Laccases for removal of recalcitrant and emerging pollutants
Introduction
Laccases (1.10.3.2, p-diphenol: dioxygen oxidoreductases), along with manganese peroxidase and lignin peroxidase, are a type of lignin-modifying enzyme (LME). Laccases have a wide substrate range, which can serve industrial purposes and/or bioremediation processes. The simple requirements of laccase catalysis (presence of substrate and O2), as well as its apparent stability and lack of inhibition (as has been observed with H2O2 for peroxidase), make this enzyme both suitable and attractive for biotechnological applications.
Laccases are monomeric, dimeric or tetrameric glycoproteins with four copper atoms (belonging to three types: 1, 2 or 3) per monomer located at the catalytic site. Type 1 (T1) copper is responsible for the oxidation of the substrate and imparts the blue color to the enzyme. Laccases use molecular oxygen to oxidize a variety of aromatic and non-aromatic hydrogen donors via a mechanism involving radicals. These radicals can undergo further laccase-catalyzed reactions and/or nonenzymatic reactions such as polymerization, hydration or hydrogen abstraction. For phenolic substrates, oxidation by laccase results in formation of an aryloxyradical, an active species that is converted to a quinone in the second stage of the oxidation. Quinone intermediates can spontaneously react with each other to form soluble or insoluble colored oligomers, depending on substrate and environmental parameters (Walker, 1988). Laccase can decarboxylate phenolic and methoxyphenolic acids (Agematu et al., 1993), and also attacks methoxyl groups through demethylation (Leonowicz et al., 1984). Dehalogenation of substituents located in the ortho or para position may also occur in the case of substituted compounds (Schultz et al., 2001).
Several reviews of the characteristics and uses of laccase have been published during the last decade. Among other features, these articles describe the ligninolytic activity of laccase in lignocellulose degradation (Leonowicz et al., 2001), the role of laccase in vivo (Mayer and Staples, 2002), their physico-chemical properties (Solomon et al., 1996, Solomon et al., 2001), and laccase inhibitors present in polluted environments (Rodriguez Couto and Toca, 2006), thus providing a wealth of information useful to the future industrial application of laccases. Baldrian (2006) describes the substrates, inhibitors and properties of purified fungal laccases characterized to date. Lignocellulosic agricultural residues used as substrates for enzyme production were summarized in another review (Ikehata et al., 2004). The biotechnological, chemical and industrial applications of laccase have been reviewed by several researchers (Mayer and Staples, 2002, Riva, 2006, Rodriguez Couto and Herrera, 2006). Immobilization of laccase for various applications was discussed extensively by Duran et al. (2002). However, waste treatment using oxidative enzymes was reviewed with no particular emphasis on laccase (Duran and Esposito, 2000, Karam and Nicell, 1997, Whiteley and Lee, 2006), and few reviews have focused on laccase for bioremediation of soil and wastewater.
Although new microorganisms producing higher amounts of laccase continue to be discovered, the most important issue precluding their practical use remains the high cost of enzyme production. Unlike nanotechnology or biosensor uses, large amounts of laccase enzyme are required for bioremediation and/or industrial applications. The cost of the enzyme could be reduced through the use of zero or negative cost substrates, such as tertiary matter, agricultural and food wastes or wastewater from the food or pulp and paper industries. Furthermore, most studies on laccase production have been conducted on a small scale in shake flasks. These processes need to be optimized and tested for production in large-scale bioreactors for industrial applications.
Until now, information on the production of laccases and parameters affecting laccase production has been, at best, sketchy. In this context, this article provides an in-depth review of laccase production, with a focus on economical production for removal of specific pollutants from wastewater.
Section snippets
Characteristics of laccases
Laccases characterized so far belong mostly to the group of wood-rotting white-rot basidiomycetes (Baldrian, 2006). Stability of laccases is usually higher at acidic pH, but optimal temperature activity and temperature stability vary considerably among enzymes from different sources. Median values of isoelectric point (pI), optimum pH, optimum temperature and molecular weight are 3.9, 3.0, 55 °C and 66 kDa, respectively. Despite numerous reports on laccases in the literature, their biochemical
Laccase substrates
Laccase can oxidize a wide range of molecules, some overlapping with substrates for monophenol mono-oxygenase tyrosinase. So far, about hundred different compounds have been identified as substrates for fungal laccase (Baldrian, 2006) and the list continues to grow. With knowledge of the X-ray structure of a specific enzyme, it is possible to use software tools to screen potential substrates for this enzyme. By using protein–ligand docking tools, Suresh et al. (2008) were able to distinguish
Mediators
The standard redox potential range for laccase activity is usually between 0.5–0.8 V, i.e., not high enough for oxidation of several xenobiotic compounds. The discovery of “mediators” – small molecules that can extend the enzymatic reactivity of laccase towards several “uncommon” substrates – stimulated interest in laccases for detoxification and industrial purposes (Bourbonnais and Paice, 1990, Call and Mucke, 1997). Mediators are easily oxidizable substrates that can act as redox intermediates
Oxidation mechanism
The redox potential of the T1 copper site is directly responsible for the catalytic capacity of the enzyme. The mechanism of interaction between a laccase T1 site and its substrate seems to be identical among fungal laccases (Smirnov et al., 2001). Nevertheless, important differences between laccase active sites have been described (Hakulinen et al., 2002, Xu, 1996). In its native state, the enzyme holds copper atoms in the monovalent state as Cu+ (Fig. 2). When dioxygen binds at the trinuclear
Sources of laccase
The first mention of this enzyme appeared near the end of the 19th century (Bertrand, 1896, Yoshida, 1883), but laccase has attracted significant attention only in the last few decades, when it came to be associated with the enzymatic arsenal secreted by white-rot fungi for wood degradation. Fungal laccases are the most studied group, with over 115 enzymes characterized so far (Baldrian, 2006). Their presence has been reported in most fungi studied, but many species, for example Phanerochaete
Laccase production by fungi
Almost all fungi studied to date possess intracellular laccase; nevertheless, extracellular laccases are produced in higher amounts, which is a prerequisite for industrial purposes. Laccase production by fungi is influenced by many parameters: species, type of cultivation, agitation (stationary or agitated culture mode), aeration and cultivation time. However, the most critical factors are the glucose and nitrogen sources, their concentration and the ratio between them, and the nature and
Production using waste material
In order to considerably reduce the cost of production, utilization of wastes from agriculture, food processing industries, and pulp and paper mills as substrates or media for laccase-producing strains has created general interest. Songulashvili et al. (2006) tested unusual food industry waste (chicken feathers, residue of ethanol production from wheat grains or mandarin peel) and obtained the highest level of laccase activity (34,000 U/l) ever reported by employing waste substrate with G.
Removal of recalcitrant compounds
Phenolic compounds in wastewater originate from industrial processes such as petroleum refining, coking and coal conversion, chemical plants, foundries, and pulp and paper plants. Black liquor from the pulp and paper industry, and stripped gas from coal plants contain lignin and its derivatives and are highly toxic. Successful bioremediation of phenolic wastewater relies on many factors, including fungal growth, growth medium composition, culture age and activity, laccase production and time of
Enzyme stability and inactivation
Although laccases are considered relatively stable enzymes, prevention of inactivation under industrial conditions remains a priority. Besides the temperature and pH of the medium, other factors may also lower laccase activity. The laccase reaction is usually performed in organic solvents as many toxic compounds of interest (e.g., PAHs, phenol, organophosphorus pesticides) are hydrophobic. Although many laccases remain stable in organic media, denaturation also occurs, in addition to changes in
Purification
Purification procedures for fungal laccases depend on species, laccase properties and the purity level required for subsequent applications. Successive column chromatography procedures are commonly used as purification techniques, for example, a combination of high pressure liquid chromatography (HPLC) and gel filtration (Rebrikov et al., 2006), or a combination of anion-exchange chromatography and gel filtration (Pozdnyakova et al., 2006b). In the beginning, or in between steps, samples are
Immobilization
Free enzymes are non-reusable and sensitive to denaturing agents. Immobilization of laccases can protect them from denaturation by organic co-solvents, increase their stability in general (D’Annibale et al., 2000), facilitate the separation of reaction products (Duran et al., 2002), and maintain good catalytic efficiency over many reaction cycles (Brandi et al., 2006, Palmieri et al., 2005). Immobilized laccases have proven effective for phenol removal in both synthetic and industrial
Heterologous expression and genetic amelioration
Heterologous expression is a common and efficient industrial process for producing appreciable amounts of a protein of interest. Recombinant laccases from diverse fungi have been expressed in yeast and in filamentous ascomycetes such as Aspergillus strains. To date, successful overexpression of recombinant laccase in an active form has not been reported (Galhaup et al., 2002, Tellez-Jurado et al., 2006), although it is possible that effective transformation may have been achieved but has been
Conclusions and future directions
Biotransformation of recalcitrant pollutants using laccase holds huge potential for the economical treatment of wastewater containing phenolic compounds, PAH, synthetic dyes, chemical pesticides and other emerging pollutants. The state of knowledge of parameters controlling laccase production in bacterial and fungal strains is still contradictory and incomplete. However, different approaches to achieving high productivity have been described in the literature. Moreover, systematic studies using
Acknowledgements
This study was financed by the Natural Sciences and Engineering Research Council of Canada (Grants A4984, STP235071, Canada Research Chair) and INRS-ETE. The authors are sincerely thankful to Helen Rothnie and Simon Barnabé (CRPP-UQTR) for their assistance in correction of this paper. The views or opinions expressed in this article are those of the authors and should not be construed as opinions of the US Environmental Protection Agency.
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