Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications
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452 ADÁNYI ET AL.
polymeric film on a spectrographic graphite electrode. Salimi et al. (2005) investi-
gated the direct voltammetry and electrocatalytic properties of CAT adsorbed onto
the surface of multi-wall carbon nanotubes. Collagen film was formed on pyrolytic
graphite electrodes, which provided a suitable microenvironment for haemoglobin
and CAT to transfer electrons directly to the underlying electrodes (Li et al.,
2005). Campanella et al. (2001) developed an OPEE by immobilizing CAT in a
-carrageenan gel and demonstrated its use in chloroform, toluene, chlorobenzene
and ethyl acetate in a stirred reactor for monitoring the hydrogen peroxide content
of extra virgin olive oil during an artificial rancidification process. Joo et al.
(1996) enhanced enzyme stability by mixing CAT with polyethylene glycol (PEG),
however, the response was dependent upon the molecular weight and concentration
of PEG. A quick analytical method was developed to indirectly monitor the water
(activator) content of various butter and margarine samples using CAT immobilized
by glutaraldehyde on a natural protein membrane in a thin-layer enzyme cell and
connected to a stopped-flow injection analyser (SFIA) system with an amperometric
detector (Adányi and Váradi, 2004).
4. OTHER APPLICATIONS
4.1. Food Microbiology and Sanitizing
The well-studied bactericide effect of the GOx/glucose system by virtue of H
2
O
2
production may be exploitable in the food industry to combat food-poisoning
microbes (Dobbenie et al., 1995; Fuglsang et al., 1995; Massa et al., 2001). It is
noteworthy that GOx affects the growth of fungi (e.g. Candida and Aspergillus spp.)
to very different extents clearly depending on the inherent antioxidant potentials of
the target organisms (Leiter et al., 2004). Combinations of the H
2
O
2
-generating and
H
2
O
2
-consuming enzymes GOx and CAT are also deleterious for aerobic micro-
organisms because of the fast depletion of available oxygen and hence can be used
as antimicrobial agents (Dondero et al., 1993; Fuglsang et al., 1995; Leiter et al.,
2004). The O
2
-scavenging effect of the GOx/CAT system has potential food-related
applications in the control of lipid peroxidation (e.g. in mayonnaises; Isaksen and
Adler-Nissen, 1997), enzymatic and non-enzymatic browning (e.g. in wine and
apple and pear purées; Ough, 1975; Parpinello et al., 2002), off-odour and off-
flavour development (e.g. in mayonnaises; Jafar et al., 1994) and colour changes
(e.g. in grape juice; Castellari et al., 2000).
The antimicrobial potential of GOx is exploited most effectively in combination
with peroxidases such as lactoperoxidases (Sandholm et al., 1988; Popper and
Knorr, 1997; Revol-Junelles et al., 2001), HRP (Hill et al., 1997) and chloroperox-
idases (Jones et al., 1998). GOx alone or encapsulated into liposomes together with
peroxidases may be used for the removal and disinfection of bacterial and fungal
biofilms from medical implants and in the oral cavity (Johansen et al., 1997; Jones
et al., 1998). Multienzymatic system encapsulation techniques are developing fast.
For example, microbeads containing all the components of the GOx-lactoperoxidase
H
2
O
2
PRODUCING AND DECOMPOSING ENZYMES 453
system (enzymes, glucose, thiocyanate) are now available, which may facilitate the
use of this system as a preservative for raw milk especially in developing countries
(Jacquot and Poncelet, 2003). CAT in immobilized form can also be used to remove
excess H
2
O
2
after ‘cold pasteurisation’ (i.e. milk treated with H
2
O
2
at a concen-
tration of 0.05–0.25%), which might be useful and applicable in areas of tropical
or hot climates (Tarhan, 1995; Akertek and Tarhan, 1995).
The increasing interest in and consumer demand for ‘low-alcohol’ wines has
triggered research into the optimisation of glucose conversion to gluconic acid in
grape must by GOx (Pickering et al., 1998, 1999) and the introduction of recom-
binant Saccharomyces cerevisiae strains into the winemaking process that harbour
the A. niger GOx gene (Malherbe et al., 2003). The use of GOx in winemaking also
improves wine stability (Gomez et al., 1995) and alcohol acid balance (Pickering
et al., 1998). An additional application for GOx is in the stabilization of beer
(Ohlmeyer, 1957).
GOx has been employed in baking to improve the strength of the gluten network
via H
2
O
2
-triggered permanent cross-linking of wheat dough proteins (Hilhorst et al.,
1999; Dunnewind et al., 2002; Primo-Martin et al., 2004; Rasiah et al., 2005),
and to reduce the stickiness of enzyme-supplemented doughs (Matinez-Anaya and
Jimenez, 1998; Collar et al., 1998). The performance of GOx is comparable to
that of chemical oxidants such as the bromates traditionally used in bakeries
(Vemulapalli et al., 1998). Combination of GOx with xylanase and -amylase
further improves dough and bread quality, e.g. dough stability and extensibility
and bread volume (Indrani et al., 2003; Primo-Martin et al., 2003). GOx may also
catalyse the formation of arabinoxylane-arabinoxylane cross-links and xylanase may
compensate this disadvantageous effect by cleaving large arabinoxylan complexes
(Primo-Martin et al., 2005).
4.2. Textile Industry
The industrial application of CAT is wide-ranging because its substrate, H
2
O
2
,is
frequently used as a strong biocide, oxidant and bleaching agent in the textile, pulp
and paper, wood, food, etc. industries (Galante and Formantici, 2003). Bleaching
with H
2
O
2
in the textile industry is performed after desizing and scouring, but
before dying. CAT is now used to decompose excess H
2
O
2
and thus contributes
to reducing water consumption (no repeated rinsing), the avoidance of loading
wastewaters with pollutants (like derivatives of Na
2
S
2
O
3
or KMnO
4
, and it enables
the dying process to be performed directly in CAT-treated rinsing baths (Amorim
et al., 2002; Galante and Formantici, 2003). To avoid unacceptable interference
by proteins (CATs) in the dyeing process (Tzanov et al., 2001), the introduction
of immobilized CATs (Costa et al., 2002; Opwis et al., 2004) or, alternatively,
immobilized thermo-alkali-stable bacterial CAT-peroxidases (Paar et al., 2001;
Fruhwirth et al., 2002) are recommended for the treatment and recycling of textile
bleaching effluents for dyeing. Two other interesting possibilities for technological
innovation in this field are the application of immobilized GOx to generate H
2
O
2
for
454 ADÁNYI ET AL.
textile bleaching (Tzanov et al., 2002) and immobilized CAT-peroxidase producer
alkalothermophilic Bacillus cells to decompose excess H
2
O
2
in bleaching effluents
(Paar et al., 2003).
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CHAPTER 26
LACCASES: BIOLOGICAL FUNCTIONS, MOLECULAR
STRUCTURE AND INDUSTRIAL APPLICATIONS
MIGUEL ALCALDE
∗
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Cantoblanco, Madrid,
Spain
∗
malcalde@icp.csic.es
1. INTRODUCTION
Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are extracellular, multi-
copper enzymes that use molecular oxygen to oxidize various aromatic and non-
aromatic compounds by a radical-catalysed reaction mechanism (Thurston, 1994).
They belong to a larger group of enzymes termed the blue-multicopper oxidase
family which includes the plant ascorbate oxidase, the mammalian plasma protein
ceruloplasmin and bilirubin oxidase, among others. The term Laccase stems from
its original identification in the exudates of the Japanese lacquer tree Rhus verni-
cifera described by Yoshida (1883). Just over a century later it was charac-
terized as being a metal-containing oxidase by Bertrand (1985). Laccases have
also been found in other plants, and also insects and bacteria, but are predom-
inant in fungi. Laccase activity has been demonstrated in more than 60 fungal
strains belonging to Basidiomycetes, Ascomycetes and Deuteromycetes, being
documented in virtually every fungus examined for it (Gianfreda et al., 1999).
Its presence in plants appears to be far more limited than in fungi. All species
of family Anacardiaceae, of which the lacquer tree is member, contain laccase
in the resin products and in the secreted resin (Huttermann et al., 2001). Reports
on the presence of laccase in other plants are however limited to Acer pseudo-
platanus, Pinus taeda, Aesculus parviflora and Populus eruamericana (Mayer and
Staples, 2002), though it is believed that they are present throughout the plant
kingdom. Polyphenol oxidases, perhaps laccase-like, have also been reported in
insects (Parkinson et al., 2003), and there is strong evidence for the widespread
distribution of laccases in prokaryotes. The first bacterial laccase to be exten-
sively studied was from Axospirillum lipoferum and the crystal structure of a
461
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 461–476.
© 2007 Springer.
462 ALCALDE
bacterial (Bacillus subtilis) laccase is now available (Enguita et al., 2003). Sequence
homology analysis suggests that laccases also occur in bacteria such as Mycobac-
terium tuberculosis (Alexandre and Zhulin, 2000). Laccase encoding genes have
been found in Gram-negative and Gram-positive bacteria, including species living
in extreme habitats, e.g. in Oceanobacillus iheyensis and Aquifex aeolicus and in the
archaeobacterium Pyrobaculum aerophilum (Claus, 2004, 2003). The development
of metagenomics in the next few years can be expected to lead to the discovery of
novel bacterial laccases from extremophile environments.
2. LACCASE SUBSTRATES AND INHIBITORS
Laccase catalyses the four electron reduction of molecular oxygen to water with
one-electron oxidation of reducing substrate, without producing hydrogen peroxide.
Although laccases preferably act on phenolic compounds their substrate spectrum
is huge, and since the range of substrates oxidized varies from one laccase to
another this complicates the formulation of a precise definition of laccase activity. In
addition there is overlap in substrate range with another type of (copper-containing)
oxidase – tyrosinase -, notionally a mono-phenol monooxygenase. Laccases
can convert o- and p-diphenols, aminophenols, methoxy-substituted phenols,
benzenethiols, polyphenols, polyamines, hydroxyindols, some aryl diamines and a
considerable range of other compounds but do not oxidize tyrosine (whereas the
tyrosinases do). Inorganic/organic metal compounds are also substrates of laccase.
Mn
2+
is oxidized to Mn
3+
and also Fe(EDTA)
2−
is accepted by the enzyme. All
known laccases catalyse the oxidation of ascorbic acid and phenol substrates with
equally high efficiencies. Simple diphenols like hydroquinone and catechol are
generally good substrates but guaicol and 2,6-dimethoxyphenol are often better.
p-Phenylene diamine is a frequently used substrate. Syringaldizine (N N
-bis(3,5-
dimethoxy-4-hydroxybenzylidene hydrazine;
525
= 65000 M
−1
cm
−1
) is a good
substrate but it has to be used in the complete absence of hydrogen peroxide since
syringaldizine is also oxidized by the manganese-dependent peroxidases produced
by many lignolytic basidiomycetes (Thurston, 1994; Gianfreda et al., 1999.)
Laccases have wide reducing substrate (DH) specificity - the K
m
DH
is in the
range of 1–10 mM -. By contrast, the enzyme displays a strong preference for O
2
as the oxidizing substrate. K
m
O2
values are around 10
−5
M and V
max
depends on
the source of the laccase 50–300 Msg
−1
.
Laccases can be very strongly inhibited by various reagents. Small anions such
as azide, halides, cyanide, thiocyanide, fluoride and hydroxide bind to the type
2 and type 3 Cu, resulting in the interruption of internal electron transfer and
inhibition of activity. Other inhibitors include metal ions (e.g. Hg
2+
), fatty acids,
sulfhydryl reagents, hydroxyglycine, kojic acid, desferal and cationic quaternary
ammonium detergents the reactions with which may involve amino acid residue
modifications, conformational changes or Cu chelation (Gianfreda et al., 1999; Call
and Mucke, 1997). Regarding conformational changes, it is known that these are
highly depended on the state of oxidation of the copper atoms. This is one of