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Biotechnology Advances 24 (2006) 500 – 513

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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) 500–513 501

1. Introduction spite that LMS has been studied extensively there are still
unsolved problems concerned with mediator recycling,
Although oxidation reactions are essential in several cost and toxicity.
industries, most of the conventional oxidation technol- Laccases have been reviewed several times in recent
ogies have the following drawbacks: non-specific or years, generally with emphasis on narrow aspects. The
undesirable side-reactions and use of environmentally reviews by Messerschmidt (1993, 1997) and by
hazardous chemicals. This has impelled the search for Solomon et al. (1996) provide excellent summaries of
new oxidation technologies based on biological systems the enzymology and electron transfer mechanism of the
such as enzymatic oxidation. These systems show the laccases and a book edited by Messerschmidt (1997)
following advantages over chemical oxidation: enzymes contains a series of articles dealing with different aspects
are specific and biodegradable catalysts and enzyme of laccase kinetics and mechanism of action and the
reactions are carried out in mild conditions. possible roles of this enzyme. The aim of this review is
Enzymatic oxidation techniques have potential within to highlight the potential industrial and biotechnological
a great variety of industrial fields including the pulp and applications of laccase enzyme.
paper, textile and food industries. Enzymes recycling on
molecular oxygen as an electron acceptor are the most 2. Potential industrial and biotechnological applica-
interesting ones. Thus, laccase (benzenediol: oxygen tions of laccase enzyme
oxidoreductase; EC 1.10.3.2) is a particularly promising
enzyme for the above-mentioned purposes. The laccase Table 1 shows different applications of laccases in
molecule is a dimeric or tetrameric glycoprotein, which the last two decades. Laccases find applications within
usually contains four copper atoms per monomer the following fields:
distributed in three redox sites (Gianfreda et al., 1999).
This enzyme catalyses the oxidation of ortho and 2.1. Food industry
paradiphenols, aminophenols, polyphenols, polyamines,
lignins and aryl diamines as well as some inorganic ions Laccases can be applied to certain processes that
coupled to the reduction of molecular dioxygen to water enhance or modify the colour appearance of food or
(Yaropolov et al., 1994; Solomon et al., 1996). beverage. In this way, an interesting application of
The reported redox potentials of laccases are lower laccases involves the elimination of undesirable phe-
than those of non-phenolic compounds, so these enzymes nolics, responsible for the browning, haze formation and
cannot oxidise such substances. However, it was shown turbidity development in clear fruit juice, beer and wine.
that in the presence of small molecules capable to act as Laccases are currently of interest in baking due to its
electron transfer mediators laccases were also able to ability to cross-link biopolymers. Thus, Selinheimo et
oxidise non-phenolic structures (Bourbonnais and Paice, al. (2006) showed that a laccase from the white-rot
1990; Call and Mücke, 1997), expanding, thus, the range fungus Trametes hirsuta increased the maximum
of compounds that can be oxidised by these enzymes. resistance of dough and decreased the dough extensi-
Laccase-mediated systems (LMS) have been applied to bility in both flour and gluten dough.
numerous processes such as pulp delignification (Bour- Recently, Minussi et al. (2002) have described the
bonnais et al., 1997; Bourbonnais et al., 1998; Crestini potential applications of laccase in different aspects of
and Argyropoulos, 1998; Li et al., 1999), oxidation of the food industry such as bioremediation, beverage
organic pollutants (Collins et al., 1996) and the processing, ascorbic acid determination, sugar beet
development of biosensors (Kulys et al., 1997; Trudeau pectin gelation, baking and as a biosensor. However,
et al., 1997; Kuznetsov et al., 2001) or biofuel cells they suggested that more studies of laccase production
(Palmore and Kim, 1999). Several organic and inorganic and immobilisation techniques at lower costs are needed
compounds have been reported as effective mediators for to improve the industrial application of this enzyme.
the above-mentioned purposes. These include thiol and
phenol aromatic derivatives, N-hydroxy compounds and 2.2. Pulp and paper industry
ferrocyanide, respectively. Claus et al. (2002) found that
the LMS enhanced dye decolourization and some dyes The industrial preparation of paper requires separa-
resistant to laccase degradation were decolourised. Lu and tion and degradation of lignin in wood pulp. Environ-
Xia (2004) have recently reviewed the applications of the mental concerns urge to replace conventional and
LMS, which comprise pulp bleaching, textile biofinishing polluting chlorine-based delignification/bleaching pro-
and environmental protection processes. However, de- 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 development of LMS delignification technologies for
al., 1997), but pre-treatments of wood pulp with kraft pulps. In addition, laccase is more readily available
ligninolytic enzymes might provide milder and cleaner and easier to manipulate than both lignin peroxidase
strategies of delignification that are also respectful of the (LiP) and manganese-dependent peroxidase (MnP) and
integrity of cellulose (Kuhad et al., 1997). LMS has already found practical applications such as
Although extensive studies have been performed to the Lignozym®-process (Call and Mücke, 1997).
develop alternative bio-bleaching systems, few enzy- Several authors applied the LMS to pulp biobleach-
matic treatments exhibit the delignification/brightening ing (see Table 1). However, all these biobleaching
capabilities of modern chemical bleaching technologies. studies were focused on wood pulps and little is known
One of the few exceptions to this generalisation is the about the efficiency of the LMS on non-wood pulps
S. Rodríguez Couto, J.L. Toca Herrera / Biotechnology Advances 24 (2006) 500–513 505

including those used for manufacturing specialty papers. processes based on laccases seems an attractive solution
In this sense, Camarero et al. (2004) explored the due to their potential in degrading dyes of diverse
potential of LMS to remove lignin-derived products chemical structure (Abadulla et al., 2000; Blánquez et
responsible for colour from a high-quality flax pulp. al., 2004; Hou et al., 2004), including synthetic dyes
They showed the feasibility of LMS to substitute currently employed in the industry (Rodríguez Couto et
chlorine-containing reagents in manufacturing of these al., 2004a, 2005).
high-price paper pulps. The use of laccase in the textile industry is growing
The capability of laccases to form reactive radicals in very fast, since besides to decolourise textile effluents as
lignin can also be used in targeted modification of wood commented above, laccase is used to bleach textiles and
fibers. For example, laccases can be used in the enzymatic even to synthetise dyes (Setti et al., 1999). Related to
adhesion of fibers in the manufacturing of lignocellulose- textile bleaching, in 1996 Novozyme (Novo Nordisk,
based composite materials such as fiberboards. Laccases Denmark) launched a new industrial application of
have been proposed to activate the fiberbound lignin laccase enzyme in denim finishing: DeniLite®, the first
during manufacturing of the composites, thus, resulting in industrial laccase and the first bleaching enzyme acting
boards with good mechanical properties without toxic with the help of a mediator molecule. Also, in 2001 the
synthetic adhesives (Felby et al., 1997; Hüttermann et al., company Zytex (Zytex Pvt. Ltd., Mumbai, India)
2001). Another possibility is to functionalise lignocellu- developed a formulation based on LMS capable of
losic fibers by laccases in order to improve the chemical or degrading indigo in a very specific way. The trade name
physical properties of the fiber products. Preliminary of the product is Zylite.
results have shown that laccases are able to graft various
phenolics acid derivatives onto kraft pulp fibers (Lund 2.4. Nanobiotechnology
and Ragauskas, 2001; Chandra and Ragauskas, 2002).
This ability could be used in the future to attach During the past two decades, bioelectrochemistry has
chemically versatile compounds to the fiber surfaces, received increased attention. Progress on bioelectrochem-
possibly resulting in fiber materials with completely novel istry has been integrated into analytical applications, e.g.
properties such as hydrophobicity or charge. in biosensors working as detectors in clinical and envi-
ronmental analysis (Haghighi et al., 2003). Since laccases
2.3. Textile industry are able to catalyse electron transfer reactions without
additional cofactors, their use has also been studied in
The textile industry accounts for two-thirds of the total biosensors to detect various phenolic compounds, oxygen
dyestuff market (Riu et al., 1998) and consumes large or azides (see Table 1). Moreover, biosensors for detection
volumes of water and chemicals for wet processing of of morphine and codeine (Bauer et al., 1999), catecho-
textiles. The chemical reagents used are very diverse in lamines (Lisdat et al., 1997; Leite et al., 2003; Ferry and
chemical composition, ranging from inorganic com- Leech, 2005), plant flavonoids (Jarosz-Wilkołazka et al.,
pounds to polymers and organic products (Mishra and 2004) and also for electroimmunoassay (Kuznetsov et al.,
Tripathy, 1993; Banat et al., 1996; Juang et al., 1996). 2001) have been developed. Nanotechnology contributes
There are more than 100,000 commercially available dyes to the development of smaller and more efficient
with over 7 × 105 t of dyestuff produced annually (Meyer, biosensors through controlled deposition and specific
1981; Zollinger, 2002). Due to their chemical structure adsorption of biomolecules on different types of surfaces,
dyes are resistant to fading on exposure to light, water and achieving micro and nanometer order. Hammond and
different chemicals (Poots and McKay, 1976; McKay, Whitesides (1995) have introduced a method to pattern
1979) and most of them are difficult to decolourise due to ultrathin ionic multilayer films with micron-sized features
their synthetic origin. onto surfaces building a patterned alkanethiol monolayer
Government legislation is becoming more and more with ionic functionality onto a gold surface. Typical mol-
stringent, especially in the more developed countries, ecules used in this process are shown in Fig. 1. Chen et al.
regarding the removal of dyes from industrial effluents (1998) showed a biotechnological application of such
(O'Neill et al., 1999). Concern arises, as several dyes are micropatterned surfaces: the production of islands of
made from known carcinogens such as benzidine and micrometer size of extracellular matrix, where the pattern
other aromatic compounds (Baughman and Perenich, of these islands could determine the position and
1988). Most currently existing processes to treat dye distribution of bovine and endothelial cells. The control
wastewater are ineffective and not economical (Cooper, of the nature and the density of the groups (e.g. alkys,
1995; Stephen, 1995). Therefore, the development of 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 on the cathode of biofuel cells that could provide power,
non-specific adsorption of proteins (Sigal et al., 1998). for example, for small transmitter systems (Chen et al.,
Regarding laccases, the immobilisation has an important 2001; Calabrese et al., 2002). Biofuel cells are extremely
influence on the biosensor sensitivity (Freire et al., 2001). attractive from an environmental point of view because
Martele et al. (2003) have shown that micropatterning is electrical energy is generated without combusting fuel,
an efficient method for the immobilisation of laccases on a thus, providing a cleaner source of energy. Fig. 2a shows
solid surface in order to develop a multi-functional bio- different functionalised flat surfaces built with polymers
sensor. Also, Roy et al. (2005) found that cross-linked and self-assembled monolayers (SAMs) that can be used
enzyme crystals (CLEC) of laccase from Trametes ver- to adsorb and immobilise proteins or other biomolecules.
sicolor could be used in biosensor applications with great The layer-by-layer technique (LbL) (Decher, 1997)
advantage over the soluble enzyme. More recently, can be used to build macromolecular structures down to
Cabrita et al. (2005) have immobilised laccase from nanometer control leading to surfaces of well-defined
Coriolus versicolor on N-Hydroxysuccinimide-terminat- thickness (see Fig. 2a). Recently, flat polyelectrolyte
ed self-assembled monolayers on gold. This procedure multilayers built by alternating adsorption of oppositely
could be useful for the further development of biosensors. charged polyelectrolytes have been used to recrystallise
In addition, an enzyme electrode based on the co-immo- bacterial proteins making the building of artificial cell
bilisation of an osmium redox polymer and a laccase from walls possible (Toca-Herrera et al., 2005). The LbL
T. versicolor on glassy carbon electrodes has been applied technique has also been used to build hollow polyelec-
to ultrasensitive amperometric detection of the catechol- trolyte capsules after core removal (Donath et al., 1998).
amine neurotransmitters dopamine, epinephrine and Further application of the sequential adsorption of
norepinephrine, attaining nanomolar detection limits oppositely charged polyelectrolytes onto enzyme crystal
(Ferry and Leech, 2005). Laccase can also be immobilised 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 lower than 6 the macromolecules permeate into the
retain 100% of its activity after incubation for 100 min capsule interior. In this way, the authors showed how to
with protease. The permeability properties of the wall open and close the capsule wall in a reversible way. This
capsule are important for the proper function of the mechanism together with the LbL encapsulation
encapsulated enzyme. Antipov et al. (2002) investigated technique permits the development of microreactors
the permeability properties of hollow polyelectrolyte Also, colloidal particles covered with polyelectrolytes
multilayer capsules as a function of pH and salt and phospholipids have been used to host and activate
concentration. It was shown that the capsule wall was rubella virus (Fischlechner et al., 2005). This type of
closed to a pH value of 8 and higher, but at pH values 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 tion of this biocatalyst in heterologous hosts and also
their modification by chemical means or protein engi-
2.5.1. Soil bioremediation neering to obtain more robust and active enzymes.
Polycyclic aromatic hydrocarbons (PAHs) together On the other hand, the development of an effective
with other xenobiotics are a major source of contami- system for laccase immobilisation also deserves great at-
nation in soil, therefore, their degradation is of great tention. Immobilisation could be achieved by chemical
importance for the environment. The catalytic properties modification of the substrates. Hence, micropatterning,
of laccases can be used to degrade such compounds. SAMs and LbL techniques can be used to functionalise flat
Thus, laccases were able to mediate the coupling of and curved surfaces in order to have specific adsorption.
reduced 2,4,6-trinitrotoluene (TNT) metabolites to an Laccase encapsulation with polyelectrolytes will be used
organic soil matrix, which resulted in detoxification of as a microreactor for catalytic reactions by changing the
the munition residue (Durán and Esposito, 2000). permeability properties of the capsule wall. Since the
Moreover, PAHs, which arise from natural oil deposits general goal is to obtain stable catalysts with long life times
and utilisation of fossil fuels, were also found to be and low cost, we think that the combination of these
degraded by laccases (Pointing, 2001). Recently, techniques will enhance: i) the adsorption of laccase on a
Nyanhongo et al. (in press) showed that a laccase suitable substrate, ii) the lifetime of the laccase activity and
from Trametes modesta was involved in immobilisation iii) reutilisation of the substrate/laccase product. Our
of TNT degradation products. research group is currently working in this direction.

2.5.2. Synthetic chemistry Acknowledgments


In the future laccases may also be of great interest in
synthetic chemistry, where they have been proposed to SRC and JLTH are Ramón y Cajal Senior Research
be applicable for oxidative deprotection (Semenov et al., Fellows. Therefore, the authors thank the Spanish
1993) and production of complex polymers and medical Ministry of Education and Science for promoting the
agents (Xu, 1999 and Refs. therein, Mai et al., 2000; Ramón y Cajal Programme.
Uyama and Kobayashi, 2002; Kurisawa et al., 2003;
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