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3 Laccase: The Blue Copper Oxidase
Deepa Thomas and A.K. Gangawane
Faculty of Applied Sciences, Parul University, Vadodara, Gujarat, India
3.1 What Is Laccase?
Laccase is an important oxidase enzyme, which is widely used industry. The multiple potential applications of laccase in these industries define this particular enzyme as a promising alternative to existing costly and polluting processes [1–4].
Laccase is one of a small group of enzymes called the large blue copper proteins or blue copper oxidases that have been the subject of study since the end of the last century. The blue oxidases have been intensively studied because they have the ability to reduce molecular oxygen to water [5, 6]. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) contain 15–30% carbohydrates and have a molecular mass of 60–90 kDa. These are glycosylated polyphenol oxidases that contain four copper ions per molecule that carry out one electron oxidation of phenolic and its related compound and reduce oxygen to water [4]. Laccases oxidize a surprisingly wide variety of organic and inorganic compounds, including diphenols, polyphenols, substituted phenols, diamines, and aromatic amines, with concomitant reduction of molecular oxygen to water [7]. Laccases exhibit a broad substrate range, which varies from one laccase to another. Although it is known that diphenol oxidase, monophenols like 2, 6‐dimethoxy phenol or guaiacol may also serve as substrates. Laccases catalyze monoelectronic oxidation of molecules to corresponding reactive radicals with the help of four copper atoms, which form the main catalytic core of the laccase, accompanied with the diminution of oxygen to water molecules and simultaneous oxidation of substrate to produce radicals (Figure 3.1). All substrates cannot be directly oxidized by laccases, either because of their large size, which restricts their penetration into the enzyme active site, or because of their particular high redox potential. To overcome this hindrance, suitable chemical mediators are used, which are oxidized by the laccase and their oxidized forms are then able to interact with high redox potential substrate targets [8]
Figure 3.1 Oxidation of substrate with laccase enzyme.
3.2 Distribution of Laccases
Laccases are widely distributed in higher plants, bacteria, fungi, and insects. In plants, laccases are found in cabbages, turnip, potatoes, pears, apples, and other vegetables. Laccases have also been present in various Ascomyceteous, Basidiomycetous, and Deuteromycteous fungi. Laccase has been found in the cuticles of many insect species, and is involved in cuticle sclerotization. In several holometabolous insects, laccase has been identified as the principal enzyme associated with cuticular hardening [9]. Plant laccases have not been characterized or used extensively despite their wide occurrence, because their detection and purification is often difficult, as the crude plant extracts contain a large number of oxidative enzymes with broad substrate specificities. The majority of laccases characterized so far have been derived from fungi especially from white‐rot basidiomycetes that are efficient lignin degraders [9]. Fungal laccases have higher redox potential than bacterial or plant laccases (up to +800 mV), and their action seems to be relevant in nature, they are also finding important applications in biotechonology. Thus, fungal laccases are involved in the degradation of lignin or in the removal of potentially toxic phenols arising during lignin degradation. In addition, fungal laccases are hypothesized to take part in the synthesis of dihydroxynaphthalene melanins, darkly pigmented polymers that organisms produce against environmental stress or in fungal morphogenesis by catalyzing the formation of extracellular pigments. Concerning their use in the biotechnology area, fungal laccases have widespread applications, ranging from effluent decoloration and detoxification to pulp bleaching, removal of phenolics from wines, organic synthesis, biosensors, synthesis of complex medical compounds, and dye transfer blocking functions in detergents and washing powders, many of which have been patented. The biotechnological use of laccase has been expanded by the introduction of laccase‐mediator systems, which are able to oxidize nonphenolic compounds that are otherwise barely or not oxidized by the enzyme alone. However large‐scale production of fungus is a still challenging [10]. Further, very little is known about the potential of bacterial laccases for bioremediation applications. Wastewater treatment involving bacteria is, however, considered to be more stable, as bacteria generally tolerate a broader range of habitats and grow faster than fungi. Moreover, in contrast to fungal laccases, some bacterial laccases can be highly active and much more stable at high temperatures, at high pH as well as at high chloride concentrations [11–13].
3.3 Application of Laccase
Laccases play an important role in: the food industry, the paper and pulp industry, the textile industry, soil bioremediation, and biodegradation of environmental phenolic pollutants. These enzymes are used for pulp delignification, pesticide or insecticide degradation, waste detoxification, textile dye transformation, food technological uses, and biosensor and analytical applications (Figure 3.2).
Figure 3.2 Applications of laccase.
Recently laccases have been efficiently applied to nanobiotechnology due to their ability to catalyze electron transfer reactions without additional cofactor. The technique for the immobilization of biomolecule such as layer‐by‐layer, micropatterning, and self‐assembled monolayer technique can be used for preserving the enzymatic activity of laccases [14]. Many micropollutants present in municipal wastewater, such as pharmaceuticals or biocides, are not easily removed in conventional biological treatments, resulting in a constant input into the aquatic environment. As these compounds are designed to be biologically active, they can affect sensitive aquatic organisms even at low concentrations. One potential means to reduce the amounts released to the environment is to improve their biodegradation in a post‐treatment step using microorganisms that produce oxidative enzymes such as laccases. Due to their wide range of substrates and the sole requirement of oxygen as the cosubstrate, laccases appear to be a promising biocatalyst to enhance the biodegradation of micropollutants in wastewater in a complementary treatment step [14].
3.3.1 Laccase in Bioremediation
One of the main environmental problems, faced by the world today, is the contamination of soil, water, and air by toxic chemicals. With the extensive use of pesticides in agriculture and industrialization, the pollution of the environment with mandate organic compounds has become a serious problem. Laccases have many possible