Malachite green decolourization and detoxification by the laccase from a newly isolated strain of Trametes sp.

Abstract

The decolourization and detoxification of the triarylmethane dye Malachite green (MG) by laccase from Trametes sp. were investigated. The laccase decolorized efficiently the dye down to 97% of 50 mg L⁻¹ initial concentration of MG when only 0.1 U mL⁻¹ of laccase was used in the reaction mixture. The effects of different physicochemical parameters were tested and optimal decolourization rates occurred at pH 6 and at temperatures between 50 and 60 °C. Decolourization of MG occurred in the presence of metal ions which could be found in textile industry effluent. 1-hydroxybenzotriazole (HBT) affected positively the decolourization of MG. The presence of some phenolic compounds namely ferulic, coumaric, gallic, and tannic acids was found to be inhibiting for the decolourization at a concentration of 10 mM.

The effect of laccase inhibitors in the decolourization of MG was tested with l-cysteine, and ethylene diamine tetra-acetic acid (EDTA) at concentrations of 0.1, 1 and 10 mM. It was demonstrated that l-cysteine and EDTA inhibited the decolourization starting from 1 mM concentration. However, for NaCl a concentration of 100 mM was needed for the inhibition of laccase. The decolourization of MG resulted in the removal of its toxicity against Phanerochaete chrysosporium.

The stability of the laccase toward temperature and HBT free radicals was also assessed during MG decolourization. It was shown that laccase was stable at 50 °C but in the presence of the laccase mediator HBT, the stability of the enzyme was severely affected resulting in a loss of 50% of the activity after 3 h incubation.

Introduction

Approximately 10,000 different dyes and pigments are produced annually worldwide and used extensively in the dye and printing industries. Several of these dyes are very stable to light, temperature and microbial attack; many are also toxic. Synthetic dyes are chemically diverse, with those commonly used in industry divided into azo, heterocyclic/polymeric structures or triphenylmethanes (Gregory, 1993).

The triphenylmethane dye malachite green (MG) is extensively used as a biocide in aquaculture worldwide. It is highly effective against important protozoan and fungal infections of farmed fish (Hoffman and Meyer, 1974, Alderman, 1985). Aquaculture industries have used malachite green extensively as a topical treatment in bath or flush methods, despite the potential for topically applied therapeutic agents to be absorbed and produce significant internal effects. Malachite green is also used as a food colouring agent, food additive, and a medical disinfectant as well as a dye in the silk, wool, jute, leather, cotton, paper and acrylic industries (Eichlerova et al., 2005). The compound has now become highly controversial, however, due to the risks it poses to consumers of treated fish (Alderman and Clifton-Hadley, 1993) including its effects on the immune system and its genotoxic carcinogenic properties (Rao, 1995). Approximately 10–14% of the total dye used in the dying process may be present in wastewater, causing serious pollution problems (Vaidya and Datye, 1982). Despite the existence of a variety of chemical and physical treatment processes, removal of the dye residues from the environment is very difficult. A number of studies have focused on microorganisms capable of decolorizing and biodegrading these dyes (Wesenberg et al., 2003). In recent years, the possible utilization of the biodegradative abilities of some white rot fungi has shown some promise. These fungi do not require preconditioning to particular pollutants and, producing non-specific extracellular free radical-based enzymatic systems, they can degrade to non detectable levels or even completely eliminate a variety of xenobiotics, including synthetic dyes.

Many white rot fungi (e.g. Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor) have been intensively studied in relation to lignolytic enzyme production and ability to decolorize complex dyes (Bumpus and Brock, 1988; Borchert and Libra, 2001, Moldes et al., 2003). This biodegradation capacity is assumed to result from the activities of numerous lignolytic and non-specific enzymes secreted by these fungi, including lignin peroxidases (EC.1.11.1.14), manganese peroxidases (EC.1.11.1.13) and laccases, of which laccases (EC 1.10.3.2) are the preferred target enzymes (Kirk and Farrell, 1987). Laccases are used in various biotechnological and environmental applications (Riva, 2006), including the removal of toxic compounds from polluted effluents through oxidative enzymatic coupling and precipitation of contaminants (Zille et al., 2005), or as biosensors for phenolic compounds (Torrecilla et al., 2008). Laccases have been extensively used in delignification, demethylation, and bleaching of wood pulp (Bajpai, 2004, Bourbonnais et al., 1997, Camarero et al., 2007). The capacity of laccases to act on chromophore compounds has lead to applications in industrial decolourisation processes (Champagne and Ramsay, 2007, Svobodova et al., 2008). The oxidation of a reducing substrate by laccase typically involves formation of a free (cation) radical after the transfer of a single electron to laccase. The efficiency of this oxidative process depends on differences in the redox potential between the reducing substrate and type 1 Cu in laccase. Due to its rather low redox potential (0.5–0.8 V), laccase is able to attack only the phenolic moieties in the lignin polymer, thus being less efficient than lignin peroxidases and manganese-dependent lignin peroxidases in delignification and bleaching of pulp. As wood lignin macromolecules are composed of phenolic (10–20%) and non-phenolic (80–90%) moieties, the cleavage of non-phenolic linkages is a necessary condition for lignin degradation. The substrate range of laccases can be expanded to include these non-phenolic compounds in the presence of small molecular weight mediators that are easily oxidized by the enzyme and in turn oxidize other substrates with redox potentials higher than laccase or are of inappropriate size to fit the active centre of the enzyme. The advantage of the mediators, apart from acting as electron shuttles between the enzyme and the substrates, is that they may follow an oxidation pathway different from that of the enzyme. Recent studies showed that laccase-mediator systems are able to oxidize non-phenolic lignin and even xenobiotic compounds (Morozova et al., 2007).

The aim of the present work was to examine the ability of crude laccase preparations from Trametes sp. to decolorize and detoxify malachite green in the presence of a mediator and to investigate the kinetics of this process. The stability of the enzyme toward temperature and HBT radical was also investigated.