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A survey on immbilized laccase systems


Enzyme and Microbial Technology 31 (2002) 907931


Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a reviewNelson Durn a,b, , Maria A. Rosa a , Alessandro DAnnibale c , Liliana Gianfreda da

Biological Chemistry Laboratory, Instituto de Quimica, Universidade Estadual de Campinas, C.P. 6154, Campinas CEP 13083-970, S.P., Brazil b Ncleo de Ci ncias Ambientais-NCA, Universidade de Mogi das Cruzes, Mogi das Cruzes, S.P., Brazil e 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 DellAmbiente, Universit 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 efcacy 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 removed 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 basic aspects of immobilized laccase and tyrosinase is still lacking.

Corresponding author. Tel.: +55-19-788-3149; fax: +55-19-3788-3023. E-mail address: (N. Dur n). a

The main purpose of this paper is to present a general picture of the results achieved in this research eld. 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 nal properties of the immobilized catalyst and the features of its action. A very short paragraph on the methods of immobilization with specic 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 categories 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 ndings have been discussed and the references listed in a chronological order. However, to help the reader to nd an enzyme of interest, we have organized the tables in alphabetical order. Finally, as the generic name phenoloxidase (PO) and polyphenoloxidase (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: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 2 1 4 - 4


N. Dur n et al. / Enzyme and Microbial Technology 31 (2002) 907931 a

1.1. Laccase (Lac) Laccase is a cuproprotein belonging to a small group of enzymes denominated blue oxidases [1]. Laccase (E.C., p-benzenediol:oxygen oxidoreductase) is an oxidoreductase able to catalyze the oxidation of various aromatic compounds (particularly phenols) with the concomitant 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 classied in three types, according to specic spectroscopic and functional characteristics [49]. The molecular genetics [10], genetic expression [11], genetic transcription [12] and cloning [13] of laccases have been exhaustively studied. The basic aspects of phenoloxidases have been reviewed, as well [4,1422]. 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 specicity and their catalytic competence varies widely depending on the source. Simple diphenols such as hydroquinone and catechols are good substrate for the majority of laccases, 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 oxidize tyrosine. N-Hydroxybenzotriazol, violuric acid and N-hydroxyacetanilide are three NOH 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, carbohydrate content) and kinetic properties [29,30]. In some fungal 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 detoxication treatment with laccase [31]. The determination of environmental pollutants using immunoassays is a continuously growing area and, within this frame, laccase proved to be an excellent alternative to peroxidase as a bioanalytical tool for monitoring polar pollutants [32].

1.1.1. Laccase active site Laccase contains four copper atoms that have been classied 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 crystallography 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) [3336] has provided insight into the catalytic reduction of oxygen to water. It has been elucidated that the Type 2 copper 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 rst is rate-determining. In this Type 2/3 bridging mode for the rst 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 CysHis linkages [37]. Previous studies [34] reported that 40% of the Type 1 and 3 that readily react with dioxygen correspond to native laccase (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 previously 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 (modied from [33]).

N. Dur n et al. / Enzyme and Microbial Technolo


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