Enzyme and Microbial Technology 31 (2002) 907–931

Review
Applications of laccases and tyrosinases (phenoloxidases)
immobilized on different supports: a review
Nelson Durán a,b,∗ , Maria A. Rosa a , Alessandro D’Annibale c , Liliana Gianfreda d
a Biological Chemistry Laboratory, Instituto de Quimica, Universidade Estadual de Campinas, C.P. 6154, Campinas CEP 13083-970, S.P., Brazil
b Núcleo de Ciências Ambientais-NCA, Universidade de Mogi das Cruzes, Mogi das Cruzes, S.P., Brazil
c Dipartimento di Agrobiologia e Agrochimica, Univerisita Degli Studi Della Tuscia, Via San Camillo de Lellis, suc. 01100 Viterbo, Italy
d Dipartimento di Scienze del Suolo, Della Pianta e Dell’Ambiente, Università di Napoli Federico II, Portici, Napoli, Italy

Received 1 August 2001; received in revised form 24 June 2002; accepted 2 July 2002

Abstract
This review summarizes all the research efforts that have been spent to immobilize laccase and tyrosinase for various applications,
including synthetic and analytical purposes, bioremediation, wastewater treatment, and must and wine stabilization. All immobilization
procedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition to
their kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linked
with epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-based
biosensors to monitor a wide range of compounds appears to be at a mature stage of technology.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: Phenoloxidases; Laccase; Tyrosine; Immobilization

1. Introduction The main purpose of this paper is to present a general

Extensive research effort have been dedicated to evaluate
the possibilities offered by enzymes in biotechnological and
environmental applications [1,2].
An effective use of enzymes, may be hampered by some
peculiar properties of the enzymatic proteins such as their
non-reusability, high sensitivity to several denaturating
agents and presence of adverse sensory or toxicological
effects. Many of these undesirable constraints may be re-
moved by the use of immobilized enzymes. This approach
has proven to be more advantageous for catalysis than the
use of free enzymes.
Among enzymes, laccases and tyrosinases are two groups
of phenoloxidases that catalyze the transformation of a large
number of phenolic and non-phenolic aromatic compounds.
In the literature, much information is available on the use
of free and immobilized phenoloxidases in several applied
areas. To date, however, an exhaustive overview of the ba-
sic aspects of immobilized laccase and tyrosinase is still
lacking.
∗Corresponding author. Tel.: +55-19-788-3149; fax: +55-19-3788-3023.
E-mail address: duran@iqm.unicamp.br (N. Durán).
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 2 1 4 - 4
picture of the results achieved in this research field. We have
approached this objective by reviewing the literature dealing
with the fundamental and applied aspects of immobilized
phenoloxidases. A brief introduction on the main aspects of
the active sites and the mechanistic characteristics of laccase
and tyrosinase function has been also added. In our opinion,
these aspects are important because it is their involvement in
enzyme immobilization that determines the final properties
of the immobilized catalyst and the features of its action.
A very short paragraph on the methods of immobilization
with specific reference to those used in the immobilization
of laccase and tyrosinase has been included, as well.
The review has been organized according to the two cate-
gories of enzymes, i.e. laccase (Lac) and tyrosinase (Tyros),
grouped by their originating sources. To give an idea of the
time-progress in this research area, the findings have been
discussed and the references listed in a chronological order.
However, to help the reader to find an enzyme of interest,
we have organized the tables in alphabetical order. Finally,
as the generic name phenoloxidase (PO) and polyphe-
noloxidase (PPO) have been utilized in many publications
to indicate both Lac and Tyros, two additional paragraphs
dedicated to these papers, have been included in this review.
908 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
1.1. Laccase (Lac)
Laccase is a cuproprotein belonging to a small group
of enzymes denominated blue oxidases [1]. Laccase (E.C.
1.10.3.2, p-benzenediol:oxygen oxidoreductase) is an
oxidoreductase able to catalyze the oxidation of various
aromatic compounds (particularly phenols) with the con-
comitant reduction of oxygen to water [1,3]. In general,
laccases exhibit four copper atoms, which play an important
role in the enzyme catalytic mechanisms. Copper atoms
are distributed in different binding sites and are classi-
fied in three types, according to specific spectroscopic and
functional characteristics [4–9].
The molecular genetics [10], genetic expression [11], ge-
netic transcription [12] and cloning [13] of laccases have
been exhaustively studied. The basic aspects of phenoloxi-
dases have been reviewed, as well [4,14–22].
In a typical laccase reaction, a phenolic substrate is
subjected to a one-electron oxidation giving rise to an
aryloxyradical. This active species can be converted to a
quinone in the second stage of the oxidation. The quinone
as well as the free radical product undergo non-enzymatic
coupling reactions leading to polymerization [23].
Laccases are characterized by low substrate specificity
and their catalytic competence varies widely depending
on the source. Simple diphenols such as hydroquinone
and catechols are good substrate for the majority of lac-
cases, but guaiacol and 2,6-dimethoxyphenol generally are
better substrates [2,5]. Laccase is also able to catalyze
the oxidation of other substituted polyphenols, aromatic
amines, benzenethiols and a series of other compounds,
but the enzyme, unlike tyrosinases, is unable to oxi-
dize tyrosine. N-Hydroxybenzotriazol, violuric acid and
N-hydroxyacetanilide are three N–OH compounds capable
of mediating a range of laccase-catalyzed biotransformation
[24].
Laccase is widely distributed in higher plants [25], in
fungi [1,4] and in some bacterial strains of Azospirillum
lipoferum [26] and Alteromonas sp. [27]. Very recently, it
has been reported that laccases are widespread in bacteria
[28].
Among fungal laccases, a great variability is observed
in the induction mechanism, degree of polymorphism, and
physico-chemical (molecular mass, isoelectric point, carbo-
hydrate content) and kinetic properties [29,30]. In some fun-
gal species, the addition of inducers to the culture medium
results in the biosynthesis of new extracellular forms.
The biological effect of polyphenols and derivatives were
tested in order to assess their environmental impact and
their use as byproducts for agriculture and industry was
evaluated after a detoxification treatment with laccase [31].
The determination of environmental pollutants using im-
munoassays is a continuously growing area and, within this
frame, laccase proved to be an excellent alternative to perox-
idase as a bioanalytical tool for monitoring polar pollutants
[32].
1.1.1. Laccase active site
Laccase contains four copper atoms that have been clas-
sified according to their electron paramagnetic resonance
(EPR) features: Type 1 or blue, Type 2 or normal and Type
3 or coupled binuclear copper site where the coppers are
antiferromagnetically coupled through a bridging ligand
(EPR undetectable) [33]. Spectroscopy combined with crys-
tallography has provided a detailed description of the active
site in laccase. Magnetic circular dichroism (MCD) and
X-rays absorption spectroscopy of laccase have shown that
the Type 2 and 3 centers combine to function as a trinuclear
copper cluster with respect to exogenous ligand interaction
including reaction with dioxygen [34]. The Type 2 center is
3-coordinate with two histidine ligands and water as ligands.
The Type 3 coppers are each 4-coordinate, having three
histidines ligands and bridging hydroxide. The structural
model of bridging between the Type 2 and 3 (Fig. 1A and B)
[33–36] has provided insight into the catalytic reduction of
oxygen to water. It has been elucidated that the Type 2 cop-
per is required for the reduction of oxygen since bridging
to this center is involved in the stabilization of the peroxide
intermediate.
Reduction of oxygen by laccase appears to occur in two
2e− steps. The first is rate-determining. In this Type 2/3
bridging mode for the first 2e− reduced, the peroxide-level
intermediate would facilitate the second 2e− reduction (from
the Type 2 and 1 centers) in that the peroxide is directly
coordinated to reduced Type 2 copper, and the reduced Type
1 is coupled to the Type 3 by the covalent Cys–His linkages
[37].
Previous studies [34] reported that 40% of the Type 1 and
3 that readily react with dioxygen correspond to native lac-
case (Fig. 2). It is clear that the Type 2 Cu is required for
dioxygen reactivity in laccase and that dioxygen reduction
occurs in the absence of the Type 1 Cu. This demonstrates
that the Type 2/3 trinuclear Cu site represents the active
site for the binding and multielectron reduction of dioxygen.
The Type 1 Cu is clearly not necessary for reactivity with
dioxygen, and in its absence, an intermediate is formed that
shares some properties with the oxygen intermediate previ-
ously described in native laccase.
Fig. 1. Two possible spectroscopically models for peroxide bridging at the
trinuclear cluster site: (A) bridging between Type 2 and one of the Type
3 copper in a ␮-1,1-hydroperoxo mode; (B) bridging all three copper in
a ␮3 (␩1 )3 (modified from [33]).
 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931 909

Fig. 2. Reactivity of laccase derivatives with oxygen (modified from [34]).

Very recently, the structural model of the Type 1 copper

protein active site was described as N2S (thiolate)-S-(thio-
ether) ligation in a Cu(II) complex [9]. Fig. 3 shows the
active site of laccase in a pictorial way (http://bioinf.leeds.
ac.uk) [38–40].
Recently, the structure of laccase from Trametes versi-
color was determined in its glycosylated, fully functional
form at 1.9 Å resolution (Fig. 4) (http://wwwbc.biol.ethz.ch).
T. versicolor laccase is a globular protein of about 500
amino acids and contains three cupredoxin-like ␤-sandwich
domains, similar to those found in ascorbate oxidase and in
ceruloplasmin.

1.1.2. Laccase catalytic cycle

Fig. 5 illustrates the catalytic cycle of laccase and the pro-
posed mechanisms for the reduction and reoxidation of the
copper sites. In this figure (center) starting from the “native
intermediate,” the substrate reduces the Type 1 site, which in
turn transfers the electron to the trinuclear cluster. Two pos-
Fig. 4. Ribbon diagram of T. versicolor laccase. The three domains are
sible mechanisms for the reduction of the trinuclear cluster shown in the figure together with the copper atoms, carbohydrate moieties
are shown: (A) the Type 1 and Type 2 sites together reduce and disulfite bridges are also depicted.
the Type 3 pair and (B) each copper in the trinuclear cluster
is sequentially reduced by electron transfer from Type 1 site,
in which case the Type 3 no longer acts as a two-electron ac-
ceptor. Slow (left) decay of the “native intermediate” leads
to the resting fully oxidized form. In this form, the Type 1

Fig. 3. Pictorial model of laccase (copper centers).

910 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931

Fig. 5. Catalytic cycle of laccase (modified from [41]).

site can still be reduced by substrate, but electron transfer to
the trinuclear site is too slow to be catalytically relevant [41].
1.2. Tyrosinase
Tyrosinase (Tyros) (E.C. 1.14.18.1, monophenol monoxy-
genase) is widely distributed throughout the phylogenetic
scale from bacteria to mammals and even present different
characteristics in different organs of the same organisms,
such as in roots and leaves of higher plants [15]. It is well
known that Tyros catalyzes two different oxygen-dependent
reactions that occur consequently: the o-hydroxylation of
monophenols to yield o-diphenols (cresolase activity) and
the subsequent oxidation of o-diphenols to o-quinones (cat-
echolase activity). The basic aspects of Tyros were previ-
ously discussed [42–45].
1.2.1. Tyrosinase active site
Chemical and spectroscopic studies of Tyros have shown
that the active site contains a coupled binuclear copper com-
plex. The Tyros exhibits a Type 3 copper center as shown
in Fig. 6.
1.2.2. Tyrosinase catalytic cycle
The oxygenated form (oxytyrosinase, Eoxy ) consists of
two tetragonal Cu(II) atoms, each coordinated by two strong
 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931 911

(Emet ), like oxy form, contains two tetragonal Cu(II) ions an-
tiferromagnetically coupled through an endogenous bridge,
although hydroxide exogenous ligands rather than peroxides
are bound to the copper site. Deoxytyrosinase (Edeoxy ) has a
bicuprous structure [Cu(I)–Cu(I)]. These three copper states
in the active site of tyrosinase suggest a structural model
for the reaction mechanism involved in the o-hydroxylation
of monophenols and oxidation of the resulting diphenols
(Fig. 7) [16]. Recently, mechanistic aspects related to its
crystal structure have been discussed [39,46].
Fig. 6. Pictorial model of tyrosinase (copper centers).

equatorial and one weaker axial NHis ligands. The exoge-
nous oxygen molecule is bound as peroxide and bridges
the two Cu centers. The cupric model complexes, which
are used to explain the electronic structures, are end-on
(cis-␮-1,2 geometry) or side-on (␮-␩2 : ␩2 ). Mettyrosinase
2. Enzyme immobilization
Many methods are available for enzyme immobilization.
Since the methods used the immobilization procedures
greatly influences the properties of the resulting biocatalyst,
the selection of an immobilization strategy determines the

Fig. 7. Catalytic cycle for the oxidation of monophenol and diphenol substrate to o-quinones by tyrosine in the presence of oxygen (modified from [16]).
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process specifications for the catalyst. They include several
parameters such as overall catalytic activity, effectiveness
of catalyst utilization, deactivation and regeneration ki-
netics, and cost. Also, toxicity of immobilization reagents
should be considered in connection with the immobiliza-
tion process, waste disposal and final application of the
immobilized enzyme catalyst [47–55].
Several techniques may be applied to immobilize enzymes
on solid supports. They are mainly based on chemical and
physical mechanisms [48]. In the following, the immobiliza-
tion processes mostly used with laccase and tyrosinase will
be briefly discussed. Their basic aspects are summarized in
Tables 1–4 [53–164].
Chemical immobilization methods mainly include: (i)
enzyme attachment to the matrix by covalent bonds, (ii)
cross-linking between enzyme and matrix, and (iii) enzyme
cross-linking by multifunctional reagents. Physical meth-
ods involve the entrapment of enzyme molecules within a
porous hollow fiber, in spun fibers or enzyme entrapment
within an insoluble gel matrix and/or enzyme entrapment
within a reverse micelle.
Enzyme immobilization by physical entrapment has the
benefit of a wide applicability and may provide relatively
small perturbation of the enzyme native structure and func-
tion. The most widely used system for enzyme entrapment
in a polymer lattice is the immobilization within a poly-
acrylamide gel, obtained by polymerization/cross-linking of
acrylamide in the presence of the enzyme.
Enzymes may be adsorbed on a variety of carriers, offer-
ing in some cases the practical convenience of simple regen-
eration by removal of deactivated enzyme and reloading with
fresh, active catalyst [49]. Another adsorption method for ty-
rosinase with pre-activation of the polyphyllosilicate (mont-
morillonite) was published [54]. An interesting method by
affinity coupling with Affi-Gel-10 and Affi-Gel-15 was also
reported.
The polystyrene-based matrices activated with nitrous
acid, phosgene or thiophosgene to give the correspond-
ing diazonium, isocyanate and isothiocyanate derivatives,
respectively, are commonly used. Polystyrene is commer-
cially available and its conversion to the active derivative
is easily accomplished. A commonly used method includes
a support pre-activation with aminopropylsilane and a sub-
sequent reaction with p-nitrobenzoyl chloride to form the
diazonium salt is also used [47].
The most commonly employed water-insoluble supports
currently used for enzyme immobilization are cyanogen
bromide-activated sepharose and sephadex. The immobiliza-
tion method is simply and reliable, and the attachment of the
enzyme to the matrix is performed under mild conditions.
The method involves the activation of the polysaccharides
with cyanogen bromide to give the reactive imidocarbonate,
which subsequently reacts with the protein [51].
The chemical activated method for sepharose CL-6B with
an epoxide is quite efficient to obtain an aldehyde activating
group in the support able to react with the enzyme [53].
Protocols for covalent enzyme immobilization often begin
with a surface modification or an activation step. Silaniza-
tion, i.e. the coating of the surface with organic functional
groups using an organofunctional silane reagent, is a widely
used strategy for initial surface modification of inorganic
supports. Such coating or native surface amino groups can be
derivatized to arylamine group using p-nitrobenzoyl chloride
or to aldehyde groups using glutaraldehyde (GLUTAL) [52].
Carbodiimide activation is used when a carboxylic group
in the support is expected to react with an amino group from
the enzyme. This activation is possible by epoxide forma-
tion in the side chain [55] or on the support surface, as for
example in the case of activated sepharose and cellulose. Be-
sides the activation of these carbohydrates, chitosan is also
used for tyrosinase immobilization by trapping the enzyme
between two chitosan gel films [50].
Both chemical and physical methods offer advantages
and disadvantages that depend on several factors. In gen-
eral, chemical immobilization methods tend to reduce the
activity of the enzyme, since the covalent bonds, formed as
a result of immobilization, may perturb the enzyme native
structure. By contrast, such covalent linkages provide strong
stable enzyme attachment and may, in some cases, reduce
enzyme deactivation rates and usefully alter enzyme speci-
ficity. However, entrapment and adsorption immobilization
methods typically perturb the enzyme much less and conse-
quently offer retention of the enzyme properties resembling
those in solution [47]. A proper choice between chemical
and physical methods depends on several factors. Usually, a
long-time applicable immobilized enzyme with a lower ini-
tial activity is preferable to that with a high level of initial
activity but with a short-time activity retention.
3. Phenoloxidases immobilized on different supports
3.1. Laccases (Lac)
Table 1 reports microbial and plant laccases immobilized
on different supports. The type of support, the method of
immobilization, and the substrate(s) used in the catalytic
process are specified. Some comments on the main features
of each example are also included.
As specified in Section 1, the examples listed in alpha-
betical order in Table 1 will be presented in the following
in a chronological order.
3.1.1. Neurospora crassa
The immobilization of Lac from N. crassa on CNBr-acti-
vated sepharose 4B by covalent attachment resulted in a re-
duction (25%) of activity yield and an increase of Km value,
probably caused by a steric hindrance to substrate diffu-
sion. By contrast, adsorption on concanavalin A-sepharose
did not affect both the kinetic parameters, thus suggesting a
non-involvement of the enzyme carbohydrate moiety in the
catalytic center [56].
 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
3.1.2. Rhizoctonia praticola
A good immobilization yield (89%) and a high trans-
forming ability (100% of methoxy-substituted phenols
transformed), similar to that of the native enzyme, were
obtained when R. praticola Lac was covalently coupled to
activated Celite® . Similarities in the relative activities of
the free and immobilized enzyme demonstrated that any
deactivating effect occurred upon immobilization [61].
3.1.3. Polyporus versicolor, T. versicolor, Coriolus
versicolor
In course of time, this fungal strain, producer of laccase,
has been indicated as Polyporus, Trametes, or C. versicolor.
According to the principle adopted in this review, we have
collected the papers by the name used to define the fungus
and discussed them separately.
3.1.3.1. Polyporus versicolor. P. versicolor Lac was im-
mobilized by entrapment and adsorption on several car-
riers (gelatin, polyurethane, sepharose 4B-Epi-IDA-Cu2 ,
i.e. a metal-chelate affinity matrix) and used for differ-
ent purposes [49,63,119,127,138,140]. The immobilization
on sepharose 4B-Epi-IDA-Cu2 gave a very high adsorp-
tion yield, showed excellent stability and resulted a fast,
environmental-friendly, inexpensive technique because of
the possible carrier reuse. The immobilized enzyme was
used for apple juice clarification (reduction of phenols,
flavonols and chromophoric compounds by 48.6, 47 and
43%, respectively) and for catechin removal (75%) in a
column packed with this carrier [119].
3.1.3.2. T. versicolor. The immobilization of T. versi-
color Lac was extensively studied for several years [67,73,
76,77,85,89,94,101,105,111,112,124,125,128,133,135,139,
141,149,163,164].
Several examples of the enzyme immobilized on different
types of glass beads activated with 3-aminopropyltriethoxy-
silane (APTES) and GLUTAL are present in the litera-
ture [67,85,89,105,139]. Usually, this support showed a very
good immobilizing capability both in terms of immobiliza-
tion yield (100%) and retained activity (90%). Often the val-
ues of kinetic parameters were not influenced, as well [67].
A specific activity higher than that of the free enzyme was
also observed, thus suggesting that some sort of purification
or other phenomena took place during the immobilization
process [105].
Lac immobilized on APTES–GLUTAL-activated glass
supports usually displayed good stability to long-term ap-
plications. When used in a batch reactor, immobilized Lac
fully retained its activity after six consecutive oxidative cy-
cles, if the reaction products were readily removed by wash-
ing steps. It was less sensitive to higher temperature than
the native counterpart, as well [67]. A similar response was
obtained in flow-injection calorimetry studies, performed
with the immobilized enzyme packed into a small polymer
column and mounted on a temperature probe housed in a
919
calorimeter [139]. Virtually unchanged performance during
4 months of operation at room temperature and exothermic
responses with several substrates, including l-ascorbate,
d-isoascorbate, 3,4-dihydroxycinammic acid and catechol,
were measured [139]. By contrast, immobilized Lac, op-
erating in a reactor, was subjected to product inactivation
and lost about 22% of activity for over 160 injections of
methylcatechol [89].
Laccase from T. versicolor was immobilized on a silt
loam soil, kaolinite and two different montmorillonites upon
pre-treatment with APTES/GLUTAL [73,105]. High per-
centages of Lac activity were measured after binding on
clays and soil (78% for Lac-soil, 94%, Lac-montmorillonite,
and 97% for Lac-kaolinite) [73]. On montmorillonite a pu-
rification process, similar to that observed with activated
glass beads, gave rise to a specific activity higher than that
of the free enzyme [105]. The enzyme immobilized on these
supports showed a very ability to remove 2,4-dichlorophenol
(2,4-DCP) from model solutions (95% for kaolinite- and
soil-immobilized Lac and 69% for montmorillonite-Lac).
The activity was quite fully retained after 4 months of stor-
age at 4 ◦ C and when tested in repeated 2 h incubation cycles
[105]. Experiments performed in the presence of soils with
different amounts of organic matter indicated that the perfor-
mance of the immobilized enzyme was strongly affected by
inorganic and organic soil constituents. These results suggest
a potential use of immobilized laccase for the clean-up of
polluted soils, but its performance can be strongly affected
by soil nature and compositions [105].
Very interesting findings were obtained with Lac immo-
bilized by entrapment in sepharose CL-6B. The gel–enzyme
association was stable in water pre-saturated solvents and
showed good stability in organic solvents as well as a high
tolerance to elevated temperatures [76]. Syringaldazine and
2,6-dimethoxyphenol (in ethyl acetate pre-saturated with
water) as well as water-insoluble organosolvent lignin,
dissolved in dioxane/water, were readily converted by
sepharose CL 6B-immobilized Lac [76]. Less than a 5%
decrease of the initial activity occurred after 36 h incubation
of Lac/complex in hydrophobic n-hexane at 30 ◦ C, while
under the same incubation conditions but in citrate buffer
more than 50% of initial activity was lost [76].
Good results in terms of storage stability (90% activity
retained versus 50% of the free enzyme after 4 months at
4 ◦ C) and substrate transformation (95–98% of 1-naphhtol,
2,6-DMP, 4-chloroaniline and vanillic acid in around 1 h)
were obtained with Lac immobilized within reverse micelles
prepared by adding small amounts of water to dioctylsulfo-
succinate in isooctane [111,128].
Within the frame of an interlaboratory project (vitro-
ceramic production, enzyme immobilization and effluent
remediation), Lac production was optimized and its im-
mobilization on different supports (i.e. ionic exchange
resin IRA-400, silica MERCK II and a vitroceramic ma-
terial) was investigated [141]. Immobilization on both
IRA-400 and silica MERCK II gave rise to a 100% yield.
920 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
Nevertheless, a high adsorption of chromophoric substances
on the surface of these matrices was evident when they
were employed for effluent treatment. In contrast, although
the immobilization yield of Lac onto vitroceramic support
was lower (around 50%), a very low adsorption of chro-
mophores on this support was observed. Consequently,
vitroceramic appeared as an efficient support to prepare
immobilized biocatalysts with a reasonable retention of
enzymatic activity, for the treatment of textile and paper in-
dustry effluents [141]. Recently, two different vitroceramic
materials were tested with a kraft E1 effluent. The best
immobilization results were obtained by combining the use
of low porosity vitroceramic with carbodiimide coupling
[124] or carbodiimide/GLUTAL activation [112].
Laccase from T. versicolor was often immobilized for a
possible use as biosensor [133,135,164]. Biosensors were
prepared on different pre-treated carbon fibres (at a poten-
tial of +0.8 V for 180 s) and utilizing different methods:
covalent binding using polyethylene bis-glycidyl ether
(PEG) as a linker of a redox polymer and Lac onto the sur-
face of glassy carbon electrode (GCE) [133,135]; physical
adsorption, carbodiimide coupling, GLUTAL activation,
combined use of carbodiimide and GLUTAL [164]. Usu-
ally, the response of the sensors (i.e. sensitivity, kinetic
parameters and amounts of substrate detected) was influ-
enced by the immobilizing methods, the enzyme loading,
electrolyte pH and ionic strength [133,135]. The carbodi-
imide/GLUTAL procedure gave the best results and the
percentage of GLUTAL was the most important influencing
parameter, since it heavily affected the properties of the
immobilized enzyme. Indeed, high degree of enzyme retic-
ulation promoted by GLUTAL probably occurred with a
resulting limited substrate access to the enzyme active site
(apparent Km value quite lower than that of the free form)
[164].
3.1.3.3. Coriolus versicolor. C. versicolor Lac and ty-
rosine Lac co-polymers entrapped in calcium alginate gel
beads [80] or Lac covalently immobilized on activated car-
bon [92] were used in the treatment of effluents from the
pulp and paper industry. The four derivatization methods
(silanization of carbon, cross-linking after binding to carbon
amino group, activation of amino groups with diimide, and
activation of carboxylic groups with diimide) used to im-
mobilize the enzyme on the activated carbon did not affect
significantly the total amount of bound protein [92]. Lac
immobilized on diimide-activated carbon showed a consid-
erably higher activity and was used in large batches for both
batch and reactor experiments. In a fluidized bed, operated
in a re-circulation mode, the average decolorization rate
was 633 colorimetric units h−1 (60 colorimetric units (lac-
case unit)−1 h−1 ) and the activity decreased from 10.5 to
8.4 ␮mol O2 min−1 g−1 carbon. Therefore, this immobiliza-
tion procedure would be suitable for treatment of phenolic
waste, but it may be inappropriate for color removal from
pulping and bleaching effluents [92,103].
3.1.4. Rhus vernicifera
In the last two decades, extensive studies on the im-
mobilization of R. vernicifera Lac were carried out
[64,66,68–70,74,78,79,121,148]. One of the first example
is the immobilization of the enzyme by entrapment in poly-
acrylamide gel [64]. Immobilized Lac was used to catalyze
the oxidative polymerization of urushiol from outmoded
Chinese lacquer. The floatability, drying properties, color,
luster value, shock strength, flexibility, and adhesion of this
laccase-treated lacquer were better than those of the native
lacquer film [64]. Several parameters and properties (i.e.
immobilization conditions, reusability, thermal stability,
Km , optimum pH and temperature) were also studied and
compared with those of the native enzyme [66,68,69].
Novel types of p-benzoquinone-activated supports
(agarose, polyvinyl alcohol, chitosan) were developed and
used for laccase immobilization (coupling of 10–95%) [74].
The high residual activity (150%), measured for immobi-
lized Lac, was ascribed to the presence of hydroquinone
groups on the support. It, as a substrate of Lac, could have
generated a simultaneous affinity retention of the enzyme
[74]. In addition, the immobilized enzyme was much less
susceptible to the inhibitory action of chloride and azide ions
than the free Lac (67 and 32% activity recovery upon dialy-
sis, respectively) and displayed a remarkably high stability
at 4 ◦ C (95% residual activity after 14 months storage) [78].
Lac was also immobilized on several transition metal ox-
ides preparations (TiCl4 , ZrCl4 , FeCl3 , CuCl2 or ZnCl2 )
[79]. The optimal pH (7.5–8.0) and temperature (5–10 ◦ C)
conditions for Lac immobilization as well as the properties
of free and immobilized Lac were determined [79,86]. Using
ZrCl2 -treated porous silica as a carrier, the activity recov-
ery and stability of immobilized Lac was higher than those
previously reported in the literature. The retained activity of
this immobilized preparation after 20 consecutive oxidative
cycles was around 75% [86]. When Lac was immobilized
on an urushiol-based resin in the presence of adsorbed metal
ions, the highest catalytic activity was obtained with alu-
minum (Al) [121]. The urushiol-salicylic acid (USR) grafted
resin proved to be a better support for Lac immobilization
than urushiol itself and, also in this case, the presence of ad-
sorbed aluminum ions gave rise to the highest activity. The
apparent Km value of USR-Al-Lac for phenol was remark-
ably lower than that of the native enzyme (4.9×10−3 versus
1.2 × 10−2 M, respectively). This was attributed to adsorp-
tion phenomena between USR and the substrate [121].
3.1.5. Geotrichum candidum, Fomes fomentarius
Very few examples on the immobilization of laccase from
G. candidum or F. fomentarius are available in the literature
[71,85]. Enzyme from G. candidum was immobilized on soil
by adsorption and used in laboratory-scale experiments for
phenol (4-methylphenol and 2,4-DCP) transformation [71].
The enzyme from F. fomentarius was coupled to 3-APTES-
controlled porosity glass (APTES-CPG) using GLUTAL.
As compared to its free counterpart, it showed an unusual
 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
higher substrate affinity but a lower catalytic velocity (lower
values of both the kinetic parameters Km and Vmax ) [85].
3.1.6. Pyricularia oryzae
Several studied were performed on the use of this im-
mobilized fungal Lac in phenol removal processes for
must and wine stabilization and for detoxification purposes
[91,93,120,158,159].
In comparative studies, Lac was immobilized on different
supports by different methods and used to decrease phenol
content in model and real phenolic solutions [91,120]. The
enzyme was linked: by covalent binding to four carriers
(e.g. CH-sepharose 4B, CNBr-sepharose 4B, VA-hydroxy
biosynth, a vinyl acetate-based co-polymer cross-linked
with divinyl-ethylene-urea and VA-epoxy) [91,120], by ad-
sorption, followed by treatment with GLUTAL, on both
silica gel and florisil, by entrapment and by radiation poly-
merization on colloidal silica [120]. Lac immobilization
on CNBr-sepharose 4B produced the most stable system,
followed by the silica immobilization [91]. Generally, when
used in catechin model solutions and in both musts and
wine, a significant decrease of phenol content (about 60, 34
and 13%, respectively, after treatment with Lac immobilized
on silica gel) was measured [91,120]. The results suggest
that the use of immobilized Lac might represent a promising
procedure to remove phenolics in wine-making technology.
An interesting application for wine stabilization was pre-
sented by using molecular sieves as laccase-immobilizing
supports [93]. Molecular sieves were ground, sieved and
degreased with ether, and then Lac was immobilized by
adsorption on the support. The enzyme-support complex
was stabilized with GLUTAL (90% yield). The same pro-
cedure was performed with alumina (80% yield) and silica
(100% yield). A catechin model solution (100 mg l−1 ),
musts (30 mg l−1 catechin) and wine (127 mg l−1 catechin)
were treated by soluble and silica-immobilized Lac. The
treatment with soluble laccase led to 68, 12 and 127 mg l−1
of residual catechin, respectively, whereas the treatment
with the immobilized form was more effective giving 40,
20 and 110 mg l−1 residual catechin, respectively [93].
Phenols were removed from aqueous solution by a two-
step treatment with co-immobilized P. oryzae Lac and mush-
room Tyros and adsorption on Polyclar (polyvinylpolypyrroli-
done). In particular, Lac and Tyros were co-immobilized on
Mikroperl in a fixed-bed tubular bioreactor by a rapid and
simple method [158]. The support immobilized 95% of the
total Lac units and 35% of the total Tyros units. A 42–90%
removal of different phenolic substances by a single passage
through the bioreactor was observed. Polyclar was used in
a second step for additional removal of phenolic substances
from the mixtures. The operational stability of the immo-
bilized system was 10–90 h depending on the substrate.
Furthermore, the biocatalyst was capable of a continuous
transformation of different phenols in mixtures [158]. Sim-
ilar co-immobilization with Tyros was previously used with
Lac from C. hirsutus. Co-immobilized enzymes were ad-
921
sorbed as a layer onto a graphite electrode and used as a
sensor in a single line flow-injection system. The bi-enzyme
sensor efficiently allowed the analytical detection of a large
group of phenolic compounds [113].
An immobilization yield of around 40% was achieved
by immobilization of Lac onto a spiral-wound asymmet-
ric polyethersulphone membrane in a reactor [159]. When
applied to the biodegradation of a model solution con-
taining many phenolic substrates (7–69% degradation), a
shift in pH and temperature optima was observed in the
membrane-immobilized laccase with respect to the soluble
counterpart (6.6 versus 6.3 ◦ C and 42 versus 35 ◦ C, respec-
tively). Immobilized Lac retained 50% of its initial activity
after 150 h of repeated runs against only 18% shown by its
free counterpart after 39 h [159].
3.1.7. Coriolus hirsutus
The main interesting example reported in the litera-
ture for this fungal Lac is its immobilization in reversed
micelle-like complexes, obtained by adding a Lac water
solution to CEPEI in benzene/n-butanol [104]. The immo-
bilized preparation lost 40% of its initial activity after 20
days storage at 4 ◦ C, whereas an 80% activity loss was ob-
served, within the same incubation period at 20 ◦ C [104].
The laccase from this fungus and from Cerrena maxima
were also applied on enzyme-linked immunoassay [103].
3.1.8. Phlebia radiata
P. radiata Lac was immobilized on APTES-CPG using
GLUTAL, with good yield in both protein (98%) and activity
(96%) binding [108]. A decrease of the enzyme affinity for
guaiacol resulted (Km values of 1.76 and 4.78 mM for the
free and immobilized forms, respectively). A remarkable
increase of both storage stability and resistance to inhibitors,
such as Cu-chelators and quinone, was evident. After 180
days storage at 4 ◦ C the immobilized enzyme lost only 4%
of its initial activity against more than 93% loss shown by
the free form [108].
3.1.9. Pleurotus eryngii
Lac was immobilized by covalent attachment to chemi-
cally-activated gels [53]. Aldehyde, amino and amino-GLU-
TAL sepharose CL-6B derivatives, prepared by traditional
methodologies, were used and gave rise to 95, 70 and 55%
coupling yields, respectively. Covalent immobilization onto
aldehyde gels markedly increased the Lac stability in 60%
N,N-dimethylformamide. The immobilized enzyme was,
however, low efficient in the treatment of a paper industry
effluent. In contrast, P. eryngii Lac immobilized by entrap-
ment within calcium alginate gels was very effective on this
effluent [53].
3.1.10. Cerrena unicolor
The catalytic capability of an extracellular laccase, iso-
lated and purified from the non-induced culture of C. uni-
color, and immobilized on GLUTAL-activated silanized
922 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
porous glass beads, was evaluated in water-miscible organic
solvents [125,130]. Immobilized Lac (94% yield of immo-
bilized protein) exhibited 100% activity. As compared with
the free enzyme, immobilized Lac showed an optimum pH
shift from 5.5 to 5.7. After about 7 months at 4 ◦ C, a 95%
retention of activity (syringaldazine as the substrate) was
observed for immobilized Lac (40% for the free form) [130].
Further studies were performed on this enzyme immobilized
on CPG by both the APTES/GLUTAL and carbonyldiimi-
dazole procedure [143]. The covering of CPG surface, prior
to enzyme attachment, with two polysaccharide layers, one
formed of cross-linked DEAE-dextran, and the other pre-
pared from cross-linked dextran and carbonyldiimidazole,
strongly increased the activity of the immobilized enzyme
in organic solvents [143].
3.1.11. Lentinus edodes (Lentinula edodes)
Two interesting practical examples of immobilized L.
edodes laccase are reported in the literature [145,154]. In
both cases the capability of the immobilized enzyme was
assessed by treatments of olive mill waste waters (OMW),
a characteristic by-product of olive oil production, very
rich of toxic phenolic compounds and largely produced in
Mediterranean countries. The enzyme was immobilized on
chitosan by adsorption and subsequent cross-linking with
GLUTAL (60% binding yield, 35% immobilization yield)
[145]. The immobilized form displayed a lower specific ac-
tivity as well as a reduced substrate (2,6-DMP) affinity than
the free enzyme (Vmax 85.5 versus 190 units mg−1 protein
and Km 0.25 versus 0.07 mM, respectively). Conversely, im-
mobilized Lac exhibited an appreciable catalytic capability
(520 units g−1 support) along with markedly improved sta-
bility properties to various deactivating parameters, such as
temperature, pH and storage time. When OMW were treated
with immobilized Lac under batch conditions, a partial de-
colorization as well as a significant abatement of polyphenol
and o-diphenol content were obtained. The dephenolization
process was also combined with a decreased toxicity of
the effluent as tested using a highly sensitive strain of the
nitrogen-fixing bacterium Rhizobium spp. [145].
The immobilization of L. edodes Lac was further op-
timized by using the epoxy-activated polyacrylic support
Eupergit® C. A catalytic capability of 340 units mg−1 sup-
port was obtained. Immobilization on this support besides
increasing pH, thermal stability and storage stability also
enhanced enzyme resistance to proteolytic attack. A 20%
decrease in the OMW dephenolization efficiency, performed
with Eupergit® C-immobilized laccase in a fluidized bed
reactor operated in a recirculation mode, was evident after
eight consecutive treatment cycles. Nevertheless, efficiency
was restored by a washing procedure with a high ionic
strength buffered solution followed by a four-bed volumes
wash with buffer alone [154]. Similar results in both activ-
ity retention and storage stability were obtained also with
Lac from Pleurotus ostreatus, covalently immobilized on
the same support [156].
The simplicity of the immobilization process, the stability
and dephenolization efficiency of the immobilized biocata-
lyst as well its hydrodynamic properties suggested the suit-
ability of Eupergit® C for wastewater treatment applications
[154,156].
3.1.12. Coriolopsis gallica (Trametes hispida), Trametes
hirsuta
A high storage and thermal stability, a good tolerance
towards some inhibitory compounds as well as the re-
duction of several dyes toxicity were measured when Lac
from C. gallica and T. hirsuta were immobilized by cova-
lent binding to Affi-Gel-15, a N-hydroxysuccinimide ester
of derivatized cross-linked agarose gel bead support, and
alumina, respectively [150,157]. The reduction by up to
80% of triarylmethane, indigoid, azo, and anthraquinonic
derivatives toxicity, as assessed by monitoring the oxygen
consumption in Pseudomonas putida liquid cultures, proved
the treatment of textile effluents with immobilized laccase
to be suitable for dyeing purposes [157].
3.1.13. Non-specified
Two examples of biosensors prepared by immobilization
of a Lac from a non-specified source are available in the lit-
erature [65,160]. The covalent immobilization on a carbon
black previously contacted with bovine serum albumin in
the presence of N-ethyl-5-phenyl-isoxazolium-3-sulphonate
(EPIS) significantly increased the thermal stability as well
as the catalytic activity of immobilized Lac [65]. A Lac im-
munoconjugated (SynectiQ Corp.) was physically adsorbed
on a pre-treated graphite ink that was deposited on a plastic
strip. Therefore, a Lac molecular layer attached to the elec-
trode surface formed an oxygen transducer. The use of a such
biosensor for immunofiltration and immunochromatography
was suggested [160].
Some further examples of immobilized non-specified
laccase, including an enzyme produced by heterologous ex-
pression of a T. versicolor Lac gene in Aspergillus sp. [155],
regard adsorption on sepharose CL-4B [72] and various
supports (glass, glass powder, silica gel, and Nylon-66
membrane) [155], and covalent binding on differently ac-
tivated polymers [160]. Nylon-66 membrane/Lac retained
a very high activity in organic solvents (diethyl ether and
methyl acetate) while exposure to methylene chloride re-
sulted in significant activity decrease regardless of the
support material [155]. Lac immobilized on polyvinyl al-
cohol, polyacrylates, and other polymers concurred with
other enzymes to a fabric softening detergent composition,
that was useful for cleaning substrates, especially fabrics
[131].
3.2. Tyrosinase (Tyros)
3.2.1. Mushroom (non-specified)
Almost all papers dealing with immobilized tyrosinase
refer to an unspecified mushroom as the enzyme source, as
 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
shown in Table 2 [57,81,88,93,95,98,100,107,109,110,113,
116–118,122,128,153,158,162].
In an old paper, Tyros entrapment within polyacrylamide
gels resulted in increased storage stability [57]. The immo-
bilized biocatalyst, retained 43 and 70% of its initial activity
when stored at room temperature or at 6 ◦ C over a 13-day
period, respectively. The results of the study indicated its
possible incorporation into a detection system for phenols
and related compounds [57].
Tyros, bound to controlled porosity glass via diazo link-
age (amino[phenyl] trimethoxysilane) and HNO2 , was ex-
ploited for continuous-flow determination of phenols [81].
The immobilized Tyros preparation retained 83% of its
initial activity throughout a 3-month period. When phenol
was used as substrate (10−4 M) the enzyme preparation
experienced a constant decrease of activity with repeated
uses, loosing 80% of its initial activity after 12 consecu-
tive oxidative cycles. In contrast, this effect was not ob-
served when using model solution with lower concentration
(10−5 M phenol). It was suggested that this immobilized
Tyros can be used in an open tubular reactor as a part
of the injector valve’s sample loop in an unsegmented
continuous-flow analyzer for the selective determination
of phenol in water samples [81]. When Tyros was immo-
bilized on APTES/GLUTAL-activated magnetite particles,
immobilization and activity yields were 80 and 70–80%,
respectively [88]. Immobilized Tyros exhibited higher stor-
age stability than the free enzyme (95 versus 30% activity
retained after 15 days incubation at 5 ◦ C, respectively) and
it oxidized phenols more rapidly than its free counterpart,
probably due to its lower susceptibility to product inactiva-
tion [88]. The procedure of phenols removal was further im-
proved by combining the enzymatic treatment with the use
of the coagulant hexamethylenediamine-epichlorohydrin
[110]. In the treatment with immobilized Tyros, more
reaction products were removed with less coagulant, as
compared with the soluble enzyme [110].
Tyros was immobilized by adsorption on several sup-
ports and used for wine stabilization [93]. Adsorption and
cross-linking with GLUTAL on DEAE-cellulose, on Affi-
Gel-10, activated CH-sepharose 4B and CNBr-sepharose,
and on GLUTAL-activated silica gel was carried out. Activ-
ity yields widely ranged from 3.5% (adsorption/cross-linking
on DEAE-cellulose) to 38% (GLUTAL-activated silica).
The silica gel/GLUTAL system was selected for further ex-
periments on must and wine stabilization. This immobilized
derivative was more effective than its soluble counterpart to
perform the removal of catechin (100 mg l−1 ) from model
solutions. Nevertheless, when experiments were conducted
on musts and wine, soluble and immobilized Tyros led
to comparable results. The immobilized Tyros showed an
operational stability of two cycles, probably due to its
sensitivity to ethanol and pH of the medium [93].
When Tyros was immobilized on Diamin WK-20 by ad-
sorption and cross-linking with 1-ethyl-3(3-dimethyl-amino
propyl)carbodiimide, an activity yield of 16.3% was ob-
923
tained [95]. After 96-h storage at 25 ◦ C, immobilized Tyros
retained 50% of the initial activity compared with 20% of
soluble Tyros. Treatment with immobilized Tyros yielded a
100% phenol removal after 2 h incubation, and only a slight
decrease in the activity was observed even after 10 repeated
treatments [95].
Tyros was immobilized by entrapment within alginate gel
and used to produce theaflavin, as well [98]. The specific
activity of immobilized Tyros was lowered by 7% during
the immobilization process, as compared with the soluble
one. A 40% decrease of Km value was also measured. The
maximum yield of theaflavin was obtained within 20–30 min
incubation with immobilized Tyros in a reactor [98].
Crude and purified Tyros were immobilized by several
methods including physical entrapment, cross-linking with
BSA-GLUTAL, covalent immobilization on both ampho-
teric PALL and carboxylic PALL membranes, and covalent
immobilization on Nylon net [100]. The resulting immobi-
lized derivatives were then tested for their use as biosensors.
Crude mushroom extracts coupled with a Clark electrode
gave a better signal stability but the biosensor life-time was
not satisfactory. Better results were obtained with biosen-
sor based on pure Tyros immobilized on a Nylon net and
the system when employed on green water from crushers
showed good reproducibility and sensitivity [100].
When Tyros was adsorbed either onto Ca-montmorillonite
(Ca-Mte) or on different hydroxyaluminum–montmorillonite
(Al(OH)x –Mte) complexes, it was found that more Tyros
molecules were adsorbed onto the former than the latter.
Tyros immobilization efficiency on Mte increased with
increasing levels of Al(OH)x coating [107,122].
In another study, factors affecting Tyros inactivation dur-
ing its immobilization on glass beads and Celite® were
investigated [109]. The degree of inactivation was depen-
dent on the enzyme loading and the carrier’s surface area.
Addition of a sacrificial protein during the immobilized
procedure offered a protective effect with increased residual
activity at comparable enzyme loading [109].
Results obtained with Tyros immobilized between two
chitosan films suggested that 45% of the activated bonding
sites were available for the adsorption. To perform a selec-
tive phenols removal from a mixture, Tyros was used to con-
vert phenols into o-quinones, which were then adsorbed onto
the amino-containing polymer chitosan gel film [116]. The
performance of enzyme-containing chitosan gels depended
on the ratio of tyrosinase-to-chitosan. In fact, within a well
defined enzyme concentration, a direct proportionality be-
tween the aforementioned ratio and system efficiency was
evident [116].
When immobilized on Nylon-66 through covalent
cross-linking, the enzyme produced 2.2 times more benzo-
quinone than soluble Tyros and was inactivated at one-tenth
the rate of soluble Tyros. Oxygen adversely affect the sta-
bility of immobilized Tyros. The inactivation rate was 3.6
times faster at 100 kPa oxygen than under 21 kPa oxy-
gen. Preliminary evidence also suggested that there is a
924 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
difference in the affinity of the inactivating species for
immobilized Tyros compared with the soluble enzyme.
The covalent cross-linking of Tyros onto Nylon-66 seemed
favorable to stabilize its catecholase activity [117].
The influence of gelatin as an additive in the formation
of Tyros-reversed micelles was investigated [118]. The en-
zyme was immobilized in a reversed micellar system con-
sisting of dioctylsulphosuccinate in isooctane and gelatine.
The amount of gelatin (2.5%) influenced both the immo-
bilization efficiency and Tyros activity. The reusability and
phenol removal efficiency of this immobilized system was
studied in a column reactor using catechol model solutions.
About 50% of catechol disappeared from the eluted solution
in each cycle and no adsorption of catechol in the column
was observed [128].
A 100% yield was obtained when Tyros was immobilized
on a polymer produced by reaction of methyl methacry-
late and glycidyl methacrylate [118]. A biosensor exhibiting
good stability properties was prepared by coating a platinum
wire electrode with the Tyros-modified polymer [118]. A
sol–gel modified Tyros biosensor was prepared by immo-
bilization on a Al2 O3 sol–gel (Al:H2 O:HCl = 1:100:0.05)
(boemite sol [␥-AlOOH]) [153]. A GCE was used as base
electrode. To test the precision of the new enzyme biosen-
sor, several assays were performed on phenol samples. Re-
sults showed a good correlation between this method and
the standard one. The method was useful for application
in real samples with good precision and accuracy, thus be-
ing an example of a reliable enzymatic biosensor at very
low costs [153]. Very recently, a new Tyros biosensor has
been developed [162]. It is based on the co-immobilization
of Tyros and a mediator, Fe(CN)6 4− , on positively charged
Al2 O3 sol–gel membranes. The stability of the encapsulated
molecules was further enhanced by the electrostatic inter-
action between the molecules and matrix. Tyros immobi-
lized in the porous and hydrophilic sol–gel matrices was
very stable and retained its functional activity to a large
extent [162].
3.2.2. Agaricus bisporus
A. bisporus Tyros was immobilized on a redox polymer-
modified electrode which was prepared using PEG as a linker
to the surface of a GCE, and allowing the subsequent hy-
drogel to dry for at least 48 h. The electron donor mediators
for these systems were Os[bpy)2 Cl2 ](Osbpy) and OsMeIm
complexes. The immobilized sensor was utilized for the de-
tection of modulators/inhibitors of enzyme activity, such as
the respiratory poison azide [135].
3.3. Polyphenoloxidases (PPO)
Table 3 reports all examples that refer to papers dealing
with the preparation and characterization of immobilized
PPO. In most of the papers, the exact nature of the enzyme,
whether it be laccase or tyrosinase, was not specified. Ac-
cording to the order used above, the papers are grouped
by the originating source and presented in a chronological
order.
3.3.1. Mushroom
PPO from mushroom was immobilized by precipitation
onto glass beads. It was shown that the enzymatic oxida-
tion of phenols performed in water resulted in negligible
yields of quinones, due to rapid enzyme inactivation, while
a quantitative conversion was achieved in chloroform. The
quinones thus produced were then non-enzymatically re-
duced to catechols [144].
PPO was immobilized on cellulose and sepharose
CL-6B using p-nitrophenylchloroformate (p-NPCF) and
p-toluensulphonyl chloride (p-TSC) [82]. The immobiliza-
tion yields were in the following order: p-NPCF-activated
sepharose > p-TSC-activated sepharose > p-TSC-activated
cellulose > p-NPCF-activated cellulose. Various properties
of the extracted enzyme and its immobilized forms, such
as optimum pH, Km , thermal stability and storage stabil-
ity, were studied and compared. Operating properties of
immobilized enzyme were studied using different reactor
configurations. Among them, a packed-bed column reactor
showed the highest conversion rates [82].
HCl-treated glass beads, zeolite and sepiolite were used
as supports for PPO adsorption and some parameters of im-
mobilized PPO-catalyzed reactions in organic solvents were
studied [84]. No inactivation phenomena occurred within the
first minutes of the reaction as it was evident for aqueous me-
dia, probably due to the transfer of the quinone product to the
organic phase. Glass beads were shown to be the best immo-
bilizing support [84]. Immobilized PPO showed an optimum
temperature at 30 ◦ C and an optimum pH at 7.0. The Km and
Vmax for o-cresol were 1.22 mM and 45 nmol min−1 mg−1
protein, respectively, whereas, for phenol Km and Vmax were
5.42 nM and 207 nmol min−1 mg−1 protein, respectively.
Further studies demonstrated that PPO underwent confor-
mational changes when exposed to organic solvents [90,96].
Thermal inactivation in toluene was decreased by the ad-
dition of a polyol [90] and the amount of retained activity
depended on the water content of the system [96].
In a successive report, PPO was immobilized in polysul-
fone capillary membranes and used in a capillary bioreactor
to transform several phenols present in synthetic and in-
dustrial effluents [142]. The immobilized enzyme-reactor
was effective in converting phenols and associated deriva-
tives, but it produced o-quinones and low molecular weight
polymers in the treated effluents. To improve the quinone
removal effectiveness, PPO was immobilized by cross-flow
circulation on the shell (outer) side of the membrane pre-
viously coated with chitosan [151]. The chitosan-coated
membrane gave rise to high phenols removal associated
with effluent decolorization. In addition, the presence of
chitosan considerably decreased the occurrence of product
inhibition which is characteristic of PPO [151].
PPO-containing plain carbon paste was prepared by mix-
ing PPO with carbon paste (60% graphite and 40% mineral
 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
oil, w/w) [152]. The electropolymerized PPO film electrode
was prepared using a platinum-disk electrode as a support.
The carbon paste entrapped PPO maintained 86% of its ini-
tial activity over a 6 h period at 80 ◦ C incubation [152].
Covalent immobilization of PPO onto epichlorohydrin-
activated carboxymethylcellulose (CMC) beads was perfor-
med leading to a 44% activity yield [55]. The temperature
optimum of the immobilized preparation was shifted to
50 ◦ C with respect to the free enzyme (40 ◦ C). Storage sta-
bility of immobilized PPO was considerably improved with
respect to the free enzyme.
3.3.2. A. bisporus
The purified PPO was immobilized by several proce-
dures including physical entrapment, cross-linking with
BSA-GLUTAL, covalent immobilization on PALL Biodyne
A and B membranes, and covalent immobilization on Nylon
net. Among the tested methods, covalent immobilization on
Nylon net provided the most stable (life-time 30 days) and
sensitive biosensor for catechol and phenol [106].
The PPO immobilization process on ␥-alumina involved a
disulfide rearrangement in the enzyme resulting in its poly-
merization in the support pores [134]. The immobilized PPO,
obtained with an activity yield of 34, retained 68% of its
initial activity after 3 months storage at 4 ◦ C and exhibited
quite similar Km values with free enzyme (2.8 × 10−4 and
4.3 × 10−4 M, respectively) suggesting that the active site
of PPO was not affected during the immobilization process
[134].
In other studies, PPO was immobilized either on Nylon
membrane or on a polyethersulfone capillary membrane
[136] resulting in 80 and 32% activity yield, respectively.
The intermediate product of PPO reaction with l-dopa,
4-methylcatechol, was isolated and identified when the reac-
tion was conducted with the former immobilized derivative.
In contrast, only the o-quinone as the final reaction product
was detected with the latter one [136]. The use of chitosan
to treat the output stream from the capillary membrane
bioreactor facilitated the removal of the colored quinone
products from the permeate as well as the recycling of the
substrate solution [142,151]. A similar approach was used
with PPO from N. crassa [137].
A bioprobe based on the concomitant use of PPO im-
mobilized on circular Nylon membranes and the compound
3-methyl-2-benzothiazolinone hydrazone (MBTH) was pro-
posed to detect phenols [146]. The bioprobe fully retained its
activity after 1 month storage at room temperature. The ap-
preciable sensitivity of the bioprobe along with its stability
could suggest the feasibility of its commercialization [146].
3.3.3. Coriolus spp.
PPO was isolated from Coriolus spp. liquid cultures and
immobilized on polyvinyl alcohol fibres. The immobilized
derivative was successfully used for purification of wastew-
ater from hydrolysis yeast industry. In fact, the treatment
reduced the lignohumic acid content and color index of the
925
wastewater by over 70 and 81–82%, respectively, and the
immobilized biocatalyst retained its activity after 15 consec-
utive treatment cycles [75].
3.3.4. Pleurotus ostreatus
A water purification process for the removal of phenolic
substances was designed by combining a treatment with im-
mobilized PPO, a flocculation step and adsorption on active
carbon. The last step was also replaced by oxidation with
ozone or ClO2 . The process was successful and the PPO
filter was not blocked by the reaction products [87].
3.3.5. Miscellaneous and non-specified
Different procedures were employed to immobilize
acetone powders of mango peel (Mangifera indica) and
tea leaves (Camellia sinesis) [58]. In particular, these
enzyme preparations were immobilized by entrapment
(polyacrylamide, calcium alginate and collagen), covalent
coupling (arylamine and alkylamine glass) or adsorption
(DEAE-sephadex A-50 and DEAE-cellulose). By com-
paring the immobilization efficiency on these supports,
adsorption on DEAE-sephadex and entrapment within poly-
acrylamide gel were selective for immobilization of PPO
from mango and tea leaves, respectively. Immobilization
led to a significant increase in the activity of tea leaf PPO
towards catechol and epicatechol. Immobilized PPO from
tea leaves obtained either by entrapment within polyacry-
lamide gel or by covalent coupling with collagen reached
their half-life after 80–90 and 60–70 consecutive oxidative
cycles, respectively. PPO immobilized on DEAE-sephadex
and on polyacrylamide gel exhibited the highest storage
stability at 4 ◦ C, retaining 85–90 and 80% of their initial
activity after 6 months [58].
Mash from yeast hydrolysis was treated at pH 4.5–6.0 at
30 ◦ C with PPO immobilized on a fiber substrate. Lignohu-
mic complex, COD and color index were reduced by 85,
56 and 82% and to 38, 34 and 21%, by immobilized and
soluble PPO, respectively [60].
In humic preparations, extracted by buffer from the
humus-accumulation horizon of sandy and loamy soils,
29.4% PPO associated with humic acid was broken by pro-
tamine sulfate. In forest litter and peat soils, 89–96% of the
PPO bound were cleaved. These findings suggest that such
humic–enzyme complexes are stabilized both by weak (e.g.
electrostatic bonds) and covalent interactions [83].
A 96% of activity yield was obtained when immobilizing
PPO from potatoes by adsorption onto chitin. Though, nei-
ther thermal stability nor kinetic constants were significantly
affected with respect to the free enzyme [97]. Similar ki-
netic and stability (pH, temperature) properties for free and
immobilized enzyme were also observed when PPO from
the same source was immobilized by adsorption on Eudragit
S100 (Röhm Pharma), chitin and chitosan [126]. Regard-
less of the support, PPO immobilized by adsorption showed
an enhancement of activity in organic co-solvent mixtures
when the concentration of the organic solvent was as low as
926 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
10–20% (v/v). It was suggested that enzyme immobilization
by adsorption on a suitable matrix could be a generally bet-
ter approach than covalent immobilization for a subsequent
use in aqueous–organic co-solvent mixture [126].
PPO from sweet potatoes (Ipomea batata (L) Lam.),
immobilized Teflon (Celgard 2400) membrane in the pres-
ence of GLUTAL, fully retained its activity after 5 months
storage at 4 ◦ C [123]. The membrane, placed onto the tip
of the oxygen electrode, was used as a biosensor, provid-
ing a linear response for catechol, pyrogallol, phenol and
p-cresol. The life-time of this electrode was excellent for 15
days (over 220 determination for each enzymic membrane).
When analyzing an effluent from a tanning industry, the
biosensor provided highly correlated quantitative data with
those obtained by a spectrophotometric method, suggesting
its suitability for the determination of phenols in industrial
wastewater [123].
The immobilization of PPO by ionotropic reticulation
in calcium alginate in the presence of GLUTAL for pre-
cross-linking or re-cross-linking proved to be an effective
method yielding a highly active and stable derivative [129].
Tissues from fruits of this palm tree present remarkably
high PPO activity. The incorporation of plant tissues in car-
bon paste matrices represents an alternative approach to the
preparation of biosensors. Paste electrodes preparation by
mixing dried tissues, graphite powder and mineral oil were
used for batch experiments and also associated with FIA.
The good sensitivity and stability of the sensor suggested
its use for various real applications [132].
An activity recovery of 76.2% and a coupling effi-
ciency of 76.3% were obtained when purified PPO from N.
tabacum leaves was immobilized by cross-linking on a Ny-
lon membrane. The thermotolerance of immobilized PPO
was markedly improved while pH stability was enhanced,
especially in the alkaline pH range [147].
3.4. Phenoloxidase (PO)
Table 4 reports all examples that refer to papers dealing
with the preparation and characterization of immobilized
PO. As specified in the previous paragraph, the papers are
grouped by the originating source and presented in a chrono-
logical order.
3.4.1. Coriolus hirsutus
PO from C. hirsutus was immobilized by ion ex-
change and covalent coupling using polvinyl alcohol,
polycaproamide and cellulose-regenerated fibres [62]. The
dependence of immobilization yield on several parameters
including enzyme loading, contact time, pH, and tempera-
ture was investigated with reference to the different matrices
employed [62].
3.4.2. Pleurotus ostreatus
PO was immobilized by entrapment in copper-alginate
gel resulting in an increase in the stability and activity of the
immobilized enzyme in comparison with those of the free
form [99,102]. Utilization of the enzyme for the removal of
toxic phenol compounds from oil mill wastewater was ex-
plored. PO immobilized in alginate in the presence of Cu2+
exhibited a greater stability than that obtained with other
divalent cations. When determining the pH-activity profile,
immobilized PO showed a shift in the optimum towards less
acidic regions. The Km and Vmax values for the immobilized
PO (ABTS) (0.3 mM and 5.7 × 10−3 mM min−1 , respec-
tively) did not significantly differ from those of the free form
(0.28 mM and 3.6 × 10−3 mM min−1 , respectively). The ap-
parent half-life at 4 ◦ C for the entrapped PO was 30 days,
while for the soluble one it was only 3 days [99,102].
PO was immobilized on CPG after silanization with
APTES by using GLUTAL [115]. It was found that a
short-time incubation of PO with proteases (papain) caused
a significant increase in PO activity (up to 240–250%).
Conversely, a longer incubation time of the enzyme with
the protease caused a decrease in PO activity. In any case,
immobilization improved the stability to proteolysis. Simi-
lar results with PO from T. versicolor and P. radiata were
obtained [115].
3.4.3. Mycelia sterilia
PO from the fungus M. sterilia was immobilized using
GLUTAL on different macroporous silica carriers. The en-
zyme immobilized on both amino-silochrome SKh-2 and
aminopropyl-silachrome 350/80 exhibited maximum activ-
ity. Soluble and immobilized PO were compared for their
catalytic properties. Immobilization considerably increased
the PO stability. Both soluble and immobilized forms of PO
catalyzed the oxidative conversion of phenolic compounds
of the green tea extract [161].
3.4.4. Non-specified
PO from apples or from potatoes immobilized by adsorp-
tion on carbon black removed pyrocatechin from aqueous
effluent from coal and petroleum processing plants. This
process can be carried out continuously or batchwisely [59].
An apparatus for the removal of dissolved oxygen from beer
with immobilized PO was recently described [114].
4. Final remarks
This review summarizes and testifies research efforts that
have been dedicated to immobilize Lac and Tyros and render
feasible their use in a variety of applications. They include
synthetic and analytical purposes, bioremediation of con-
taminated soils, wastewater treatment, and must and wine
stabilization. Among them, wastewater treatment, and wine
and beverages stabilization appear to be very promising and,
with regard to this aspect, it is worthwhile to make some
considerations.
The life-time as well as the efficiency of immobilized
copper oxidases, regardless of the reactor configuration,
 N. Durán et al. / Enzyme and Microbial Technology 31 (2002) 907–931
could be negatively affected by two concurrent phenom-
ena, i.e. the generation of chromophoric products and the
insolubilization of laccase reaction products. With respect
to the first phenomenon, the adsorption of choromophoric
oxidized products on the surface of the immobilization
support often leads to enzyme inactivation phenomena.
This effect, when reversible, can be partially removed by
using washing procedures with high ionic strength buffered
solutions, as shown by some literature reports [145,154].
A feasible alternative is to select appropriate supports,
whose surface chemistry avoids the occurrence of ad-
sorption phenomena, such as vitroceramic materials [112,
124,141].
The formation of insoluble laccase reaction products,
due to non-enzymatic reactions (oxidative coupling) is an-
other important technical constraint to the development of
Lac- and Tyros-based reactors. In fact, a prolonged and
repeated use of an immobilized laccase, whether it be in
a packed-bed, a fluidized bed or in a membrane reactor,
could result in the accumulation of precipitate on the outlet
filter of the reactor (fouling) leading to significant reduc-
tions in the flow rates or, directly, to complete plugging.
This is a drawback which is intrinsically connected with
the fate of laccase-generated free radicals and quinones. In
this regard, a particularly promising approach is to combine
the use of immobilized laccase with cationic polymers,
such as chitosan, polyethyleneimine and hexamethylene-
diamine cross-linked with epichlorohydrin, which are able
to promote the coagulation of oxidized reaction products
[110]. In particular, the outlet stream of the reactor could
be subjected to a flocculation step or, alternatively, could be
directed to a column containing the aforementioned poly-
mers. In both cases, this combined approach could exert
several positive effects such as (i) decolorization of the ef-
fluent stream, (ii) possibility to perform effluent recycling
inside the laccase reactor to improve removal yields of the
target pollutants, (iii) improved detoxification due to the re-
moval of quinonoid products whose toxicity is often higher
than that of the parent compounds.
The development of Lac- and Tyros-based biosensors to
monitor a wide range of compounds appears to be at a ma-
ture stage of technology. In fact, several biosensor systems
based on copper oxidases allowed a high selectivity and sen-
sitivity with reduced assay times and showed a good stability
over time. The set-up of reagentless enzyme electrodes has
extended the range of detected compounds to activators or
inhibitors of enzyme activity, such as the respiratory poison
azide.
Acknowledgments
Support from FAPESP and CNPq (Brazil) is acknowl-
edged. For any request for ordering and buying of reprints
to Università di Napoli Federico II, Portici (Napoli, Italy)
indicates: DiSSPA Contribution No. 0011.
927
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