Enzymes are crucial in biotechnological applications due to their multifunctional characteristic. Laccase is an enzyme that is multifunctional and is used in various biotechnological applications.¹

Laccases belong to the multi-copper oxidases enzyme group and are classified as benzenediol oxygen reductases. It is also known as urushiol oxidases and p-diphenol oxidases. They are viewed as versatile proteins fit for oxidizing enormous numbers of particles such as non-phenolic and phenolic compounds because of their broad substrate specificity. Laccases, being a flexible biocatalyst and with eco-friendly enzymatic degradation, produces water as a by-product and utilize molecular oxygen as a concluding acceptor of electrons which results in increased biotechnological interest among the science community. Usually, they are present in insects, plants, and microorganisms including fungi and archaea, along with other oxidative enzymes that degrade lignin. The same is used in a vast range of bioremediation processes for protecting ecosystems from damages incurred through effluents of industries. In recent years, researchers are intensively focusing on laccase, much of it due to the large variety of laccases, their usefulness, and their enzymology, which is very important. ^2-4

This group of enzymes is used for several purposes, including manufacturing of food additives, in the production process of beverages, for medical uses, and as a pretreatment agent in the biofuel production process. They have also been extensively researched for implantable biosensors and biofuel cells in nanobiotechnology. In addition, their ability to convert complex xenobiotics makes them useful enzymatic bioremediation biocatalysts. The broad substrate specificity, high catalytic constants, and the utilization of atmospheric oxygen as the second substrate offer the basis for the application of laccase in the synthesis of organic compounds, biosensors, and immunoassays.⁵ Various biotechnological applications of laccase is shown in Figure 1.

Laccase was first discovered in 1883 in the latex of the lacquer tree Rhus vernicifera. Both Bertrand and Laborde established for the first time in 1896 that laccase was found in fungi. It is found in higher plants, fungi, bacteria, and in various insects, as well as in archaea.

Catalytic Mechanism of Laccase
Laccases are generally known to be monomeric, but also exist in homodimeric, heterodimeric, and multimeric forms. Some laccases, such as fungal laccases, are glycosylated. The enzyme’s carbohydrate moiety will make up 10-45 pounds (86-91%) of the protein molecule by weight. It includes carbohydrates such as hexosamine, glucose, mannose, galactose, fucose, and arabinose. Depending upon the source of the laccases, the molecular masses can differ and, the probable reason for the variation could be the variation in its structure. Laccases are MCOs and are known to have four copper atoms in special oxidation states: one type-1, one type-2, and two type-3s, together which form the catalytic site.⁶

Usually, the laccase spectrum shows the property of absorption at 280 and 605 nm but some laccases do not follow the mentioned range. They belong to the group of multicopper oxidase with varying content of glycosylation. Mostly, laccases occur as monomeric and polymeric glycoproteins.

It catalyzes oxidation of a vast range of compounds integrated with 4 electrons reduction of molecular oxygen to water. The biological functionality for laccases depends upon the sources of origin and life stages of the producing organisms. Laccases can oxidize various aromatic compounds including phenol derivatives, amine derivatives, thiols, and also some inorganic compounds such as iodine and ferrocyanide ions.⁷

The action mechanism of laccases includes two individual sites that bind the reducing substrate and O2 with four catalytic copper atoms. Two individual sites connecting decreasing substrate and oxygen with 4 catalytic copper atoms form the laccase mechanism of action: the paramagnetic type 1 copper (T1Cu) responsible for the distinctive blue color of the protein in the decreased resting state where the oxidation of the substrate takes place; the T2Cu and the two T3Cu clustered 12Å away from the T1Cu. This trinuclear cluster is where O2 is reduced to two water molecules, receiving four consecutive electrons through a strictly conserved His-Cys- His electron transfer route from four separate mono-oxidation reactions at the T1Cu site. X-ray crystallography resolved approximately 90 laccase structures to date.^8,9

The graphical representation of the catalytic mechanism of laccase is shown in Figure 2.

As a novel industrial enzyme, Laccase received huge attention due to its much higher abilities for catalyzing the oxidation of aromatic compounds that worked on the catalytic activity of laccase hosted in reversed micelles.¹⁰ Researchers have successfully heightened the catalytic capacity of the laccase enzyme to degrade the diclofenac through carbon nanotubes.¹¹ Some compounds have been shown to have a capacity to inhibit laccases, such as halides, acetate, and so on.

Laccase production
Laccase is an enzyme released in the extracellular medium by numerous fungi¹²   through secondary metabolism, however, not every fungal species, such as Chytridiomycetes and Zygomycetes produce laccase.¹³ Laccase synthesis by soil and some freshwater Ascomycete species is mentioned in the literature.^14-18

Various sources of laccase and the organism producing it, are listed in Table 1.

Table (1):
Various sources of laccase and the organism producing it. It is found in higher plants, fungi, bacteria, and in various insects, as well as in archaea.

Laccase production has also been discovered in Magnaporthe grisea, Ophiostoma novo-ulmi, Gaeumannomyces graminis, Monocillium indicum, Neurospora crassa, and Podospora anserina, Marginella, Melanocarpus albomyces.¹ Botryosphaeria develops a real laccase-like dimethoxyphenol oxidising enzyme. Laccase genes that oxidise syringaldazine are found in Ascomycetes that contribute to plant biomass decomposition.¹⁹ The Basidiomycete yeast Cryptococcus neoformans generates laccase and oxidises phenols and aminophenols but not tyrosine. Only the Saccharomyces cerevisiae plasma membrane-bound multicopper oxidase has similarity with fungal laccase.

Various well-known species which produce a significant number of laccases in variable quantities are Basidiomycetes and Saprotrophic fungi.²⁰ Laccases produced by Pycnoporus cinnabarinus are known to be a ligninolytic enzymes that can degrade lignin. However, ability of brown-rot fungi for producing laccases is unknown, and no laccases are being purified. Brown-rot Coniophora puteana fungus²¹ has been discovered to oxidise syringaldazine and assist in oxidation of ABTS in Laetiporus sulphureus.²² Laccase production is influenced by several parameters, including cultivation type (submerging or in state of solid), limitation of carbon and source of nitrogen.²³ Various bacteria of various genera are used to produce laccases. Bacillus, Geobacillus, Streptomyces, Rhodococcus, Staphylococcus, Azospirillum, Lysinibacillus, and Aquisalibacillus are examples of Gram-positive bacteria.^24,25 For maximum laccase production, literature suggests that each type of fermentation (submerged and solid) is suitable.²⁴

Types of Cultivation
Laccase is produced primarily via submerged and solid-state fermentation methods. In various cultivation techniques (Table 2), wild-type filamentous fungi are employed to produce laccase on a big scale.

Table (2):
Laccase production by various microorganisms as reported in the literature, the cultivation conditions required for laccase production, inducers used, and the laccase activity detected.

Solid State Fermentation
Solid state fermentation (SSF) is well suited for enzyme production by using natural substrates such as agriculture residue.^26,27 The sugar-rich lignin, cellulose, and hemicellulose enhance the growth of fungus in the fermentor and makes such a process highly cost-effective.²⁸ Many bacteria are common for producing laccases extra-cellularly, on the other hand, many bacteria are not able to secrete laccases outward from the cell.⁵ Solid-state fermentation is shown as a cost-effective method to produce laccase by bacteria. The nearby absence or absence of water that is free-flowing, causes the process to occur.⁴ Solid state fermentation requires a moisture content of roughly 15 percent. Sawdust, wheat bran, cereal grains, wood shavings, and a variety of other plants and animal products are the most often utilized solid substrates for SSF.²⁹ Conditions in which microorganisms are cultivated under SSF are comparable to those in their native habitat. The bioreactor design has a severe flaw in that heat and mass transport are narrower. Various bioreactor configurations for laccase production are investigated, including immersion, extended bed, tray, inert (nylon), and non-inert (barley bran) support, with the design of tray yielding the good results.³¹ Grape seed and orange peels are used as substrates in a comparison of tray and immersion laccase production.³² Rice bran has a higher laccase production rate during both solid state and submerged fermentation than other substrates. Inducers are found to promote laccase production. The addition of veratryl alcohol or MnO2 to the cultures seemed to induce laccase activity production. Cycloheximide is also known to induce laccase biosynthesis. Based on phenolic compounds such as vanillic acid and ferulic acid, rice bran has the ability to induce laccase production. A wide variety of agricultural wastes are also used for laccase production, including seeds of grapes, wastewater, barley bran, cotton stalk, and wheat bran. This is because laccase production was not maximized in either solid-state or submerged fermentation.²³

Submerged Fermentation
In submerged fermentation, the microorganism is cultivated in a liquid nutrient medium that contains a high level of oxygen concentration. When it comes to fungal submerged fermentation, the main problem is the viscosity of broth. Because oxygen and mass transfer are limited because of mycelium formation, impeller action is hindered. Diverse strategies have been employed to combat this issue. For maximum efficiency, the bioreactor must run continuously. Trametes versicolor is used to decolorize synthetic dye, and a pulse system was developed for this purpose.³³ Cell immobilisation also solves problems with broth viscosity, mass transfer and oxygen. Because Neurospora crassa is immobilised on membrane, Luke and Burton report that regular laccase production occurs for four months without enzyme deactivation. As part of the bioremediation of pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP) nylon mesh is used to compare free cell cultures of T. versicolor having an immobilised culture of these chemicals.

Multifunctional Biotechnological Applications of Laccase
Due to its multiple signature functions, laccase can be employed as a multifunctional enzyme, relying on the cell type and the conditions under which its isozymes are expressed (intracellular or extracellular). Figure 3 shows the various plausible mechanism for shifting multiple functions of laccases.

As biosensors or bioreporters, multiple oxidoreductases can be added. A miscellaneous and limitless supply of energy sources may be used by enzymatic biofuel cells, which has encouraged scientists to concentrate on small, implantable power devices in living systems.³⁴

Laccase in Food and Bioremediation Technology
Without a costly investment, laccase could improve production, performance, and quality of food products and has the advantage of being a moderate technology. Laccase is one of the most studied enzymes for bioremediation; credited for its ability to decompose phenolic compounds. Laccase helps in the bioremediation of food industry wastewater. Various researchers recorded that laccase has been immobilized on organogel support and was able to extract aqueous suspensions from naturally occurring and xenobiotic aromatic compounds. In a model wastewater solution, with the help of polyethersulphone the laccase was immobilized on it through adsorption which is capable for reducing phenol concentration.³⁵ In food industries, laccase is the choice because of its capability to expedite homo- and hetero- polymerization reactions. It may be used in the stabilization of wine and beer, processing of fruit juices, baking process, improvement of the sensory parameter of food, and gelation of sugar beet pectin.³⁶ Owing to its huge polyphenol contents and dark-blown colors, beer-factory wastewater is a significant environmental concern. The white-rot fungi producer of laccases, Coriolopsis gallica, is used by researchers for degrading its high-tannin-containing wastewater.³⁷

When it comes to the food industry, the major application of laccase is for wine stabilization. While the aroma of wine is caused by alcohol and organic acids, different wine types may be determined through the enrichment of wine with the various compound of phenolic which plays a crucial role in facilitating distinct color and taste to it. To preserve color and flavor in wine, different methods have been used, such as the elimination of polyvinylpolypyrrolidone (PVPP) phenolic groups, alternatively, oxidizers are blocked using sulfur dioxide. Fungal laccase (e.g., derived from Trametes versicolor) is well-suited for polyphenol removal from wines but requires a set of conditions essential for proper functioning, such as medium acid stability with an ideal pH range around 2.5-4.0 and reversible sulfite inhibition.³⁸

Beer polyphenols can be oxidized and removed through the application of laccases (eg. Laccase derived from strains of Aspergillus, Neurospora etc.) ― filtration or other separating processes are preferred for the removal of formed polyphenol complexes. Laccases were also preferred for the removal of O2 from beer at the end of the production process, thus enhancing the storage life of beer.³⁹ Excessive phenolic oxidation has almost always been considered unfavorable for apple and grape juices. By delaying the onset of protein-polyphenol haze formation, laccase has also been used to stabilize fruit juices in several tests.⁴⁰

Various other applications such as sugar beet pectin gelation, improvement of food sensory parameters, and ascorbic acid determination, where fungal laccases were proved to be an extremely potential and propitious enzyme for the food industries.

The xenobiotic may be oxidized by laccase or Laccase-mediator System (LMS), which may be readily taken away through a practical or mechanical process to produce a product that is less toxic or has greater bioavailability. Laccase has been reported to oxidize numerous hazardous compounds alone or through a redox mediator. Laccase enzyme may be employed for detoxifying pollutants ― the toxins are oxidized into free radicals or quinones, as a result, it undergoes polymerization and partial precipitation. In their insoluble shape, the contaminants are less harmful and can be separated from water through physical procedures.⁴¹ To form reactive aryloxy radicals, both laccase and peroxidase oxidize phenolics, which automatically polymerize for forming an insoluble complex. Such complexity may be eliminated with the application of physical methods like precipitations, filtrations, or centrifugations.⁴¹ Laccase from Trametes versicolor can potentially decompose pesticides (insecticides, fungicides, and herbicides). While trinitrotoluene does not serve as a substrate for an oxidative enzyme such as laccases, whereas, partial degradation is possible and it does cause the accumulation of reduction of metabolites such as aminodinitrotoluenes (ADNT), azoxies compound, and diamino nitrotoluenes.⁴²

Laccase in Textile and Paper Technology
Laccases are enzymes that have been applied to bleach textiles, modify cloth surfaces, and synthesize dyes in addition to de-colorize textile effluents. Therefore, various chemicals, energies, and water-consuming textile operations could be replaced by laccase-based processes. Laccase-based processes seem to be an appealing alternative due to their ability for degrading dye of various chemical structures, which also include synthetic dyes which are being used in the industry.^{4, 43}

Different aromatic compounds (which includes phenol and aniline) may also be oxidized by laccase to simultaneously facilitate non- enzymatic homo and hetero-coupling reaction, resulting in color palettes of various useful textile dyes (which include phenoxazine and azo dye).⁴⁴

In the paper pulp industry, laccases are favored over peroxidases (lignin, manganese, and flexible peroxidases) because unlike peroxidases, they utilize O2 instead of H₂O₂ and the co-substrate does not affect their functioning. There have been records of enzymatic bleaching of flax pulps along with laccase of various fungi and redox mediators of pure and synthetic origin.⁴⁴ Various other applications are there including some fungal and bacterial laccase-induced grafting of phenol to flax fiber for the production of paper.^{45, 46, 47}

Laccase in Biosensors and Nanobiotechnology
Enzyme immobilization is useful in biomedicine. Enrico et al. (year?) worked with the production of cheaper carriers of nylons 6 film and nanofiber and immobilization on nylon carriers by laccase, which helped to develop new biocatalytic material for industrial use.⁴⁸

Chauhan et al.⁵ made a laccase (Coriolus Versicolor) immobilized biosensor on a polyethers sulfon membranes at pH value 4.5, which is added to the base electrode of Universal Sensors. Reduction of the oxidation product of several laccase catalyzed polyphenols was performed at a potential for which the polyphenol of interest has been found to react. Catechol reductions have been found to occur at the potential of -200 mV, sometimes referring to the polyphenolic biosensor literature.⁴⁹ researched the production of the catechol biosensor, and laccase enzymes were used from two separate sources, namely tree laccases from Rhus vernicifera and fungal laccases from Coriolus hirsutus. Laccase, through glutaraldehyde coupling, is immobilized on amine termination thiol monolayer on the surface which is golden. Based on sensitiveness, stability, reproducibility, and various electrochemical properties of enzyme electrodes and many monolayers which are investigated, Cystamine has been found much optimal^49,50 worked to make a novel laccases-modified screen-printing electrodes and these bio nanocomposite sensors were optimized in terms of enzyme loading, pH, and applied potential. It was assessed for Bisphenol A (BPA) determination.⁵⁰

A laccase biosensor can be used for electrochemical estimation of the polyphenol index in wines using a laccase biosensor. It was reported to be used for rapid and reliable amperometric estimation of the total content of polyphenolic compounds in wines.⁵¹ By inter-linking with the glutaraldehyde on a glassy carbon electrode, the enzyme was immobilized. To carry out such an estimation, caffeic and gallic acid were chosen as a compound that is standard.

Laccase in Biofuel and other Applications
Laccase plays a major role in the biodegradation of lignin, and its potential uses as an agent in the development of biofuels and as biocatalysts for the removal of growth of yeast inhibitors (majorly phenolics) for the consecutive enzymatic process is therefore being studied. Laccases are the enzyme, the investigation of which is done not for potential use as a pre-treatment agent in developing biofuels, primarily as enzyme delignify, on the other hand, also as a biotechnological method to extract (mainly phenolic) inhibitors from subsequent enzymatic processes.⁵² Researchers developed laccase–glucose oxidase biofuel cells with designs and analysis of biofuel cells consisting of an anode basis on glucose oxidase (Aspergillus niger) and laccases-basis of cathodes (Trametes versicolor) using redox polymers basis of osmium as a mediator of electrons transferring by biocatalysts to graphite electrodes surface.⁵³ Laccases-based biocathode for bio electrocatalytic oxygen reduction and its use of advanced electrode material, like nanoparticle and nanowire is researched for commercial applications.⁵⁴

Laccase is very promising as a biocatalyst in the synthesis of bioactive compounds. Their response requires molecules of oxygen only and as only by-products, releases water. To generate reactive radicals, Laccase catalysis requires abstracting one electron through its substrate. Subsequently, free radical is subjected to homo & hetero-coupling for forming oligomeric, polymeric, dimeric, and cross-coupling products through practical consequences for organic synthesis.⁵⁵

Laccases can be used in the formulation of hair dyes to substitute H₂O₂ as an oxidizing agent. The potential for real phenols for forming products beneficial in hair dyeing is explored by laccase-catalyzed polymerization.⁵⁶ By use of mixtures of two phenolic monomers, 15 phenol test yields colored product after treatment of lacquer, and diversity of color has been achieved. The study shows that for the production of new cosmetic pigments, laccases- catalyze polymerizations of natural phenol are applicable.⁵⁶

Various industrial applications of laccase and its innumerable usages are listed in Table 3.

Table (3):
Laccase industrial applications and their innumerable usages. The wide range of applications is in the industries of food bioremediation, nanotechnology, textile, pulp, and paper industry, dye synthesis and dye decoloration, petroleum chemistry, biosensor, etc.