Biotechnology Advances 24 (2006) 500 – 513

www.elsevier.com/locate/biotechadv

Research review paper

Industrial and biotechnological applications of laccases: A review
Susana Rodríguez Couto ⁎, José Luis Toca Herrera ⁎
Department of Chemical Engineering, Rovira i Virgili University, Av. Països Catalans 26, 43007 Tarragona, Spain
Received 20 January 2006; received in revised form 29 March 2006; accepted 1 April 2006
Available online 18 April 2006

Abstract

Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and non-
phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their
application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the
paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up
herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as
catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove
xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the
applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Food industry; Industrial applications; Laccase; Nanobiotechnology; Pulp and paper industry; Textile industry

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
2. Potential industrial and biotechnological applications of laccase enzyme . . . . . . . . . . . . . . . . . . . . . . . 501
2.1. Food industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
2.2. Pulp and paper industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
2.3. Textile industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
2.4. Nanobiotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
2.5. Other laccase applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
2.5.1. Soil bioremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
2.5.2. Synthetic chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
2.5.3. Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
3. Future outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508

⁎ Corresponding authors. Tel.: +34 977 55 9617; fax: +34 977 55 9667.
E-mail addresses: susana.rodriguez@urv.net (S. Rodríguez Couto), joseluis.toca@urv.net (J.L. Toca Herrera).

0734-9750/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2006.04.003
 S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513
1. Introduction
Although oxidation reactions are essential in several
industries, most of the conventional oxidation technol-
ogies have the following drawbacks: non-specific or
undesirable side-reactions and use of environmentally
hazardous chemicals. This has impelled the search for
new oxidation technologies based on biological systems
such as enzymatic oxidation. These systems show the
following advantages over chemical oxidation: enzymes
are specific and biodegradable catalysts and enzyme
reactions are carried out in mild conditions.
Enzymatic oxidation techniques have potential within
a great variety of industrial fields including the pulp and
paper, textile and food industries. Enzymes recycling on
molecular oxygen as an electron acceptor are the most
interesting ones. Thus, laccase (benzenediol: oxygen
oxidoreductase; EC 1.10.3.2) is a particularly promising
enzyme for the above-mentioned purposes. The laccase
molecule is a dimeric or tetrameric glycoprotein, which
usually contains four copper atoms per monomer
distributed in three redox sites (Gianfreda et al., 1999).
This enzyme catalyses the oxidation of ortho and
paradiphenols, aminophenols, polyphenols, polyamines,
lignins and aryl diamines as well as some inorganic ions
coupled to the reduction of molecular dioxygen to water
(Yaropolov et al., 1994; Solomon et al., 1996).
The reported redox potentials of laccases are lower
than those of non-phenolic compounds, so these enzymes
cannot oxidise such substances. However, it was shown
that in the presence of small molecules capable to act as
electron transfer mediators laccases were also able to
oxidise non-phenolic structures (Bourbonnais and Paice,
1990; Call and Mücke, 1997), expanding, thus, the range
of compounds that can be oxidised by these enzymes.
Laccase-mediated systems (LMS) have been applied to
numerous processes such as pulp delignification (Bour-
bonnais et al., 1997; Bourbonnais et al., 1998; Crestini
and Argyropoulos, 1998; Li et al., 1999), oxidation of
organic pollutants (Collins et al., 1996) and the
development of biosensors (Kulys et al., 1997; Trudeau
et al., 1997; Kuznetsov et al., 2001) or biofuel cells
(Palmore and Kim, 1999). Several organic and inorganic
compounds have been reported as effective mediators for
the above-mentioned purposes. These include thiol and
phenol aromatic derivatives, N-hydroxy compounds and
ferrocyanide, respectively. Claus et al. (2002) found that
the LMS enhanced dye decolourization and some dyes
resistant to laccase degradation were decolourised. Lu and
Xia (2004) have recently reviewed the applications of the
LMS, which comprise pulp bleaching, textile biofinishing
and environmental protection processes. However, de-
501
spite that LMS has been studied extensively there are still
unsolved problems concerned with mediator recycling,
cost and toxicity.
Laccases have been reviewed several times in recent
years, generally with emphasis on narrow aspects. The
reviews by Messerschmidt (1993, 1997) and by
Solomon et al. (1996) provide excellent summaries of
the enzymology and electron transfer mechanism of the
laccases and a book edited by Messerschmidt (1997)
contains a series of articles dealing with different aspects
of laccase kinetics and mechanism of action and the
possible roles of this enzyme. The aim of this review is
to highlight the potential industrial and biotechnological
applications of laccase enzyme.
2. Potential industrial and biotechnological applica-
tions of laccase enzyme
Table 1 shows different applications of laccases in
the last two decades. Laccases find applications within
the following fields:
2.1. Food industry
Laccases can be applied to certain processes that
enhance or modify the colour appearance of food or
beverage. In this way, an interesting application of
laccases involves the elimination of undesirable phe-
nolics, responsible for the browning, haze formation and
turbidity development in clear fruit juice, beer and wine.
Laccases are currently of interest in baking due to its
ability to cross-link biopolymers. Thus, Selinheimo et
al. (2006) showed that a laccase from the white-rot
fungus Trametes hirsuta increased the maximum
resistance of dough and decreased the dough extensi-
bility in both flour and gluten dough.
Recently, Minussi et al. (2002) have described the
potential applications of laccase in different aspects of
the food industry such as bioremediation, beverage
processing, ascorbic acid determination, sugar beet
pectin gelation, baking and as a biosensor. However,
they suggested that more studies of laccase production
and immobilisation techniques at lower costs are needed
to improve the industrial application of this enzyme.
2.2. Pulp and paper industry
The industrial preparation of paper requires separa-
tion and degradation of lignin in wood pulp. Environ-
mental concerns urge to replace conventional and
polluting chlorine-based delignification/bleaching pro-
cedures (Kuhad et al., 1997). Oxygen delignification
502 S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513

Table 1
Different laccase applications
Application Laccase source Reference
Decolourization of Aspergillus (genetically modified) Soares et al. (2001a)
dyes Aspergillus (genetically modified) Soares et al. (2001b)
Aspergillus niger Soares et al. (2002)
Cerrena unicolor Michniewicz et al. (2003)
Coriolopsis gallica Reyes et al. (1999)
Coriolopsis rigida Gómez et al. (2005)
Funalia trogii Ünyayar et al. (2005)
Irpex lacteus Kasinath et al. (2003)
Myceliophthora thermophila, Polyporus pinsitus, Trametes versicolor Claus et al. (2002)
Pleurotus eryngii, Pycnoporus cinnabarinus, T. versicolor Camarero et al. (2004)
Pleurotus ostreatus Hou et al. (2004)
P. ostreatus Palmieri et al. (2005)
P. cinnabarinus Mccarthy et al. (1999)
P. cinnabarinus Schliephake et al. (2000)
Sclerotium rolfsii, Trametes hirsuta Campos et al. (2001)
Streptomyces cyaneus Arias et al. (2003)
T. hirsuta Abadulla et al. (2000)
T. hirsuta Domínguez et al. (2005)
T. hirsuta Moldes et al. (2003)
T. hirsuta Rodríguez Couto et al.
(2004a)
T. hirsuta Rodríguez Couto et al.
(2004c)
T. hirsuta Rodríguez Couto et al. (2005)
T. hirsuta Rodríguez Couto et al. (2006)
T. hirsuta Rodríguez Couto and
Sanromán (2006)
T. hirsuta Rodríguez Couto and
Sanromán (2005)
T. hirsuta, T. versicolor Rodríguez Couto et al.
(2004b)
Trametes modesta Nyanhongo et al. (2002)
T. modesta Rehorek et al. (2004)
Trametes trogii Levin et al. (2005)
T. versicolor Maceiras et al. (2001)
T. versicolor Lorenzo et al. (2002)
T. versicolor Rodríguez Couto et al. (2002)
T. versicolor Peralta-Zamora et al. (2003)
T. versicolor Blánquez et al. (2004)
T. versicolor Tavares et al. (2004)
Trametes villosa Zille et al. (2003)
T. villosa Knutson and Ragauskas
(2004)
Degradation of strain I-4 of the family Chaetomiaceae Saito et al. (2004)
xenobiotics Cladosporium sphaerospermum Potin et al. (2004)
Coprinus cinereus, Myceliophthora thermophila, P. pinsitus, Rhizoctonia solani Kulys et al. (2003)
C. gallica Pickard et al. (1999)
C. gallica Vandertol-Vanier et al. (2002)
Coriolus hirsutus Cho et al. (2002)
Coriolus versicolor Itoh et al. (2000)
C. versicolor Okazaki et al. (2002)
Myceliophtora thermophyla, Trametes pubescens Nicotra et al. (2004)
Panus tigrinus Zavarzina et al. (2004)
P. osteratus Eggen (1999)
P. ostreatus Hublik and Schinner (2000)
P. ostreatus, T. versicolor Keum and Li (2004)
P. cinnabarinus Mougin et al. (2002)
Pyricularia oryzae Lante et al. (2000)
 S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513 503

Table 1 (continued )
Application Laccase source Reference
P. oryzae Carunchio et al. (2001)
Rhus vernicifera Moeder et al. (2004)
T. hirsuta Niku-Paavola and Viikari
(2000)
T. hirsuta D10 Böhmer et al. (1988)
Trametes sp. Tanaka et al. (2001)
Trametes sp. Tanaka et al. (2003)
T. versicolor Collins et al. (1996)
T. versicolor Johannes et al. (1998)
T. versicolor Majcherczyk et al. (1998)
T. versicolor Johannes and Majcherczyk
(2000)
T. versicolor Majcherczyk and Johannes
(2000)
T. versicolor Castro et al. (2003)
T. versicolor Dodor et al. (2004)
T. villosa Fabbrini et al. (2001)
T. villosa Fukuda et al. (2001)
T. villosa Kang et al. (2002)
T. villosa Cantarella et al. (2003)
Trichophyton sp. LKY-7 Jung et al. (2003)
unspecified Zhang et al. (2002)
Biosensors Agaricus bisporus, A. niger, T. versicolor Timur et al. (2004)
Agaricus bisporus, R. vernicifera Rigidoporus lignosus, T. versicolor Vianello et al. (2004)
Aspergillus oryzae, Myceliophtora Thermophila, P. pinsitus Kulys and Vidziunaite (2003)
C. unicolor Jarosz-Wilkołazka et al.
(2004)
C. unicolor Jarosz-Wilkołazka et al.
(2005)
C. hirsutus Marko-Varga et al. (1995)
C. hirsutus Lisdat et al. (1997)
C. hirsutus Bauer et al. (1999)
C. hirsutus Kuznetsov et al. (2001)
C. hirsutus Freire et al. (2002)
C. hirsutus, R. vernicifera Gupta et al. (2003)
C. versicolor Gomes and Rebelo (2003)
P. ostreatus Leite et al. (2003)
P. oryzae Palmore and Kim (1999)
R. vernicifera Gardiol et al. (1996)
T. versicolor Leech and Daigle (1998)
T. versicolor Freire et al. (2001)
T. versicolor Gomes and Rebelo (2003)
T. versicolo Haghighi et al. (2003)
T.. versicolor Gomes et al. (2004)
T. versicolor Roy et al. (2005)
T. versicolor Ferry and Leech (2005)
Effluent treatment C. gallica Calvo et al. (1998)
Gliocladium virens Murugesan (2003)
Lentinula edodes D'Annibale et al. (1999)
L. edodes D'Annibale et al. (2000)
L. edodes Casa et al. (2003)
P. tigrinus D'Annibale et al. (2004)
P. ostreatus Aggelis et al. (2003)
Pleurotus spp. Tsioulpas et al. (2002)
Pycnoporus coccineus Jaouani et al. (2005)
R. vernicifera Durante et al. (2004)
Trametes sp. strain AH28-2 Xiao et al. (2003)
T. versicolor Jolivalt et al. (2000)
(continued on next page)
504 S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513

Table 1 (continued )
Application Laccase source Reference
T. versicolor Edwards et al. (2002)
T. versicolor Lucas et al. (2003)
Biopulping Fomes fomentarius, Ganoderma collosum, Lentinus edades, Merulius tremellosus, Phlebia Bourbonnais et al. (1997)
radiata, P. ostreatus T. versicolor
C. versicolor Call and Mücke (1997)
(Lignozym®-process)
Peniophora sp., Pycnoporus sanguineus, T. hirsuta, T. versicolor Kandioller and Christov
(2001)
T. versicolor Archibald et al. (1997)
T. versicolor Crestini and Argyropoulos
(1998)
unspecified Jacob et al. (1999)
unspecified Sealey et al. (1999)
unspecified Chakar and Ragauskas (2001)
unspecified Poppius-Levlin et al. (2001)
unspecified Tamminen et al. (2003)
Organic synthesis C. hirsuta Baker et al. (1996)
C. hirsutus Karamyshev et al. (2003)
P. cinnabarinus Mikolasch et al. (2002)
P. coccineus Uyama and Kobayashi (2002)
P. oryzae Setti et al. (1999)
T. versicolor Fritz-Langhals and Kunath
(1998)
T. versicolor Akta et al. (2001)
T. versicolor Schäfer et al. (2001)
T. versicolor Akta and Tanyolaç (2003)
T. villosa Uchida et al. (2001)
Food industry Chinese rhus lacquer Huang et al. (1995)
Myceliophtora thermophili, P. pinsitius Micard and Thibault (1999)
P. cinnabarinus Georis et al. (2003)
T. hirsuta Kuuva et al. (2003)
T. versicolor Crecchio et al. (1995)
unspecified Mathiasen (1996)
unspecified Petersen and Mathiasen
(1997)
unspecified Norsker et al. (2000)
Biobleaching C. versicolor Balakshin et al. (2001)
P. eryngii, P. cinnabarinus, T. versicolor Camarero et al. (2004)
P. cinnabarinus Georis et al. (2003)
T. versicolor Paice et al. (1995)
T. versicolor Archibald et al. (1997)
unspecified Balakshin et al. (2001)
unspecified Han et al. (2002)
Denim bleaching T. versicolor Pazarlıoglu et al. (2005)
unspecified Vinod (2001)

processes have been industrially introduced (Carter et
al., 1997), but pre-treatments of wood pulp with
ligninolytic enzymes might provide milder and cleaner
strategies of delignification that are also respectful of the
integrity of cellulose (Kuhad et al., 1997).
Although extensive studies have been performed to
develop alternative bio-bleaching systems, few enzy-
matic treatments exhibit the delignification/brightening
capabilities of modern chemical bleaching technologies.
One of the few exceptions to this generalisation is the
development of LMS delignification technologies for
kraft pulps. In addition, laccase is more readily available
and easier to manipulate than both lignin peroxidase
(LiP) and manganese-dependent peroxidase (MnP) and
LMS has already found practical applications such as
the Lignozym®-process (Call and Mücke, 1997).
Several authors applied the LMS to pulp biobleach-
ing (see Table 1). However, all these biobleaching
studies were focused on wood pulps and little is known
about the efficiency of the LMS on non-wood pulps
 S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513
including those used for manufacturing specialty papers.
In this sense, Camarero et al. (2004) explored the
potential of LMS to remove lignin-derived products
responsible for colour from a high-quality flax pulp.
They showed the feasibility of LMS to substitute
chlorine-containing reagents in manufacturing of these
high-price paper pulps.
The capability of laccases to form reactive radicals in
lignin can also be used in targeted modification of wood
fibers. For example, laccases can be used in the enzymatic
adhesion of fibers in the manufacturing of lignocellulose-
based composite materials such as fiberboards. Laccases
have been proposed to activate the fiberbound lignin
during manufacturing of the composites, thus, resulting in
boards with good mechanical properties without toxic
synthetic adhesives (Felby et al., 1997; Hüttermann et al.,
2001). Another possibility is to functionalise lignocellu-
losic 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
phenolics acid derivatives onto kraft pulp fibers (Lund
and Ragauskas, 2001; Chandra and Ragauskas, 2002).
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.
2.3. Textile industry
The textile industry accounts for two-thirds of the total
dyestuff market (Riu et al., 1998) and consumes large
volumes of water and chemicals for wet processing of
textiles. The chemical reagents used are very diverse in
chemical composition, ranging from inorganic com-
pounds to polymers and organic products (Mishra and
Tripathy, 1993; Banat et al., 1996; Juang et al., 1996).
There are more than 100,000 commercially available dyes
with over 7 × 105 t of dyestuff produced annually (Meyer,
1981; Zollinger, 2002). Due to their chemical structure
dyes are resistant to fading on exposure to light, water and
different chemicals (Poots and McKay, 1976; McKay,
1979) and most of them are difficult to decolourise due to
their synthetic origin.
Government legislation is becoming more and more
stringent, especially in the more developed countries,
regarding the removal of dyes from industrial effluents
(O'Neill et al., 1999). Concern arises, as several dyes are
made from known carcinogens such as benzidine and
other aromatic compounds (Baughman and Perenich,
1988). Most currently existing processes to treat dye
wastewater are ineffective and not economical (Cooper,
1995; Stephen, 1995). Therefore, the development of
505
processes based on laccases seems an attractive solution
due to their potential in degrading dyes of diverse
chemical structure (Abadulla et al., 2000; Blánquez et
al., 2004; Hou et al., 2004), including synthetic dyes
currently employed in the industry (Rodríguez Couto et
al., 2004a, 2005).
The use of laccase in the textile industry is growing
very fast, since besides to decolourise textile effluents as
commented above, laccase is used to bleach textiles and
even to synthetise dyes (Setti et al., 1999). Related to
textile bleaching, in 1996 Novozyme (Novo Nordisk,
Denmark) launched a new industrial application of
laccase enzyme in denim finishing: DeniLite®, the first
industrial laccase and the first bleaching enzyme acting
with the help of a mediator molecule. Also, in 2001 the
company Zytex (Zytex Pvt. Ltd., Mumbai, India)
developed a formulation based on LMS capable of
degrading indigo in a very specific way. The trade name
of the product is Zylite.
2.4. Nanobiotechnology
During the past two decades, bioelectrochemistry has
received increased attention. Progress on bioelectrochem-
istry has been integrated into analytical applications, e.g.
in biosensors working as detectors in clinical and envi-
ronmental analysis (Haghighi et al., 2003). Since laccases
are able to catalyse electron transfer reactions without
additional cofactors, their use has also been studied in
biosensors to detect various phenolic compounds, oxygen
or azides (see Table 1). Moreover, biosensors for detection
of morphine and codeine (Bauer et al., 1999), catecho-
lamines (Lisdat et al., 1997; Leite et al., 2003; Ferry and
Leech, 2005), plant flavonoids (Jarosz-Wilkołazka et al.,
2004) and also for electroimmunoassay (Kuznetsov et al.,
2001) have been developed. Nanotechnology contributes
to the development of smaller and more efficient
biosensors through controlled deposition and specific
adsorption of biomolecules on different types of surfaces,
achieving micro and nanometer order. Hammond and
Whitesides (1995) have introduced a method to pattern
ultrathin ionic multilayer films with micron-sized features
onto surfaces building a patterned alkanethiol monolayer
with ionic functionality onto a gold surface. Typical mol-
ecules used in this process are shown in Fig. 1. Chen et al.
(1998) showed a biotechnological application of such
micropatterned surfaces: the production of islands of
micrometer size of extracellular matrix, where the pattern
of these islands could determine the position and
distribution of bovine and endothelial cells. The control
of the nature and the density of the groups (e.g. alkys,
amides, alcohols) of a surface built with assembled
506 S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513

a) Polyanions Polycations

* *
n*
n*

NH 3+

SO 3 - PAH

PSS
* n*

* N+
n*

COO - H3 C CH3

PAA PDADMAC

b)
Phospholipids Alkanethiols

O
O
O +

SH
O P OCH2CH2NH3
O O–

Fig. 1. a) Polylectrolytes are currently used to build multilayers due to their different versatility. PSS: poly(styrene sulfonate); PAA: Poly(acrylic
acid), PAH: poly(allylamine hydrochlorid), PDADMAC: poly(diallyldimethylammonium chloride). b) Phospholipid and alkanethiols have the ability
to form bilayers and self-assembled monolayers.

monolayers has been used succesfully to investigate the
non-specific adsorption of proteins (Sigal et al., 1998).
Regarding laccases, the immobilisation has an important
influence on the biosensor sensitivity (Freire et al., 2001).
Martele et al. (2003) have shown that micropatterning is
an efficient method for the immobilisation of laccases on a
solid surface in order to develop a multi-functional bio-
sensor. Also, Roy et al. (2005) found that cross-linked
enzyme crystals (CLEC) of laccase from Trametes ver-
sicolor could be used in biosensor applications with great
advantage over the soluble enzyme. More recently,
Cabrita et al. (2005) have immobilised laccase from
Coriolus versicolor on N-Hydroxysuccinimide-terminat-
ed self-assembled monolayers on gold. This procedure
could be useful for the further development of biosensors.
In addition, an enzyme electrode based on the co-immo-
bilisation of an osmium redox polymer and a laccase from
T. versicolor on glassy carbon electrodes has been applied
to ultrasensitive amperometric detection of the catechol-
amine neurotransmitters dopamine, epinephrine and
norepinephrine, attaining nanomolar detection limits
(Ferry and Leech, 2005). Laccase can also be immobilised
on the cathode of biofuel cells that could provide power,
for example, for small transmitter systems (Chen et al.,
2001; Calabrese et al., 2002). Biofuel cells are extremely
attractive from an environmental point of view because
electrical energy is generated without combusting fuel,
thus, providing a cleaner source of energy. Fig. 2a shows
different functionalised flat surfaces built with polymers
and self-assembled monolayers (SAMs) that can be used
to adsorb and immobilise proteins or other biomolecules.
The layer-by-layer technique (LbL) (Decher, 1997)
can be used to build macromolecular structures down to
nanometer control leading to surfaces of well-defined
thickness (see Fig. 2a). Recently, flat polyelectrolyte
multilayers built by alternating adsorption of oppositely
charged polyelectrolytes have been used to recrystallise
bacterial proteins making the building of artificial cell
walls possible (Toca-Herrera et al., 2005). The LbL
technique has also been used to build hollow polyelec-
trolyte capsules after core removal (Donath et al., 1998).
Further application of the sequential adsorption of
oppositely charged polyelectrolytes onto enzyme crystal
templates would permit their encapsulation. Caruso et
 S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513 507

a)
Enzymes, Proteins

Polyelectrolyte
cushion

Si, Au, mica, glass Substrate

Patterning:
Polymer
Enzyme, protein
Enzyme, protein

SAM
S S S S S S S S S S

Si, Au, mica, glass Au Substrate

b)
Molecules to degrade

Enzyme

Polyelectrolyte wall

Protein layer

Enzyme, biomolecules

Polyelectrolyte wall
Colloidal particle

Fig. 2. a) 2D supramacromolecular structures that can be used to immobilise biomolecules. Several structures are suitable: polylectrolyte multilayer,
micropatterning and self-assembled monolayers (SAMs). b) 3D supramacromolecular structures that can be used to build microreactors and
immobilise biomolecules. In the first case, hollow polelectrolyte shells can host proteins inside, permitting the diffusion of molecules through the
shell wall. A colloidal particle covered by polyelectrolytes (and phospholipids) can host proteins and/or other types of functional molecules.

al. (2000) showed that the encapsulated enzyme could
retain 100% of its activity after incubation for 100 min
with protease. The permeability properties of the wall
capsule are important for the proper function of the
encapsulated enzyme. Antipov et al. (2002) investigated
the permeability properties of hollow polyelectrolyte
multilayer capsules as a function of pH and salt
concentration. It was shown that the capsule wall was
closed to a pH value of 8 and higher, but at pH values
lower than 6 the macromolecules permeate into the
capsule interior. In this way, the authors showed how to
open and close the capsule wall in a reversible way. This
mechanism together with the LbL encapsulation
technique permits the development of microreactors
Also, colloidal particles covered with polyelectrolytes
and phospholipids have been used to host and activate
rubella virus (Fischlechner et al., 2005). This type of
system is shown in Fig. 2b.
508 S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513
2.5. Other laccase applications
2.5.1. Soil bioremediation
Polycyclic aromatic hydrocarbons (PAHs) together
with other xenobiotics are a major source of contami-
nation in soil, therefore, their degradation is of great
importance for the environment. The catalytic properties
of laccases can be used to degrade such compounds.
Thus, laccases were able to mediate the coupling of
reduced 2,4,6-trinitrotoluene (TNT) metabolites to an
organic soil matrix, which resulted in detoxification of
the munition residue (Durán and Esposito, 2000).
Moreover, PAHs, which arise from natural oil deposits
and utilisation of fossil fuels, were also found to be
degraded by laccases (Pointing, 2001). Recently,
Nyanhongo et al. (in press) showed that a laccase
from Trametes modesta was involved in immobilisation
of TNT degradation products.
2.5.2. Synthetic chemistry
In the future laccases may also be of great interest in
synthetic chemistry, where they have been proposed to
be applicable for oxidative deprotection (Semenov et al.,
1993) and production of complex polymers and medical
agents (Xu, 1999 and Refs. therein, Mai et al., 2000;
Uyama and Kobayashi, 2002; Kurisawa et al., 2003;
Nicotra et al., 2004). Recently, Mustafa et al. (2005)
synthetised phenolic colourants by using an industrial
laccase named Suberase® (Novo Nordisk A/S, Bags-
vaerdt, Denmark).
2.5.3. Cosmetics
The cosmetic world has not been indifferent to the
application of laccase: for example, laccase-based hair
dyes are less irritant and easier to handle than current hair
dyes, since laccases replace H2O2 as an oxidising agent in
the dye formulation (Roure et al., 1992; Aaslyng et al.,
1996; Lang and Cotteret, 1999). More recently, cosmetic
and dermatological preparations containing proteins for
skin lightening have also been developed (Golz-Berner et
al., 2004).
3. Future outlook
The most important obstacles to commercial appli-
cation of laccases are the lack of sufficient enzyme stocks
and the cost of redox mediators. Marked progress has
been made over the last years to solve these problems and
it is expected that laccases will be able to compete with
other processes such as elemental chlorine-free (ECF)
and totally chlorine-free (TCF) bleaching. Thus, efforts
have to be made in order to achieve cheap overproduc-
tion of this biocatalyst in heterologous hosts and also
their modification by chemical means or protein engi-
neering to obtain more robust and active enzymes.
On the other hand, the development of an effective
system for laccase immobilisation also deserves great at-
tention. Immobilisation could be achieved by chemical
modification of the substrates. Hence, micropatterning,
SAMs and LbL techniques can be used to functionalise flat
and curved surfaces in order to have specific adsorption.
Laccase encapsulation with polyelectrolytes will be used
as a microreactor for catalytic reactions by changing the
permeability properties of the capsule wall. Since the
general goal is to obtain stable catalysts with long life times
and low cost, we think that the combination of these
techniques will enhance: i) the adsorption of laccase on a
suitable substrate, ii) the lifetime of the laccase activity and
iii) reutilisation of the substrate/laccase product. Our
research group is currently working in this direction.
Acknowledgments
SRC and JLTH are Ramón y Cajal Senior Research
Fellows. Therefore, the authors thank the Spanish
Ministry of Education and Science for promoting the
Ramón y Cajal Programme.
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