholar
  132. Marzorati M, Danieli B, Haltrich D, Riva S: Selective laccase-mediated oxidation of sugars derivatives. Green Chem. 2005, 7: 310-315.Google Scholar
  133. Baratto L, Candido A, Marzorati M, Sagui F, Riva S, Danieli B: Laccase-mediated oxidation of natural glycosides. J Mol Cat B: Enzym. 2006, 39: 3-8.Google Scholar
  134. Ncanana S, Baratto L, Roncaglia L, Riva S, Burton SG: Laccase-mediated oxidation of totarol. Adv Synth Catal. 2007, 349: 1507-1513.Google Scholar
  135. Suderman RJ, Dittmer NT, Kanost MR, Kramer KJ: Model reactions for insect cuticle sclerotization: Crosslinking of recombinant proteins upon their laccase-catalyzed oxidative conjugation with catechols. Insect Biochem Mol Biol. 2006, 36: 353-365.Google Scholar
  136. Kurisawa M, Chung JE, Uyama H, Kobayashi S: Laccase-catalyzed synthesis and antioxidant property of poly(catechin). Macromol Biosci. 2003, 3: 758-764.Google Scholar
  137. Aktas N, Kibarer G, Tanyolaç A: Effects of reaction conditions on laccase-catalysed 1-naphthol polymerisation. J Chem Technol Biotechnol. 2000, 75: 840-846.Google Scholar
  138. Aktas N, Tanyolaç A: Kinetics of laccase-catalyzed oxidative polymerization of catechol. J Mol Catal B: Enzym. 2003, 22: 61-69.Google Scholar
  139. Ceylan H, Kubilay S, Aktas N, Sahiner N: An approach for prediction of optimum reaction conditions for laccase-catalyzed bio-transformation of 1-naphthol by response surface methodology (RSM). Bioresour Technol. 2008, 99: 2025-2031.Google Scholar
  140. Intra A, Nicotra S, Riva S, Danieli B: Significant and unexpected solvent influence on the selectivity of laccase-catalyzed coupling of tetrahydro-2-naphthol derivatives. Adv Synth Catal. 2005, 347: 973-977.Google Scholar
  141. Dodrick JS, Marletta MA, Klibanov AM: Polymerization of phenols catalyzed by peroxidase in nonaqueous media. Biotechnol Bioeng. 1987, 30: 31-36.Google Scholar
  142. Wariishi H, Nonaka D, Nishihashi S, Hirahashi T, Ito K: Enzymic preparation of polyphenylne oxides. Patent JP2006280259 A2. 2006Google Scholar
  143. An ES, Kim SC, Kim YH, Park SY, Ryu JY, Song BK, Song JK: Production of phenolic polymers using bio-catalysts for polymerization. Patent KR2005011958A. 2005Google Scholar
  144. Takahara J: Enzymic manufacture of polyphenylene oxide (PPO). Patent JP2004313057 A2. 2004Google Scholar
  145. Ikeda R, Sugihara J, Uyama H, Kobayashi S: Enzymatic oxidative polymerization of 2, 6-dimethylphenol[J]. Macromol. 1996, 29: 8702-8705.Google Scholar
  146. Ikeda R, Tanaka H, Uyama H, Kobayashi S: Laccase-catalyzed polymerization of acrylamide. Macromol Rapid Commun. 1998, 19: 423-425.Google Scholar
  147. Ikeda R, Sugihara J, Uyama H, Kobayashi S: Enzymatic oxidative polymerization of 4-hydroxybenzoic acid derivatives to poly(phenylene oxide)s[J]. Polym Int. 1998, 47: 295-301.Google Scholar
  148. Kobayashi S, Ikeda R, Oyabu H, Tanaka H, Kobayashi S: Artificial Urushi: Design, synthesis, and enzymatic curing of new urushiol analogues. Chem Lett. 2000, 29: 1214-1215.Google Scholar
  149. Kobayashi S, Uyama H, Ikeda R: Artificial urushi. Chemistry. 2001, 7 (22): 4754-4760.Google Scholar
  150. Ikeda R, Tsujimoto T, Tanaka H, Oyabu H, Uyama H, Kobayashi S: Man-made Urushi. Preparation of crosslinked polymeric films from renewable resources via air-oxidation processes. Proc Jpn Acad. 2000, 76B: 155-160.Google Scholar
  151. Ikeda R, Tanaka H, Oyabu H, Uyama H, Kobayashi S: Preparation of artificial urushi via an environmentally benign process. Bull Chem Soc Jpn. 2001, 74: 1067-1073.Google Scholar
  152. Uyama H, Kobayashi S: Enzymatic synthesis of polyesters via polycondensation. Adv Polym Sci. 2006, 194: 133-158.Google Scholar
  153. Gübitz GM, Paulo AC: New substrates for reliable enzymes: enzymatic modification of polymers. Curr Opin Biotechnol. 2003, 14 (6): 577-582.Google Scholar
  154. Gronqvist S, Rantanen K, Alen R, Mattinen ML, Buchert J, Viikari L: Laccase-catalysed functionalisation of TMP with tyramine. Holzforschung. 2006, 60: 503-508.Google Scholar
  155. Felby C, Pedersen LS, Nielsen BR: Enhanced autoadhesion of wood fibers using phenol oxidases. Holzforschung. 1997, 51: 281-286.Google Scholar
  156. Huttermann A, Mai C, Kharazipour A: Modification of lignin for the production of new compound and materials. Appl Microbiol Biotechnol. 2001, 55: 387-394.Google Scholar
  157. Lund M, Ragauskas AJ: Enzymatic modification of kraft lignin through oxidative coupling with water-soluble phenols. Appl Microbiol Biotechnol. 2001, 55: 699-703.Google Scholar
  158. Chandra RP, Ragauskas AJ: Evaluating laccase-facilitated coupling of phenolic acids to high-yield kraft pulps. Enzyme Microb Technol. 2002, 30: 855-861.Google Scholar
  159. Bragd PL, van Bekkum H, Besemer AC: TEMPO-mediated oxidation of polysaccharides: survey of methods and applications. Topics in Catal. 2004, 27: 49-66.Google Scholar
  160. Monti D, Candido D, Manuel Cruz Silva M, Køen V, Riva S, Danieli B: Biocatalyzed generation of molecular diversity: selective modification of the saponin asiaticoside. Adv Synth Catal. 2005, 347: 1168-1174.Google Scholar
  161. Burton S: Laccases and phenol oxidases in organic synthesis. Curr Org Chem. 2003, 7: 1317-1331.Google Scholar
  162. Ossiadacz J, Al-Adhami AJH, Bajraszewska D, Fischer P, Peczynska-Czoch W: On the use of Trametes versicolor laccase for the conversion of 4-methyl-3-hydroxyanthranilic acid to actinocin chromophore. J Biotechnol. 1999, 72: 141-149.Google Scholar
  163. Pilz R, Hammer E, Schauer F, Kragl U: Laccase-catalyzed synthesis of coupling products of phenolic substrates in different reactors. Appl Microbiol Biotechnol. 2003, 60: 708-712.Google Scholar
  164. Stahl P, Kissau L, Mazitschek R, Giannis A, Waldmann H: Natural product derived receptor tyrosin kinase inhibitors: Identification of IGF1R-, Tie-2 and VEGFR3 inhibitors. Angew Chem Int Ed Engl. 2002, 41 (7): 1174-1178.Google Scholar
  165. Honma Y, Kasukabe T, Hozumi M, Shibata K, Omura S: Effects of herbimycin A derivatives on growth and differentiation of K562 human leukemic cells. Anticancer Res. 1992, 12: 189-192.Google Scholar
  166. Mathew AE, Zee-Cheng KY, Cheng CC: Amino-substituted p-benzoquinones. J Med Chem. 1986, 29: 1792-1795.Google Scholar
  167. Zee-Cheng KY, Cheng CC: Preparation and the results of antitumor screening of some substituted amino-, azido-, halogeno- and hydroxy-p-benzoquinones. J Med Chem. 1970, 13: 264-268.Google Scholar
  168. Pöckel D, Niedermeyer THJ, Pham HTL, Mikolasch A, Mundt S, Lindequist U, Lalk M, Werz O: Inhibition of human 5-lipoxygenase and anti-neoplastic effects by 2-amino-1, 4-benzoquinones. Med Chem. 2006, 2: 591-595.Google Scholar
  169. Timo HJN, Michael L: Nuclear amination catalyzed by fungal laccases: Comparison of laccase catalyzed amination with known chemical routes to aminoquinones. J Mol Catal B: Enzym. 2007, 45: 113-117.Google Scholar
  170. Molino BF, Haydar SN, Yang Z, Michels PC, Hemenway MS, Rich JO, Khmelnitsky Y: Preparation of novel cyclosporins. Patent WO2004082629 A2. 2004Google Scholar
  171. Hosny M, Rosazza JPN: Novel oxidations of (+)-catechin by horseradish peroxidase and laccase. J Agric Food Chem. 2002, 50: 5539-5545.Google Scholar
  172. Intra A: Ossidazione di derivati fenolici ad opera di laccasi. Tesi di laurea, Universita' di Milano. 2003Google Scholar
  173. Shiba T, Xiao L, Miyakoshi T, Chen C-L: Oxidation of isoeugenol and coniferyl alcohol catalyzed by laccase isolated from Rhus vernicifera Stokes and Pycnoporus coccineus. J Mol Catal B: Enzym. 2000, 10: 605-615.Google Scholar
  174. Niedermeyer THJ, Mikolasch A, Lalk M: Nuclear amination catalyzed by fungal laccases: reaction products of p-hydroquinones and primary aromatic amines. J Org Chem. 2005, 70: 2002-2008.Google Scholar
  175. Manda K, Hammer E, Mikolasch A, Niedermeyer T, Dec J, Daniel Jones A, Benesi AJ, Schauer F, Bollag JM: Laccase-induced cross-coupling of 4-aminobenzoic acid with para-dihydroxylated compounds 2, 5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 2, 5-dihydroxybenzoic acid methyl esters. J Mol Catal B: Enzym. 2005, 35: 86-92.Google Scholar


© Kunamneni et al; licensee BioMed Central Ltd. 2008

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.