review immobilized laccases

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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 Ciˆ encias 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 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: [email protected] (N. Dur´ an). The main purpose of this paper is to present a general 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. 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0141-0229(02)00214-4

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A survey on immbilized laccase systems

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Page 1: REview immobilized laccases

Enzyme and Microbial Technology 31 (2002) 907–931

Review

Applications of laccases and tyrosinases (phenoloxidases)immobilized on different supports: a review

Nelson Durána,b,∗, Maria A. Rosaa, Alessandro D’Annibalec, Liliana Gianfredada Biological Chemistry Laboratory, Instituto de Quimica, Universidade Estadual de Campinas, C.P. 6154, Campinas CEP 13083-970, S.P., Brazil

b Núcleo de Ciencias Ambientais-NCA, Universidade de Mogi das Cruzes, Mogi das Cruzes, S.P., Brazilc 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 immobilizationprocedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition totheir kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linkedwith epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-basedbiosensors 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

Extensive research effort have been dedicated to evaluatethe possibilities offered by enzymes in biotechnological andenvironmental applications[1,2].

An effective use of enzymes, may be hampered by somepeculiar properties of the enzymatic proteins such as theirnon-reusability, high sensitivity to several denaturatingagents and presence of adverse sensory or toxicologicaleffects. Many of these undesirable constraints may be re-moved by the use of immobilized enzymes. This approachhas proven to be more advantageous for catalysis than theuse of free enzymes.

Among enzymes, laccases and tyrosinases are two groupsof phenoloxidases that catalyze the transformation of a largenumber of phenolic and non-phenolic aromatic compounds.In the literature, much information is available on the useof free and immobilized phenoloxidases in several appliedareas. To date, however, an exhaustive overview of the ba-sic aspects of immobilized laccase and tyrosinase is stilllacking.

∗ Corresponding author. Tel.:+55-19-788-3149; fax:+55-19-3788-3023.E-mail address:[email protected] (N. Duran).

The main purpose of this paper is to present a generalpicture of the results achieved in this research field. We haveapproached this objective by reviewing the literature dealingwith the fundamental and applied aspects of immobilizedphenoloxidases. A brief introduction on the main aspects ofthe active sites and the mechanistic characteristics of laccaseand tyrosinase function has been also added. In our opinion,these aspects are important because it is their involvement inenzyme immobilization that determines the final propertiesof the immobilized catalyst and the features of its action.A very short paragraph on the methods of immobilizationwith specific reference to those used in the immobilizationof 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 thetime-progress in this research area, the findings have beendiscussed 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 publicationsto indicate both Lac and Tyros, two additional paragraphsdedicated to these papers, have been included in this review.

0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.PII: S0141-0229(02)00214-4

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1.1. Laccase (Lac)

Laccase is a cuproprotein belonging to a small groupof enzymes denominated blue oxidases[1]. Laccase (E.C.1.10.3.2, p-benzenediol:oxygen oxidoreductase) is anoxidoreductase able to catalyze the oxidation of variousaromatic compounds (particularly phenols) with the con-comitant reduction of oxygen to water[1,3]. In general,laccases exhibit four copper atoms, which play an importantrole in the enzyme catalytic mechanisms. Copper atomsare distributed in different binding sites and are classi-fied in three types, according to specific spectroscopic andfunctional characteristics[4–9].

The molecular genetics[10], genetic expression[11], ge-netic transcription[12] and cloning[13] of laccases havebeen exhaustively studied. The basic aspects of phenoloxi-dases have been reviewed, as well[4,14–22].

In a typical laccase reaction, a phenolic substrate issubjected to a one-electron oxidation giving rise to anaryloxyradical. This active species can be converted to aquinone in the second stage of the oxidation. The quinoneas well as the free radical product undergo non-enzymaticcoupling reactions leading to polymerization[23].

Laccases are characterized by low substrate specificityand their catalytic competence varies widely dependingon the source. Simple diphenols such as hydroquinoneand catechols are good substrate for the majority of lac-cases, but guaiacol and 2,6-dimethoxyphenol generally arebetter substrates[2,5]. Laccase is also able to catalyzethe oxidation of other substituted polyphenols, aromaticamines, benzenethiols and a series of other compounds,but the enzyme, unlike tyrosinases, is unable to oxi-dize tyrosine.N-Hydroxybenzotriazol, violuric acid andN-hydroxyacetanilide are three N–OH compounds capableof mediating a range of laccase-catalyzed biotransformation[24].

Laccase is widely distributed in higher plants[25], infungi [1,4] and in some bacterial strains ofAzospirillumlipoferum [26] and Alteromonassp. [27]. Very recently, ithas been reported that laccases are widespread in bacteria[28].

Among fungal laccases, a great variability is observedin the induction mechanism, degree of polymorphism, andphysico-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 mediumresults in the biosynthesis of new extracellular forms.

The biological effect of polyphenols and derivatives weretested in order to assess their environmental impact andtheir use as byproducts for agriculture and industry wasevaluated after a detoxification treatment with laccase[31].The determination of environmental pollutants using im-munoassays is a continuously growing area and, within thisframe, laccase proved to be an excellent alternative to perox-idase as a bioanalytical tool for monitoring polar pollutants[32].

1.1.1. Laccase active siteLaccase 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 Type3 or coupled binuclear copper site where the coppers areantiferromagnetically coupled through a bridging ligand(EPR undetectable)[33]. Spectroscopy combined with crys-tallography has provided a detailed description of the activesite in laccase. Magnetic circular dichroism (MCD) andX-rays absorption spectroscopy of laccase have shown thatthe Type 2 and 3 centers combine to function as a trinuclearcopper cluster with respect to exogenous ligand interactionincluding reaction with dioxygen[34]. The Type 2 center is3-coordinate with two histidine ligands and water as ligands.The Type 3 coppers are each 4-coordinate, having threehistidines ligands and bridging hydroxide. The structuralmodel of bridging between the Type 2 and 3 (Fig. 1A and B)[33–36]has provided insight into the catalytic reduction ofoxygen to water. It has been elucidated that the Type 2 cop-per is required for the reduction of oxygen since bridgingto this center is involved in the stabilization of the peroxideintermediate.

Reduction of oxygen by laccase appears to occur in two2e− steps. The first is rate-determining. In this Type 2/3bridging mode for the first 2e− reduced, the peroxide-levelintermediate would facilitate the second 2e− reduction (fromthe Type 2 and 1 centers) in that the peroxide is directlycoordinated to reduced Type 2 copper, and the reduced Type1 is coupled to the Type 3 by the covalent Cys–His linkages[37].

Previous studies[34] reported that 40% of the Type 1 and3 that readily react with dioxygen correspond to native lac-case (Fig. 2). It is clear that the Type 2 Cu is required fordioxygen reactivity in laccase and that dioxygen reductionoccurs in the absence of the Type 1 Cu. This demonstratesthat the Type 2/3 trinuclear Cu site represents the activesite for the binding and multielectron reduction of dioxygen.The Type 1 Cu is clearly not necessary for reactivity withdioxygen, and in its absence, an intermediate is formed thatshares some properties with the oxygen intermediate previ-ously described in native laccase.

Fig. 1. Two possible spectroscopically models for peroxide bridging at thetrinuclear cluster site: (A) bridging between Type 2 and one of the Type3 copper in a�-1,1-hydroperoxo mode; (B) bridging all three copper ina �3 (�1)3 (modified from[33]).

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

Very recently, the structural model of the Type 1 copperprotein active site was described as N2S (thiolate)-S-(thio-ether) ligation in a Cu(II) complex[9]. Fig. 3 shows theactive site of laccase in a pictorial way (http://bioinf.leeds.ac.uk) [38–40].

Recently, the structure of laccase fromTrametes versi-color was determined in its glycosylated, fully functionalform at 1.9 Å resolution (Fig. 4) (http://wwwbc.biol.ethz.ch).T. versicolor laccase is a globular protein of about 500amino acids and contains three cupredoxin-like�-sandwichdomains, similar to those found in ascorbate oxidase and inceruloplasmin.

1.1.2. Laccase catalytic cycleFig. 5illustrates the catalytic cycle of laccase and the pro-

posed mechanisms for the reduction and reoxidation of thecopper sites. In this figure (center) starting from the “nativeintermediate,” the substrate reduces the Type 1 site, which inturn transfers the electron to the trinuclear cluster. Two pos-sible mechanisms for the reduction of the trinuclear clusterare shown: (A) the Type 1 and Type 2 sites together reducethe Type 3 pair and (B) each copper in the trinuclear clusteris 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” leadsto the resting fully oxidized form. In this form, the Type 1

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

Fig. 4. Ribbon diagram ofT. versicolor laccase. The three domains areshown in the figure together with the copper atoms, carbohydrate moietiesand disulfite bridges are also depicted.

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

site can still be reduced by substrate, but electron transfer tothe 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 phylogeneticscale from bacteria to mammals and even present differentcharacteristics in different organs of the same organisms,such as in roots and leaves of higher plants[15]. It is wellknown that Tyros catalyzes two different oxygen-dependentreactions that occur consequently: theo-hydroxylation ofmonophenols to yieldo-diphenols (cresolase activity) and

the subsequent oxidation ofo-diphenols too-quinones (cat-echolase activity). The basic aspects of Tyros were previ-ously discussed[42–45].

1.2.1. Tyrosinase active siteChemical 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 shownin Fig. 6.

1.2.2. Tyrosinase catalytic cycleThe oxygenated form (oxytyrosinase, Eoxy) consists of

two tetragonal Cu(II) atoms, each coordinated by two strong

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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 bridgesthe two Cu centers. The cupric model complexes, whichare used to explain the electronic structures, are end-on(cis-�-1,2 geometry) or side-on (�-�2: �2). Mettyrosinase

Fig. 7. Catalytic cycle for the oxidation of monophenol and diphenol substrate too-quinones by tyrosine in the presence of oxygen (modified from[16]).

(Emet), like oxy form, contains two tetragonal Cu(II) ions an-tiferromagnetically coupled through an endogenous bridge,although hydroxide exogenous ligands rather than peroxidesare bound to the copper site. Deoxytyrosinase (Edeoxy) has abicuprous structure [Cu(I)–Cu(I)]. These three copper statesin the active site of tyrosinase suggest a structural modelfor the reaction mechanism involved in theo-hydroxylationof monophenols and oxidation of the resulting diphenols(Fig. 7) [16]. Recently, mechanistic aspects related to itscrystal structure have been discussed[39,46].

2. Enzyme immobilization

Many methods are available for enzyme immobilization.Since the methods used the immobilization proceduresgreatly influences the properties of the resulting biocatalyst,the selection of an immobilization strategy determines the

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process specifications for the catalyst. They include severalparameters such as overall catalytic activity, effectivenessof catalyst utilization, deactivation and regeneration ki-netics, and cost. Also, toxicity of immobilization reagentsshould be considered in connection with the immobiliza-tion process, waste disposal and final application of theimmobilized enzyme catalyst[47–55].

Several techniques may be applied to immobilize enzymeson solid supports. They are mainly based on chemical andphysical mechanisms[48]. In the following, the immobiliza-tion processes mostly used with laccase and tyrosinase willbe briefly discussed. Their basic aspects are summarized inTables 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) enzymecross-linking by multifunctional reagents. Physical meth-ods involve the entrapment of enzyme molecules within aporous hollow fiber, in spun fibers or enzyme entrapmentwithin an insoluble gel matrix and/or enzyme entrapmentwithin a reverse micelle.

Enzyme immobilization by physical entrapment has thebenefit of a wide applicability and may provide relativelysmall perturbation of the enzyme native structure and func-tion. The most widely used system for enzyme entrapmentin a polymer lattice is the immobilization within a poly-acrylamide gel, obtained by polymerization/cross-linking ofacrylamide 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 withfresh, active catalyst[49]. Another adsorption method for ty-rosinase with pre-activation of the polyphyllosilicate (mont-morillonite) was published[54]. An interesting method byaffinity coupling with Affi-Gel-10 and Affi-Gel-15 was alsoreported.

The polystyrene-based matrices activated with nitrousacid, 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 derivativeis easily accomplished. A commonly used method includesa support pre-activation with aminopropylsilane and a sub-sequent reaction withp-nitrobenzoyl chloride to form thediazonium salt is also used[47].

The most commonly employed water-insoluble supportscurrently used for enzyme immobilization are cyanogenbromide-activated sepharose and sephadex. The immobiliza-tion method is simply and reliable, and the attachment of theenzyme to the matrix is performed under mild conditions.The method involves the activation of the polysaccharideswith cyanogen bromide to give the reactive imidocarbonate,which subsequently reacts with the protein[51].

The chemical activated method for sepharose CL-6B withan epoxide is quite efficient to obtain an aldehyde activatinggroup in the support able to react with the enzyme[53].

Protocols for covalent enzyme immobilization often beginwith a surface modification or an activation step. Silaniza-tion, i.e. the coating of the surface with organic functionalgroups using an organofunctional silane reagent, is a widelyused strategy for initial surface modification of inorganicsupports. Such coating or native surface amino groups can bederivatized to arylamine group usingp-nitrobenzoyl chlorideor to aldehyde groups using glutaraldehyde (GLUTAL)[52].

Carbodiimide activation is used when a carboxylic groupin the support is expected to react with an amino group fromthe enzyme. This activation is possible by epoxide forma-tion in the side chain[55] or on the support surface, as forexample in the case of activated sepharose and cellulose. Be-sides the activation of these carbohydrates, chitosan is alsoused for tyrosinase immobilization by trapping the enzymebetween two chitosan gel films[50].

Both chemical and physical methods offer advantagesand disadvantages that depend on several factors. In gen-eral, chemical immobilization methods tend to reduce theactivity of the enzyme, since the covalent bonds, formed asa result of immobilization, may perturb the enzyme nativestructure. By contrast, such covalent linkages provide strongstable enzyme attachment and may, in some cases, reduceenzyme deactivation rates and usefully alter enzyme speci-ficity. However, entrapment and adsorption immobilizationmethods typically perturb the enzyme much less and conse-quently offer retention of the enzyme properties resemblingthose in solution[47]. A proper choice between chemicaland physical methods depends on several factors. Usually, along-time applicable immobilized enzyme with a lower ini-tial activity is preferable to that with a high level of initialactivity but with a short-time activity retention.

3. Phenoloxidases immobilized on different supports

3.1. Laccases (Lac)

Table 1reports microbial and plant laccases immobilizedon different supports. The type of support, the method ofimmobilization, and the substrate(s) used in the catalyticprocess are specified. Some comments on the main featuresof each example are also included.

As specified inSection 1, the examples listed in alpha-betical order inTable 1will be presented in the followingin a chronological order.

3.1.1. Neurospora crassaThe immobilization of Lac fromN. crassaon CNBr-acti-

vated sepharose 4B by covalent attachment resulted in a re-duction (25%) of activity yield and an increase ofKm value,probably caused by a steric hindrance to substrate diffu-sion. By contrast, adsorption on concanavalin A-sepharosedid not affect both the kinetic parameters, thus suggesting anon-involvement of the enzyme carbohydrate moiety in thecatalytic center[56].

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3.1.2. Rhizoctonia praticolaA good immobilization yield (89%) and a high trans-

forming ability (100% of methoxy-substituted phenolstransformed), similar to that of the native enzyme, wereobtained whenR. praticolaLac was covalently coupled toactivated Celite®. Similarities in the relative activities ofthe free and immobilized enzyme demonstrated that anydeactivating effect occurred upon immobilization[61].

3.1.3. Polyporus versicolor, T. versicolor, Coriolusversicolor

In course of time, this fungal strain, producer of laccase,has been indicated asPolyporus, Trametes, or C. versicolor.According to the principle adopted in this review, we havecollected the papers by the name used to define the fungusand discussed them separately.

3.1.3.1. Polyporus versicolor. P. versicolorLac 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 immobilizationon sepharose 4B-Epi-IDA-Cu2 gave a very high adsorp-tion yield, showed excellent stability and resulted a fast,environmental-friendly, inexpensive technique because ofthe possible carrier reuse. The immobilized enzyme wasused for apple juice clarification (reduction of phenols,flavonols and chromophoric compounds by 48.6, 47 and43%, respectively) and for catechin removal (75%) in acolumn 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 differenttypes 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 verygood 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 wasalso observed, thus suggesting that some sort of purificationor other phenomena took place during the immobilizationprocess[105].

Lac immobilized on APTES–GLUTAL-activated glasssupports usually displayed good stability to long-term ap-plications. When used in a batch reactor, immobilized Lacfully 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 thanthe native counterpart, as well[67]. A similar response wasobtained in flow-injection calorimetry studies, performedwith the immobilized enzyme packed into a small polymercolumn and mounted on a temperature probe housed in a

calorimeter[139]. Virtually unchanged performance during4 months of operation at room temperature and exothermicresponses with several substrates, includingl-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 inactivationand lost about 22% of activity for over 160 injections ofmethylcatechol[89].

Laccase fromT. versicolor was immobilized on a siltloam soil, kaolinite and two different montmorillonites uponpre-treatment with APTES/GLUTAL[73,105]. High per-centages of Lac activity were measured after binding onclays 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 activatedglass beads, gave rise to a specific activity higher than thatof the free enzyme[105]. The enzyme immobilized on thesesupports showed a very ability to remove 2,4-dichlorophenol(2,4-DCP) from model solutions (95% for kaolinite- andsoil-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 withdifferent amounts of organic matter indicated that the perfor-mance of the immobilized enzyme was strongly affected byinorganic and organic soil constituents. These results suggesta potential use of immobilized laccase for the clean-up ofpolluted soils, but its performance can be strongly affectedby soil nature and compositions[105].

Very interesting findings were obtained with Lac immo-bilized by entrapment in sepharose CL-6B. The gel–enzymeassociation was stable in water pre-saturated solvents andshowed good stability in organic solvents as well as a hightolerance to elevated temperatures[76]. Syringaldazine and2,6-dimethoxyphenol (in ethyl acetate pre-saturated withwater) as well as water-insoluble organosolvent lignin,dissolved in dioxane/water, were readily converted bysepharose CL 6B-immobilized Lac[76]. Less than a 5%decrease of the initial activity occurred after 36 h incubationof Lac/complex in hydrophobicn-hexane at 30◦C, whileunder the same incubation conditions but in citrate buffermore than 50% of initial activity was lost[76].

Good results in terms of storage stability (90% activityretained versus 50% of the free enzyme after 4 months at4◦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 micellesprepared 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 effluentremediation), Lac production was optimized and its im-mobilization on different supports (i.e. ionic exchangeresin IRA-400, silica MERCK II and a vitroceramic ma-terial) was investigated[141]. Immobilization on bothIRA-400 and silica MERCK II gave rise to a 100% yield.

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Nevertheless, a high adsorption of chromophoric substanceson the surface of these matrices was evident when theywere employed for effluent treatment. In contrast, althoughthe immobilization yield of Lac onto vitroceramic supportwas lower (around 50%), a very low adsorption of chro-mophores on this support was observed. Consequently,vitroceramic appeared as an efficient support to prepareimmobilized biocatalysts with a reasonable retention ofenzymatic activity, for the treatment of textile and paper in-dustry effluents[141]. Recently, two different vitroceramicmaterials were tested with a kraft E1 effluent. The bestimmobilization results were obtained by combining the useof low porosity vitroceramic with carbodiimide coupling[124] or carbodiimide/GLUTAL activation[112].

Laccase fromT. versicolorwas often immobilized for apossible use as biosensor[133,135,164]. Biosensors wereprepared 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]; physicaladsorption, carbodiimide coupling, GLUTAL activation,combined use of carbodiimide and GLUTAL[164]. Usu-ally, the response of the sensors (i.e. sensitivity, kineticparameters 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 thepercentage of GLUTAL was the most important influencingparameter, since it heavily affected the properties of theimmobilized enzyme. Indeed, high degree of enzyme retic-ulation promoted by GLUTAL probably occurred with aresulting limited substrate access to the enzyme active site(apparentKm value quite lower than that of the free form)[164].

3.1.3.3. Coriolus versicolor. C. versicolorLac and ty-rosine Lac co-polymers entrapped in calcium alginate gelbeads[80] or Lac covalently immobilized on activated car-bon [92] were used in the treatment of effluents from thepulp and paper industry. The four derivatization methods(silanization of carbon, cross-linking after binding to carbonamino group, activation of amino groups with diimide, andactivation of carboxylic groups with diimide) used to im-mobilize the enzyme on the activated carbon did not affectsignificantly the total amount of bound protein[92]. Lacimmobilized on diimide-activated carbon showed a consid-erably higher activity and was used in large batches for bothbatch and reactor experiments. In a fluidized bed, operatedin a re-circulation mode, the average decolorization ratewas 633 colorimetric units h−1 (60 colorimetric units (lac-case unit)−1 h−1) and the activity decreased from 10.5 to8.4�mol O2 min−1 g−1 carbon. Therefore, this immobiliza-tion procedure would be suitable for treatment of phenolicwaste, but it may be inappropriate for color removal frompulping and bleaching effluents[92,103].

3.1.4. Rhus verniciferaIn 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 exampleis the immobilization of the enzyme by entrapment in poly-acrylamide gel[64]. Immobilized Lac was used to catalyzethe oxidative polymerization of urushiol from outmodedChinese lacquer. The floatability, drying properties, color,luster value, shock strength, flexibility, and adhesion of thislaccase-treated lacquer were better than those of the nativelacquer film [64]. Several parameters and properties (i.e.immobilization conditions, reusability, thermal stability,Km, optimum pH and temperature) were also studied andcompared with those of the native enzyme[66,68,69].

Novel types of p-benzoquinone-activated supports(agarose, polyvinyl alcohol, chitosan) were developed andused for laccase immobilization (coupling of 10–95%)[74].The high residual activity (150%), measured for immobi-lized Lac, was ascribed to the presence of hydroquinonegroups on the support. It, as a substrate of Lac, could havegenerated a simultaneous affinity retention of the enzyme[74]. In addition, the immobilized enzyme was much lesssusceptible to the inhibitory action of chloride and azide ionsthan the free Lac (67 and 32% activity recovery upon dialy-sis, respectively) and displayed a remarkably high stabilityat 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 propertiesof free and immobilized Lac were determined[79,86]. UsingZrCl2-treated porous silica as a carrier, the activity recov-ery and stability of immobilized Lac was higher than thosepreviously reported in the literature. The retained activity ofthis immobilized preparation after 20 consecutive oxidativecycles was around 75%[86]. When Lac was immobilizedon an urushiol-based resin in the presence of adsorbed metalions, the highest catalytic activity was obtained with alu-minum (Al) [121]. The urushiol-salicylic acid (USR) graftedresin proved to be a better support for Lac immobilizationthan urushiol itself and, also in this case, the presence of ad-sorbed aluminum ions gave rise to the highest activity. TheapparentKm value of USR-Al-Lac for phenol was remark-ably lower than that of the native enzyme (4.9×10−3 versus1.2 × 10−2 M, respectively). This was attributed to adsorp-tion phenomena between USR and the substrate[121].

3.1.5. Geotrichum candidum, Fomes fomentariusVery few examples on the immobilization of laccase from

G. candidumor F. fomentariusare available in the literature[71,85]. Enzyme fromG. candidumwas immobilized on soilby adsorption and used in laboratory-scale experiments forphenol (4-methylphenol and 2,4-DCP) transformation[71].The enzyme fromF. fomentariuswas coupled to 3-APTES-controlled porosity glass (APTES-CPG) using GLUTAL.As compared to its free counterpart, it showed an unusual

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higher substrate affinity but a lower catalytic velocity (lowervalues of both the kinetic parametersKm andVmax) [85].

3.1.6. Pyricularia oryzaeSeveral studied were performed on the use of this im-

mobilized fungal Lac in phenol removal processes formust and wine stabilization and for detoxification purposes[91,93,120,158,159].

In comparative studies, Lac was immobilized on differentsupports by different methods and used to decrease phenolcontent in model and real phenolic solutions[91,120]. Theenzyme was linked: by covalent binding to four carriers(e.g. CH-sepharose 4B, CNBr-sepharose 4B, VA-hydroxybiosynth, a vinyl acetate-based co-polymer cross-linkedwith divinyl-ethylene-urea and VA-epoxy)[91,120], by ad-sorption, followed by treatment with GLUTAL, on bothsilica gel and florisil, by entrapment and by radiation poly-merization on colloidal silica[120]. Lac immobilizationon CNBr-sepharose 4B produced the most stable system,followed by the silica immobilization[91]. Generally, whenused in catechin model solutions and in both musts andwine, a significant decrease of phenol content (about 60, 34and 13%, respectively, after treatment with Lac immobilizedon silica gel) was measured[91,120]. The results suggestthat the use of immobilized Lac might represent a promisingprocedure to remove phenolics in wine-making technology.

An interesting application for wine stabilization was pre-sented by using molecular sieves as laccase-immobilizingsupports[93]. Molecular sieves were ground, sieved anddegreased with ether, and then Lac was immobilized byadsorption on the support. The enzyme-support complexwas 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. Thetreatment with soluble laccase led to 68, 12 and 127 mg l−1

of residual catechin, respectively, whereas the treatmentwith 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-immobilizedP. oryzaeLac and mush-room Tyros and adsorption on Polyclar (polyvinylpolypyrroli-done). In particular, Lac and Tyros were co-immobilized onMikroperl in a fixed-bed tubular bioreactor by a rapid andsimple method[158]. The support immobilized 95% of thetotal Lac units and 35% of the total Tyros units. A 42–90%removal of different phenolic substances by a single passagethrough the bioreactor was observed. Polyclar was used ina second step for additional removal of phenolic substancesfrom 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 continuoustransformation of different phenols in mixtures[158]. Sim-ilar co-immobilization with Tyros was previously used withLac from C. hirsutus. Co-immobilized enzymes were ad-

sorbed as a layer onto a graphite electrode and used as asensor in a single line flow-injection system. The bi-enzymesensor efficiently allowed the analytical detection of a largegroup of phenolic compounds[113].

An immobilization yield of around 40% was achievedby immobilization of Lac onto a spiral-wound asymmet-ric polyethersulphone membrane in a reactor[159]. Whenapplied to the biodegradation of a model solution con-taining many phenolic substrates (7–69% degradation), ashift in pH and temperature optima was observed in themembrane-immobilized laccase with respect to the solublecounterpart (6.6 versus 6.3◦C and 42 versus 35◦C, respec-tively). Immobilized Lac retained 50% of its initial activityafter 150 h of repeated runs against only 18% shown by itsfree counterpart after 39 h[159].

3.1.7. Coriolus hirsutusThe main interesting example reported in the litera-

ture for this fungal Lac is its immobilization in reversedmicelle-like complexes, obtained by adding a Lac watersolution to CEPEI in benzene/n-butanol[104]. The immo-bilized preparation lost 40% of its initial activity after 20days 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 fromCerrena maximawere also applied on enzyme-linked immunoassay[103].

3.1.8. Phlebia radiataP. 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 forguaiacol resulted (Km values of 1.76 and 4.78 mM for thefree and immobilized forms, respectively). A remarkableincrease of both storage stability and resistance to inhibitors,such as Cu-chelators and quinone, was evident. After 180days storage at 4◦C the immobilized enzyme lost only 4%of its initial activity against more than 93% loss shown bythe free form[108].

3.1.9. Pleurotus eryngiiLac was immobilized by covalent attachment to chemi-

cally-activated gels[53]. Aldehyde, amino and amino-GLU-TAL sepharose CL-6B derivatives, prepared by traditionalmethodologies, were used and gave rise to 95, 70 and 55%coupling yields, respectively. Covalent immobilization ontoaldehyde gels markedly increased the Lac stability in 60%N,N-dimethylformamide. The immobilized enzyme was,however, low efficient in the treatment of a paper industryeffluent. In contrast,P. eryngiiLac immobilized by entrap-ment within calcium alginate gels was very effective on thiseffluent[53].

3.1.10. Cerrena unicolorThe catalytic capability of an extracellular laccase, iso-

lated and purified from the non-induced culture ofC. uni-color, and immobilized on GLUTAL-activated silanized

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porous glass beads, was evaluated in water-miscible organicsolvents[125,130]. Immobilized Lac (94% yield of immo-bilized protein) exhibited 100% activity. As compared withthe free enzyme, immobilized Lac showed an optimum pHshift from 5.5 to 5.7. After about 7 months at 4◦C, a 95%retention of activity (syringaldazine as the substrate) wasobserved for immobilized Lac (40% for the free form)[130].Further studies were performed on this enzyme immobilizedon CPG by both the APTES/GLUTAL and carbonyldiimi-dazole procedure[143]. The covering of CPG surface, priorto enzyme attachment, with two polysaccharide layers, oneformed of cross-linked DEAE-dextran, and the other pre-pared from cross-linked dextran and carbonyldiimidazole,strongly increased the activity of the immobilized enzymein organic solvents[143].

3.1.11. Lentinus edodes (Lentinula edodes)Two interesting practical examples of immobilizedL.

edodeslaccase are reported in the literature[145,154]. Inboth cases the capability of the immobilized enzyme wasassessed by treatments of olive mill waste waters (OMW),a characteristic by-product of olive oil production, veryrich of toxic phenolic compounds and largely produced inMediterranean countries. The enzyme was immobilized onchitosan by adsorption and subsequent cross-linking withGLUTAL (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 thanthe free enzyme (Vmax 85.5 versus 190 units mg−1 proteinandKm 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 astemperature, pH and storage time. When OMW were treatedwith immobilized Lac under batch conditions, a partial de-colorization as well as a significant abatement of polyphenolando-diphenol content were obtained. The dephenolizationprocess was also combined with a decreased toxicity ofthe effluent as tested using a highly sensitive strain of thenitrogen-fixing bacteriumRhizobiumspp.[145].

The immobilization ofL. edodesLac was further op-timized by using the epoxy-activated polyacrylic supportEupergit® C. A catalytic capability of 340 units mg−1 sup-port was obtained. Immobilization on this support besidesincreasing pH, thermal stability and storage stability alsoenhanced enzyme resistance to proteolytic attack. A 20%decrease in the OMW dephenolization efficiency, performedwith Eupergit® C-immobilized laccase in a fluidized bedreactor operated in a recirculation mode, was evident aftereight consecutive treatment cycles. Nevertheless, efficiencywas restored by a washing procedure with a high ionicstrength buffered solution followed by a four-bed volumeswash with buffer alone[154]. Similar results in both activ-ity retention and storage stability were obtained also withLac from Pleurotus ostreatus, covalently immobilized onthe same support[156].

The simplicity of the immobilization process, the stabilityand 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), Trameteshirsuta

A high storage and thermal stability, a good tolerancetowards some inhibitory compounds as well as the re-duction of several dyes toxicity were measured when Lacfrom C. gallica andT. hirsutawere immobilized by cova-lent binding to Affi-Gel-15, aN-hydroxysuccinimide esterof derivatized cross-linked agarose gel bead support, andalumina, respectively[150,157]. The reduction by up to80% of triarylmethane, indigoid, azo, and anthraquinonicderivatives toxicity, as assessed by monitoring the oxygenconsumption inPseudomonas putidaliquid cultures, provedthe treatment of textile effluents with immobilized laccaseto be suitable for dyeing purposes[157].

3.1.13. Non-specifiedTwo 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 carbonblack previously contacted with bovine serum albumin inthe presence ofN-ethyl-5-phenyl-isoxazolium-3-sulphonate(EPIS) significantly increased the thermal stability as wellas the catalytic activity of immobilized Lac[65]. A Lac im-munoconjugated (SynectiQ Corp.) was physically adsorbedon a pre-treated graphite ink that was deposited on a plasticstrip. Therefore, a Lac molecular layer attached to the elec-trode surface formed an oxygen transducer. The use of a suchbiosensor for immunofiltration and immunochromatographywas suggested[160].

Some further examples of immobilized non-specifiedlaccase, including an enzyme produced by heterologous ex-pression of aT. versicolorLac gene inAspergillussp.[155],regard adsorption on sepharose CL-4B[72] and varioussupports (glass, glass powder, silica gel, and Nylon-66membrane)[155], and covalent binding on differently ac-tivated polymers[160]. Nylon-66 membrane/Lac retaineda very high activity in organic solvents (diethyl ether andmethyl acetate) while exposure to methylene chloride re-sulted in significant activity decrease regardless of thesupport material[155]. Lac immobilized on polyvinyl al-cohol, polyacrylates, and other polymers concurred withother 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

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shown inTable 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 polyacrylamidegels resulted in increased storage stability[57]. The immo-bilized biocatalyst, retained 43 and 70% of its initial activitywhen stored at room temperature or at 6◦C over a 13-dayperiod, respectively. The results of the study indicated itspossible incorporation into a detection system for phenolsand 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 itsinitial activity throughout a 3-month period. When phenolwas used as substrate (10−4 M) the enzyme preparationexperienced a constant decrease of activity with repeateduses, 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 immobilizedTyros can be used in an open tubular reactor as a partof the injector valve’s sample loop in an unsegmentedcontinuous-flow analyzer for the selective determinationof 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% activityretained after 15 days incubation at 5◦C, respectively) andit 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 useof the coagulant hexamethylenediamine-epichlorohydrin[110]. In the treatment with immobilized Tyros, morereaction products were removed with less coagulant, ascompared with the soluble enzyme[110].

Tyros was immobilized by adsorption on several sup-ports and used for wine stabilization[93]. Adsorption andcross-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-linkingon DEAE-cellulose) to 38% (GLUTAL-activated silica).The silica gel/GLUTAL system was selected for further ex-periments on must and wine stabilization. This immobilizedderivative was more effective than its soluble counterpart toperform the removal of catechin (100 mg l−1) from modelsolutions. Nevertheless, when experiments were conductedon musts and wine, soluble and immobilized Tyros ledto comparable results. The immobilized Tyros showed anoperational stability of two cycles, probably due to itssensitivity 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-aminopropyl)carbodiimide, an activity yield of 16.3% was ob-

tained[95]. After 96-h storage at 25◦C, immobilized Tyrosretained 50% of the initial activity compared with 20% ofsoluble Tyros. Treatment with immobilized Tyros yielded a100% phenol removal after 2 h incubation, and only a slightdecrease in the activity was observed even after 10 repeatedtreatments[95].

Tyros was immobilized by entrapment within alginate geland used to produce theaflavin, as well[98]. The specificactivity of immobilized Tyros was lowered by 7% duringthe immobilization process, as compared with the solubleone. A 40% decrease ofKm value was also measured. Themaximum yield of theaflavin was obtained within 20–30 minincubation with immobilized Tyros in a reactor[98].

Crude and purified Tyros were immobilized by severalmethods including physical entrapment, cross-linking withBSA-GLUTAL, covalent immobilization on both ampho-teric PALL and carboxylic PALL membranes, and covalentimmobilization on Nylon net[100]. The resulting immobi-lized derivatives were then tested for their use as biosensors.Crude mushroom extracts coupled with a Clark electrodegave a better signal stability but the biosensor life-time wasnot satisfactory. Better results were obtained with biosen-sor based on pure Tyros immobilized on a Nylon net andthe system when employed on green water from crushersshowed 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 Tyrosmolecules were adsorbed onto the former than the latter.Tyros immobilization efficiency on Mte increased withincreasing levels of Al(OH)x coating[107,122].

In another study, factors affecting Tyros inactivation dur-ing its immobilization on glass beads and Celite® wereinvestigated[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 immobilizedprocedure offered a protective effect with increased residualactivity at comparable enzyme loading[109].

Results obtained with Tyros immobilized between twochitosan films suggested that 45% of the activated bondingsites were available for the adsorption. To perform a selec-tive phenols removal from a mixture, Tyros was used to con-vert phenols intoo-quinones, which were then adsorbed ontothe amino-containing polymer chitosan gel film[116]. Theperformance of enzyme-containing chitosan gels dependedon the ratio of tyrosinase-to-chitosan. In fact, within a welldefined enzyme concentration, a direct proportionality be-tween the aforementioned ratio and system efficiency wasevident[116].

When immobilized on Nylon-66 through covalentcross-linking, the enzyme produced 2.2 times more benzo-quinone than soluble Tyros and was inactivated at one-tenththe rate of soluble Tyros. Oxygen adversely affect the sta-bility of immobilized Tyros. The inactivation rate was 3.6times faster at 100 kPa oxygen than under 21 kPa oxy-gen. Preliminary evidence also suggested that there is a

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difference in the affinity of the inactivating species forimmobilized Tyros compared with the soluble enzyme.The covalent cross-linking of Tyros onto Nylon-66 seemedfavorable to stabilize its catecholase activity[117].

The influence of gelatin as an additive in the formationof 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 andphenol removal efficiency of this immobilized system wasstudied in a column reactor using catechol model solutions.About 50% of catechol disappeared from the eluted solutionin each cycle and no adsorption of catechol in the columnwas observed[128].

A 100% yield was obtained when Tyros was immobilizedon a polymer produced by reaction of methyl methacry-late and glycidyl methacrylate[118]. A biosensor exhibitinggood stability properties was prepared by coating a platinumwire electrode with the Tyros-modified polymer[118]. Asol–gel modified Tyros biosensor was prepared by immo-bilization on a Al2O3 sol–gel (Al:H2O:HCl = 1:100:0.05)(boemite sol [�-AlOOH]) [153]. A GCE was used as baseelectrode. 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 andthe standard one. The method was useful for applicationin real samples with good precision and accuracy, thus be-ing an example of a reliable enzymatic biosensor at verylow costs[153]. Very recently, a new Tyros biosensor hasbeen developed[162]. It is based on the co-immobilizationof Tyros and a mediator, Fe(CN)6

4−, on positively chargedAl2O3 sol–gel membranes. The stability of the encapsulatedmolecules was further enhanced by the electrostatic inter-action between the molecules and matrix. Tyros immobi-lized in the porous and hydrophilic sol–gel matrices wasvery stable and retained its functional activity to a largeextent[162].

3.2.2. Agaricus bisporusA. bisporusTyros was immobilized on a redox polymer-

modified electrode which was prepared using PEG as a linkerto the surface of a GCE, and allowing the subsequent hy-drogel to dry for at least 48 h. The electron donor mediatorsfor these systems were Os[bpy)2Cl2](Osbpy) and OsMeImcomplexes. The immobilized sensor was utilized for the de-tection of modulators/inhibitors of enzyme activity, such asthe respiratory poison azide[135].

3.3. Polyphenoloxidases (PPO)

Table 3reports all examples that refer to papers dealingwith the preparation and characterization of immobilizedPPO. 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 chronologicalorder.

3.3.1. MushroomPPO from mushroom was immobilized by precipitation

onto glass beads. It was shown that the enzymatic oxida-tion of phenols performed in water resulted in negligibleyields of quinones, due to rapid enzyme inactivation, whilea quantitative conversion was achieved in chloroform. Thequinones thus produced were then non-enzymatically re-duced to catechols[144].

PPO was immobilized on cellulose and sepharoseCL-6B using p-nitrophenylchloroformate (p-NPCF) andp-toluensulphonyl chloride (p-TSC) [82]. The immobiliza-tion yields were in the following order:p-NPCF-activatedsepharose> p-TSC-activated sepharose> p-TSC-activatedcellulose> p-NPCF-activated cellulose. Various propertiesof the extracted enzyme and its immobilized forms, suchas optimum pH,Km, thermal stability and storage stabil-ity, were studied and compared. Operating properties ofimmobilized enzyme were studied using different reactorconfigurations. Among them, a packed-bed column reactorshowed the highest conversion rates[82].

HCl-treated glass beads, zeolite and sepiolite were usedas supports for PPO adsorption and some parameters of im-mobilized PPO-catalyzed reactions in organic solvents werestudied[84]. No inactivation phenomena occurred within thefirst minutes of the reaction as it was evident for aqueous me-dia, probably due to the transfer of the quinone product to theorganic phase. Glass beads were shown to be the best immo-bilizing support[84]. Immobilized PPO showed an optimumtemperature at 30◦C and an optimum pH at 7.0. TheKm andVmax for o-cresol were 1.22 mM and 45 nmol min−1 mg−1

protein, respectively, whereas, for phenolKm andVmax were5.42 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 activitydepended 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 bioreactorto transform several phenols present in synthetic and in-dustrial effluents[142]. The immobilized enzyme-reactorwas effective in converting phenols and associated deriva-tives, but it producedo-quinones and low molecular weightpolymers in the treated effluents. To improve the quinoneremoval effectiveness, PPO was immobilized by cross-flowcirculation on the shell (outer) side of the membrane pre-viously coated with chitosan[151]. The chitosan-coatedmembrane gave rise to high phenols removal associatedwith effluent decolorization. In addition, the presence ofchitosan considerably decreased the occurrence of productinhibition 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

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oil, w/w) [152]. The electropolymerized PPO film electrodewas 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 temperatureoptimum of the immobilized preparation was shifted to50◦C with respect to the free enzyme (40◦C). Storage sta-bility of immobilized PPO was considerably improved withrespect to the free enzyme.

3.3.2. A. bisporusThe purified PPO was immobilized by several proce-

dures including physical entrapment, cross-linking withBSA-GLUTAL, covalent immobilization on PALL BiodyneA and B membranes, and covalent immobilization on Nylonnet. Among the tested methods, covalent immobilization onNylon net provided the most stable (life-time 30 days) andsensitive biosensor for catechol and phenol[106].

The PPO immobilization process on�-alumina involved adisulfide 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 itsinitial activity after 3 months storage at 4◦C and exhibitedquite similarKm values with free enzyme (2.8 × 10−4 and4.3 × 10−4 M, respectively) suggesting that the active siteof PPO was not affected during the immobilization process[134].

In other studies, PPO was immobilized either on Nylonmembrane or on a polyethersulfone capillary membrane[136] resulting in 80 and 32% activity yield, respectively.The intermediate product of PPO reaction withl-dopa,4-methylcatechol, was isolated and identified when the reac-tion was conducted with the former immobilized derivative.In contrast, only theo-quinone as the final reaction productwas detected with the latter one[136]. The use of chitosanto treat the output stream from the capillary membranebioreactor facilitated the removal of the colored quinoneproducts from the permeate as well as the recycling of thesubstrate solution[142,151]. A similar approach was usedwith PPO fromN. crassa[137].

A bioprobe based on the concomitant use of PPO im-mobilized on circular Nylon membranes and the compound3-methyl-2-benzothiazolinone hydrazone (MBTH) was pro-posed to detect phenols[146]. The bioprobe fully retained itsactivity after 1 month storage at room temperature. The ap-preciable sensitivity of the bioprobe along with its stabilitycould suggest the feasibility of its commercialization[146].

3.3.3. Coriolus spp.PPO was isolated fromCoriolus spp. liquid cultures and

immobilized on polyvinyl alcohol fibres. The immobilizedderivative was successfully used for purification of wastew-ater from hydrolysis yeast industry. In fact, the treatmentreduced the lignohumic acid content and color index of the

wastewater by over 70 and 81–82%, respectively, and theimmobilized biocatalyst retained its activity after 15 consec-utive treatment cycles[75].

3.3.4. Pleurotus ostreatusA 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 activecarbon. The last step was also replaced by oxidation withozone or ClO2. The process was successful and the PPOfilter was not blocked by the reaction products[87].

3.3.5. Miscellaneous and non-specifiedDifferent procedures were employed to immobilize

acetone powders of mango peel (Mangifera indica) andtea leaves (Camellia sinesis) [58]. In particular, theseenzyme preparations were immobilized by entrapment(polyacrylamide, calcium alginate and collagen), covalentcoupling (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 PPOfrom mango and tea leaves, respectively. Immobilizationled to a significant increase in the activity of tea leaf PPOtowards catechol and epicatechol. Immobilized PPO fromtea leaves obtained either by entrapment within polyacry-lamide gel or by covalent coupling with collagen reachedtheir half-life after 80–90 and 60–70 consecutive oxidativecycles, respectively. PPO immobilized on DEAE-sephadexand on polyacrylamide gel exhibited the highest storagestability at 4◦C, retaining 85–90 and 80% of their initialactivity after 6 months[58].

Mash from yeast hydrolysis was treated at pH 4.5–6.0 at30◦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 andsoluble PPO, respectively[60].

In humic preparations, extracted by buffer from thehumus-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 thePPO bound were cleaved. These findings suggest that suchhumic–enzyme complexes are stabilized both by weak (e.g.electrostatic bonds) and covalent interactions[83].

A 96% of activity yield was obtained when immobilizingPPO from potatoes by adsorption onto chitin. Though, nei-ther thermal stability nor kinetic constants were significantlyaffected with respect to the free enzyme[97]. Similar ki-netic and stability (pH, temperature) properties for free andimmobilized enzyme were also observed when PPO fromthe same source was immobilized by adsorption on EudragitS100 (Röhm Pharma), chitin and chitosan[126]. Regard-less of the support, PPO immobilized by adsorption showedan enhancement of activity in organic co-solvent mixtureswhen the concentration of the organic solvent was as low as

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10–20% (v/v). It was suggested that enzyme immobilizationby adsorption on a suitable matrix could be a generally bet-ter approach than covalent immobilization for a subsequentuse 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 monthsstorage at 4◦C [123]. The membrane, placed onto the tipof the oxygen electrode, was used as a biosensor, provid-ing a linear response for catechol, pyrogallol, phenol andp-cresol. The life-time of this electrode was excellent for 15days (over 220 determination for each enzymic membrane).When analyzing an effluent from a tanning industry, thebiosensor provided highly correlated quantitative data withthose obtained by a spectrophotometric method, suggestingits suitability for the determination of phenols in industrialwastewater[123].

The immobilization of PPO by ionotropic reticulationin calcium alginate in the presence of GLUTAL for pre-cross-linking or re-cross-linking proved to be an effectivemethod yielding a highly active and stable derivative[129].

Tissues from fruits of this palm tree present remarkablyhigh PPO activity. The incorporation of plant tissues in car-bon paste matrices represents an alternative approach to thepreparation of biosensors. Paste electrodes preparation bymixing dried tissues, graphite powder and mineral oil wereused for batch experiments and also associated with FIA.The good sensitivity and stability of the sensor suggestedits 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 fromN.tabacumleaves was immobilized by cross-linking on a Ny-lon membrane. The thermotolerance of immobilized PPOwas markedly improved while pH stability was enhanced,especially in the alkaline pH range[147].

3.4. Phenoloxidase (PO)

Table 4reports all examples that refer to papers dealingwith the preparation and characterization of immobilizedPO. As specified in the previous paragraph, the papers aregrouped by the originating source and presented in a chrono-logical order.

3.4.1. Coriolus hirsutusPO from C. hirsutus was immobilized by ion ex-

change and covalent coupling using polvinyl alcohol,polycaproamide and cellulose-regenerated fibres[62]. Thedependence of immobilization yield on several parametersincluding enzyme loading, contact time, pH, and tempera-ture was investigated with reference to the different matricesemployed[62].

3.4.2. Pleurotus ostreatusPO 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 freeform [99,102]. Utilization of the enzyme for the removal oftoxic 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 otherdivalent cations. When determining the pH-activity profile,immobilized PO showed a shift in the optimum towards lessacidic regions. TheKm andVmax values for the immobilizedPO (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 withAPTES by using GLUTAL [115]. It was found that ashort-time incubation of PO with proteases (papain) causeda significant increase in PO activity (up to 240–250%).Conversely, a longer incubation time of the enzyme withthe protease caused a decrease in PO activity. In any case,immobilization improved the stability to proteolysis. Simi-lar results with PO fromT. versicolorandP. radiata wereobtained[115].

3.4.3. Mycelia steriliaPO from the fungusM. sterilia was immobilized using

GLUTAL on different macroporous silica carriers. The en-zyme immobilized on both amino-silochrome SKh-2 andaminopropyl-silachrome 350/80 exhibited maximum activ-ity. Soluble and immobilized PO were compared for theircatalytic properties. Immobilization considerably increasedthe PO stability. Both soluble and immobilized forms of POcatalyzed the oxidative conversion of phenolic compoundsof the green tea extract[161].

3.4.4. Non-specifiedPO from apples or from potatoes immobilized by adsorp-

tion on carbon black removed pyrocatechin from aqueouseffluent from coal and petroleum processing plants. Thisprocess can be carried out continuously or batchwisely[59].An apparatus for the removal of dissolved oxygen from beerwith immobilized PO was recently described[114].

4. Final remarks

This review summarizes and testifies research efforts thathave been dedicated to immobilize Lac and Tyros and renderfeasible their use in a variety of applications. They includesynthetic and analytical purposes, bioremediation of con-taminated soils, wastewater treatment, and must and winestabilization. Among them, wastewater treatment, and wineand beverages stabilization appear to be very promising and,with regard to this aspect, it is worthwhile to make someconsiderations.

The life-time as well as the efficiency of immobilizedcopper oxidases, regardless of the reactor configuration,

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could be negatively affected by two concurrent phenom-ena, i.e. the generation of chromophoric products and theinsolubilization of laccase reaction products. With respectto the first phenomenon, the adsorption of choromophoricoxidized products on the surface of the immobilizationsupport often leads to enzyme inactivation phenomena.This effect, when reversible, can be partially removed byusing washing procedures with high ionic strength bufferedsolutions, 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 ofLac- and Tyros-based reactors. In fact, a prolonged andrepeated use of an immobilized laccase, whether it be ina packed-bed, a fluidized bed or in a membrane reactor,could result in the accumulation of precipitate on the outletfilter 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 withthe fate of laccase-generated free radicals and quinones. Inthis regard, a particularly promising approach is to combinethe use of immobilized laccase with cationic polymers,such as chitosan, polyethyleneimine and hexamethylene-diamine cross-linked with epichlorohydrin, which are ableto promote the coagulation of oxidized reaction products[110]. In particular, the outlet stream of the reactor couldbe subjected to a flocculation step or, alternatively, could bedirected to a column containing the aforementioned poly-mers. In both cases, this combined approach could exertseveral positive effects such as (i) decolorization of the ef-fluent stream, (ii) possibility to perform effluent recyclinginside the laccase reactor to improve removal yields of thetarget pollutants, (iii) improved detoxification due to the re-moval of quinonoid products whose toxicity is often higherthan that of the parent compounds.

The development of Lac- and Tyros-based biosensors tomonitor a wide range of compounds appears to be at a ma-ture stage of technology. In fact, several biosensor systemsbased on copper oxidases allowed a high selectivity and sen-sitivity with reduced assay times and showed a good stabilityover time. The set-up of reagentless enzyme electrodes hasextended the range of detected compounds to activators orinhibitors of enzyme activity, such as the respiratory poisonazide.

Acknowledgments

Support from FAPESP and CNPq (Brazil) is acknowl-edged. For any request for ordering and buying of reprintsto Università di Napoli Federico II, Portici (Napoli, Italy)indicates: DiSSPA Contribution No. 0011.

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