Biocatalytic Versatilities and Biotechnological Prospects of Laccase for a Sustainable Industry

Laccases are multicopper-containing enzymes that have the ability to oxidize a wide variety of substrates with a single electron transfer reaction. These are environmentally benign versatile biocatalysts that have gained great interest in the biotechnological community since they utilize molecular oxygen as the last electron acceptor and only produce water as a byproduct. This family of enzymes has been widely used in a broad variety of applications, ranging from food additives and beverage processing to biological diagnostics and even as crosslinking agents in the furniture construction and manufacture of biofuels. Considering the benefits of enzyme immobilization, there has been a dramatic increase in applying immobilized laccases in recent years. Despite the impressive biotechnological promise, the use of laccases in the real world is still constrained by cost–benefit analysis, particularly in terms of practically large-scale production. The enzyme industry is booming research on laccase production, and use neglects to include the economic impact of the operations. Because of their ability to metabolize complex xenobiotics, they are also useful biocatalysts in enzymatic bioremediation processes, such as wastewater treatment. This study discusses the most important and recent breakthroughs in the biocatalytic attributes, sources, and exploitation of laccases in biotechnology for a sustainable industry.

pollution remediation. As a result of their biodegradability and absence of unwanted side effects, laccases have been highlighted in the scientific literature as potential substitutes to catalyze synthetic chemical processes [4,172,199,249].
The usage of soluble laccases is restricted by enzyme activity loss, which may be caused by their poor constancy and performance under certain operating conditions. Most processes, particularly those on a large scale, are influenced by these considerations. Enzymatic immobilization techniques have been investigated to preserve or increase laccase catalytic capabilities to avoid these limitations. Using immobilization, the biocatalyst may be reused, and the operational stability is improved, lowering the cost of using enzymes in 1 3 biotechnology operations [19,20,158,160,175]. Recently, laccases have been extensively used in food, pulp and paper, forest product, grafting reactions, and organic synthesis. Furthermore, extensive studies have recently concentrated on engineering laccases for usage in various biotechnological and biomedical applications [39].

Structural Organization and Catalytic Properties
Laccases (EC 1. 10.3.2) are multicopper oxidases that are members of the cupredoxin superfamily. These glycoproteins have a catalytic region that contains four copper (Cu) atoms per molecule in their natural state. Copper sites are categorized into three types: Type -1 (one copper, T1), Type-2 (one copper, T2), and Type 3 (two coppers, T3). The spectroscopic and paramagnetic features of the copper sites determine which type they are. When it comes to the initial stage of substrate oxidation, type-1 copper is blue paramagnetic. The trigonal orientation of this site is linked with a cysteine-conserved equatorial molecule and two histidine ligands, as well as an axial ligand that changes depending on the methionine in enzymes of different types. Laccases with a wavelength of around 600 nm are known as blue laccases. In T1, white or yellow laccases are enzymes that lack the typical absorption band at around 600 nm [152]. White laccases include copper ions, iron ions, and two zinc ions, among other elements. The species also display an absorption band at 400 nm, which various authors have discovered in the species Pleurotus ostreatus, Trametes hirsuta, and Myrothecium verrucaria [247]. The yellow laccases are distinguished because they are a variation of the blue laccases and include copper in a changed oxidation state [152]. Type-2 copper is connected to two histidines and one molecule of water, with no evident absorption capabilities [245]. Type-3 absorbs light at 330 nm. Each copper atom in T3 is covalently linked to three histidine molecules in a symmetrical manner, which is joined by a hydroxyl group that preserves the copper atoms' antiferromagnetic coupling [5]. T2 and T3 copper sites constitute a trinuclear center (T2/ T3) at a distance of 12 from T1. Three cupredoxin domains contribute to the structure of common laccases. The T1 site is found in domain 3; the T2/T3 trinuclear center is placed between domains 1 and 3 and includes the copper atom coordination residues. Domain 2 is required for establishing a trinuclear center and includes residues that help substrate binding at the enzyme catalytic region ( [5,237]. The redox potential (E0) of copper T1 is one of the laccases' most critical properties. At the commencement of enzymatic catalysis, this value represents the energy to extract electrons from the reducing substrate [15]. Laccases are characterized as having a high, medium, or low redox potential according to E0 of copper T1. Fungi have high and medium E0 laccases, whereas bacteria and plants have laccases with low redox potential. Laccase has defined redox potentials (Eo) at its Cu sites as an oxidoreductase. Some laccases have a "low" Eo of 0.4-0.5 V for the T1 and T3 Cu (compared to the standard hydrogen electrode), while others have a "high" Eo of 0.7-0.8 V [243].
For both the low-and high-Eo laccase groups, the Eo of the T2 Cu appears to be 0.4 V [205], but a few bacterial and fungal laccases, as well as several enzyme-substrate complexes, have had their high-resolution crystallographic structures identified [29,68,78,90,176]. The backbone polypeptide chain folds into three different domains in most cases. The structural knowledge acquired might help us better comprehend and create laccases when combined with protein engineering research [35,166].

Novel Laccase Catalytic Systems and Genome Sequencing
When purified, some of the isolated laccases-whose sequences are quite similar to those of ordinary multi-Cu oxidases-appear "yellow" or "white," denoting the absence of the characteristic "blue" T1Cu site. The site may be altered by oxidized substrates, as suggested for the "yellow" laccases from Pleurotus ostreatus or Panus tigrinus [180], or it may include other metal ions (such as Zn 2+ or Fe 3+ ), as suggested for the "white" laccase from P. ostreatus [167,169]. A "yellow" or "white" laccase frequently has a better pH or temperature profile than its "blue" counterpart and may oxidize non-phenolic substances without the need for a mediator. A dimeric (43 kDa subunit) Tricholoma giganteum (mushroom) oxidase that exhibits anti-HIV-1 reverse transcriptase activity was recently described as a laccase analog, as was a dimeric (40 kDa subunit) Tellima grandiflora (plant) oxidase and an 85 kDa Nephotettix cincticeps (insect) oxidase. However, the N terminus of these oxidases is either unknown or not like the N-terminus of conventional laccases. Therefore, more structural and enzymological data are required to establish if these oxidases are members of the laccase (or multi-Cu oxidases) family.
The growing number of genomes that have been sequenced and the genetic information that has been made public are driving the identification of new laccases. Due to the vast amount of data, technologies for computer analysis to connect sequences have been developed with biological performance. The same technologies have also been used to analyze the traditional laccases. There were over 100 laccase sequences from fungi and plants to find laccase signature sequences; sequence alignment was used to examine the data [224]. More than 350 multi-Cu oxidases were included in a phylogenetic study cited in different research [98,99]. Fungal laccases, the biggest group of enzymes in the later research, clearly split into two groups of sequences: sequences from ascomycetes and basidiomycetes.
Four conserved Cu-oxidase consensus patterns were used to select the sequences for the investigation, which also contained a number of bacterial protein sequences. Compared to all the eukaryotic proteins, these bacterial sequences formed their own distinct cluster. A few bacterial genomes were also discarded because they could not be aligned in the phylogenetic analysis without losing resolution. The example highlights how challenging it is to categorize the bacterial laccase sequences that have evolved among the multicopper oxidases in recent years. Multiple protein alignments and professional-HMMs of these are contained in the Pfam database, which is a collection of protein families and domains [25]. The multicopper oxidases are a clan of seven Pfam members in Pfam. At least three members of this clan are represented in the standard fungal laccases. Several novel bacterial laccases' domain organization is entirely different from this model. The addition of bacterial members to the laccase enzyme class gives both tremendous potential and a challenge to the field ( [7,41,58]. Azospirillum, Bacillus, Escherichia, Marinomonas, Pseudomonas, Streptomyces, Thermus, and Xanthomonas genera have bacterial laccase-like enzymes. Despite having a signal-like sequence in many bacterial laccase genes, little is known about the cellular location and biological function of these enzymes. Some genes have a Tat-signal sequence that marks the gene product for translocation via the Tat-pathway, such as the laccases from Streptomyces coelicolor and Thermus thermophilus [126,145]. When overexpressed homologously, S. coelicolor laccase activity has been found in the media. Other times, laccase activity is associated with the synthesis of pigment that provides UV radiation protection on the surface of spores, such as those of Bacillus subtilis [68].
In conclusion, the subgroup of the laccase enzyme class made up of bacterial laccases is extremely varied. Diverse physical characteristics of the enzymes, such as molecular weight, pI, and Eo, are naturally reflective of the variety at the amino acid sequence level. The bacterial laccases are more temperature stable than their fungal counterparts. Additionally, they often have a good action above neutral pH. Their affinity for aromatic phenolic substrates is similar to fungal laccases in terms of substrate specificity. Because of genome sequencing, more bacterial laccases with characteristics distinct from those of fungal laccases will likely be found.

Mechanism of Action
The oxidation-reduction potential of laccases is mediated by copper atoms. All copper ions exist in the 2 + oxidation state at rest. The initial step in enzymatic catalysis is substrate oxidation by mononuclear copper T1, which works as an electron acceptor and converts the oxidation state of Cu 2+ to Cu + [237]. A cationic radical that is unstable is formed when an electron is taken from the substrate, which may be oxidized by a second enzymatic step or perform nonenzymatic processes, i.e., polymerization or hydration. At the T1 site, electrons are taken from the substrate and transferred to the T2/T3 center, where O 2 is reduced to H 2 O. Four molecules of reducing substrate are needed to completely reduce molecular oxygen in the water. The contrast in redox potential between the substrate and the Cu T1 influences laccase's catalytic effectiveness in substrate oxidation). Supplementation of redox mediators to the reaction medium boosts the catalytic ability of the laccase for substrates with E0 greater than those provided by the enzyme. Figure 1 portrays a mechanistic insight into laccase-mediator based catalytic model [32].
If the substrate is enormous to enter the enzyme catalytic site, mediators are supplied to aid the substrate in its oxidative degradation [245]. Natural mediators, such as syringaldehyde, as well as synthetic mediators, such as HBT and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) are often used. Even though artificial mediators are effective, their usage is restricted owing to the high cost and toxicity of the compounds used in their production. On the other hand, natural laccase mediators propose a costeffective and ecologically beneficial solution to expand the range of biocatalytic applications available.

Bioprocess for Laccase
Enzymes are often produced on a large scale by the organism's strains that have the potential to overproduce the enzyme under environmentally friendly conditions. It is the first stage in manufacturing any kind of enzyme to culture the organisms that generate the necessary enzymes. Afterward, the microorganisms are optimized under various Fig. 1 Mechanistic insight into laccase-mediator based catalytic model Reprinted from Bilal and Iqbal [32] with permission from Elsevier fermentation settings to obtain excessive amounts of the desired enzyme [10,114].

Type of Cultivation
Laccase production has been executed by using submerged and solid-state fermentation processes. Wild-type filamentous fungus are used to make laccase in large numbers on a commercial basis, and a variety of growth techniques are used to achieve this.

Submerged Fermentation
Submerged fermentation is the microorganisms cultivation in a liquid medium with a high concentration of oxygen and nutrients. As a result of the viscosity of the broth, one of the most severe problems that might occur during fungal submerged fermentations occurs. When a fungal cell matures, mycelium is produced, which interferes with the impeller's ability to rotate, resulting in a reduction in oxygen and mass transfer due to this restriction. To properly deal with this problem, a variety of approaches have been used. To attain optimal efficiency, the bioreactor must be run continuously. This procedure uses a strain of Trametes versicolor, which decolorizes the synthetic dye by the action of the bacteria. Cell immobilization may be employed to address challenges associated with mass transfer, oxygenation, and broth viscosity. Neurospora crassa immobilization on the membrane leads to the continuous production of laccase without inactivation for 4 months [123]. Nylon mesh was used to degrade pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4 DCP) by T. versicolor-free cell culture with immobilized cultures to compare the bioremediation of PCP and 2,4 DCP by T. versicolor free cell culture with immobilized cultures. Laccase is manufactured using the fed-batch technique, which is the most efficient method since it produces the most laccase activity per unit of time.

Solid-State Fermentation
Because SSF replicates the conditions under which fungi thrive in their natural habitat, it is a suitable alternative for enzyme production from natural substrates such as agricultural waste. Because of the presence of lignin, cellulose, and hemicelluloses, all of which have a high sugar concentration, fungal growth in the fermentor is encouraged, which lowers the cost of the process. The most common snag is the bioreactor design, which has limited mass and heat movement. Various types of bioreactor designs, including immersion configuration, extended bed configuration, tray configuration, and inert (nylon) and noninert (barley bran) support materials, have been examined to produce laccase [46]. It has been shown that the tray layout provides the best reaction time available. When grape seeds and orange peel are employed as substrates, it is examined whether a tray or immersion arrangement is optimal for producing laccase [45,193].
Solid-state and submerged fermentation of rice bran produce more laccase than other substrates, indicating that rice bran is a more productive substrate for laccase production due to the presence of phenolic compounds (i.e., vanillic acid and ferulic acid) [154]. It is possible to use a range of agricultural waste products as substrates to synthesize laccase in various applications. Grape seeds and stalks, barley bran [121], cotton stalk and molasses wastewater [105], and wheat bran [214], are examples of trash that may be recycled. It was discovered that while laccase production rose significantly in solid-state and submerged fermentations, it did not reach full potential; consequently, further culture is required.

Laccase Inducing Factors
Laccases are glycoproteins that may be found in a variety of forms, the most common of which are monomeric, dimeric, or tetrameric. In many activities, such as copper retention, heat stability, proteolytic degradation susceptibility, and the release of chemicals, glycosylation is essential. When laccase enzymes are separated and purified, they demonstrate tremendous diversity. Regarding glycosylation, the number and content of glycoproteins vary depending on the type of growth media used.

Carbon and Nitrogen Source
Lactose, sucrose, maltose, glycerol, fructose, and glucose are carbon-containing carbohydrates. Fructose has been demonstrated to increase laccase synthesis in Pleurotus sajor-caju, cellobiose in Toxicodendron pubescens, and glycerol or lactose synthesis in Pseudotrametes gibbosa, Fomes fomentarius, and Coriolus versicolor. Higher glucose levels repressed laccase synthesis in a variety of fungal strains, and a high sucrose concentration lowered laccase production to the constitutive level in the same strains. Polymeric substrates, such as cellulose, have a different behavior throughout the culture process and have been shown to increase enzyme production.
Nitrogen depletion is the most common trigger for producing fungal laccases, but laccase activity in certain strains is not linked to nitrogen content. Low carbon to nitrogen ratio was found to be beneficial in some studies. When fungi were grown in a nitrogen-rich medium rather than a nitrogen-limited medium, laccase production was shown to be higher in both [171].
Arthopyrenia sp. and Nigrospora sp. produced laccase in a liquid culture medium at varied copper sulfate concentrations, which was observed in both species [173]. Increasing the concentration of a copper ion in water from 1 to 10 mM/L boosted laccase activity in Setosphaeria turcica [124], which correspond with a large increase in Agrobacterium laccase activity [206]. Despite this, it has been shown that the copper ion inhibits Fusarium solani even at concentrations as low as 0.5 mM [239]. Bacillus halodurans laccase was found to be an ideal biocatalyst for the production of paper at alkaline pH [194].
The element type and concentration, various cations, and anions have varying impacts on laccase activity. As a result of Fe 3+ and Mn 2+ inclusion in Setosphaeria turcica laccase activity expressed in Escherichia coli, the activity increased by approximately 434.8% at 10 mM/L and 5 mM/L, respectively, whereas Na + increased activity by approximately 434.8% at 1 mM/L, but impeded activity at 5 and 10 mM/L. At 1 mM/L, SDS had a growing impact on laccase activity; but at 5 and 10 mM/L, SDS suppressed laccase activity [124].

Influence of Other Inducers
Several investigations have shown that fungal growth has a major impact on laccase production. During the secondary metabolic phase, the presence of nitrogen in the environment activates and triggers the activation of ligninolytic mechanisms. Generally, laccases are formed in low titers by fungal strains, but greater concentrations have been achieved by supplementing media with different additives, such as xenobiotic chemicals, which have been shown to enhance laccase titer. Even in the absence of any supplements, laccase-producing fungi are able to produce small amounts of laccases. When aromatic substances (i.e., veratryl alcohol, lignin and 2,5-xylidine,) are added to cultures of laccase activity, the activity of the enzyme increases [240]. Increased laccase production is caused by adding veratryl alcohol to the cultivation medium. Veratryl alcohol is a chemical compound that has an aromatic odor. After 24 h of culture, the inclusion of 2,5-xylidine resulted in the largest induction of laccase activity as well as the most significant increase in laccase activity, which was nine times more than the baseline. This was due to the toxicity of the 2,5-xylidine at higher dosages, which had a limiting effect on the proliferation of bacteria [65]. Following the inclusion of an inducer, it is seen that the quantities of a certain laccase enzyme have increased. Lee et al. [117] observed that alcohol boosted laccase activity more than xylidine, indicating it is a very cost-effective means of enhancing laccase output. Cellobiose, which is present in certain Trametes species, enhances laccase activity in the plant by increasing the number of branches [70]. Adding modest levels of Cu +2 to culture media increases laccase synthesis by 50-times when compared to the culture medium used as a control [168]. Using o-toluidine at concentrations of 0.5 mM and 6 mM, Trametes sp. 420 was found to produce laccase in cellobiose and glucose medium after being cultured with the compounds [220]. D'Souza-Ticlo et al. [53] carried out a series of tests to examine the effect of inhibitors on Lac-II activity and observed that the Lac-II activity was inhibited by 32-37% in the presence of Ag, Hg, and Sn but that the activity of Lac-II was inhibited by 56 and 48% in the presence of Cr and W, respectively.

Thermodynamic Stability
It has been shown that the laccase from Tricholoma matsutake has high thermal stability, with most of its activity remaining active at 20 to 80 °C [244]. There are several optimal temperatures for laccase activity depending on the strain. Fruiting body growth and laccase synthesis occur at 25 °C by incubating cultures in the light. However, when the cultures are incubated in the dark, the temperature increases to 30 °C for laccase production [171].
Pleurotus ostreatus maintains laccase activity at temperatures ranging from 40 to 60 °C, with the maximum activity at 50 °C. The ideal pH value for laccase biosynthesis varies with the substrate since different substrates result in various laccase reactions. According to various studies, laccase activity is characterized by a bell shape chromatogram profile. Increasing the pH value may cause the phenolic substrate to oxidize more quickly while the hydroxide anion (OH) binds to the T2/T3 copper core. By assessing enzyme activity [44] studied the impact of pH on enzyme activity in the pH range of 3.0-8.0 in the presence of syringaldazine as a substrate. When it comes to L1 (the isozyme of laccase), the best pH value was 4.0, but when it came to L2, the optimal pH value was 5.0. [91] isolated laccase from Trametes versicolor, which displayed a robust enzyme activity in diverse temperature and pH ranges, with the maximum activity being reported at pH 3.0 and 50 °C. Most studies have discovered that pH levels between 4.5 and 6.0 are ideal for maximum enzyme biosynthesis.

Purification of Laccase
Laccases are decontaminated from plants using tissue or sap extracts. Laccases from fungi are isolated and purified from the fermentation broths by a single step or multistep procedure. The entire downstream procedure was done in two parts; the first step involved solid/liquid separation by filtration and centrifugation, and the second step involved filtering and centrifugation. The use of ammonium sulfate as a solvent for enzyme purification has increased dramatically in recent decades. On the other hand, researchers have identified far more successful processes, such as ammonium sulfate fractionation, desalting, anion exchange, and gel filtration chromatography. With the use of celite chromatography, it is possible to purify laccase from Neurospora crassa in a single step, and it has been shown to achieve 54-fold purification with a specific activity of 333 U mg −1 [85]. Gel filtration might be utilized to purify the laccase isolated from LLP13, which had previously been purified by column chromatography [108,109]. Using ethanol precipitation, Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography, T. versicolor laccase has been purified from its natural environment in the laboratory. Ninety-one thousand four hundred forty-three units per milligram of protein are the specific activity of this monomeric laccase [97]. Purifying T. versicolor laccase by ion-exchange chromatography following gel filtration led to a specific activity of 101 units per millilitre of solution and a purifying efficiency of 34.8 times [44]. The use of ammonium sulphate followed by column chromatography (Sephadex G-100) was shown to be effective in purifying laccase from the bacterium Stereum ostrea by a factor of 70 [229].
Allos extracted the laccase enzyme from Bacillus cereus using a variety of techniques. The crude laccase filter produced from B. cereus was treated with ammonium sulfate before centrifuged at 6000 rpm for 20 min at 4 °C. A potassium phosphate buffer solution was used to dissolve the pellets before they could be collected. A dialysis tube with the same buffer was then used to dialyze for a prolonged time against the same buffer. More product purification was obtained using an ion-exchange chromatography technique combined with a DEAE cellulose column. The laccase enzyme was eluted from the solution at a rate of 30 mL/h using a constant-flow system. It was decided to conduct a gel filtration chromatography technique utilizing Sephacryl S-200 to purify further the laccase generated. In this experiment, laccase was eluted out at a rate of 3 mL/fraction using the same buffer as before. SDS-PAGE was performed in the presence of SDS to assess the enzyme's concentration (). It took roughly 4 h for the gel to run at a temperature of 4 °C after the sample was loaded onto it in a volume of 20 mL [9].

Critical Properties for Laccase Functionalities
In addition to their extensive substrate range and usage of molecular oxygen as a final electron acceptor, laccases possess several additional properties critical for biological functions. Copper T1 is the first electron acceptor because it is situated in the cavity adjacent to the enzyme surface [208]. Reduced copper T1 is the rate-limiting step in laccase-catalyzed reactions. Because of its low redox potential (ranging from 420 to 790 millivolts (mV) when compared to the conventional hydrogen electrode (NHE), the laccase substrate array is restricted to molecules containing phenolic moieties [81]. A phenolic compound is converted into phenoxyl radicals by the enzyme laccase. These radicals can polymerize through coupling or radical rearrangement, depending on their structural composition. It is seen in the presence of phenoxyl radical stability that there is redox reversibility, as defined by the oxidation of a specific substrate. The connection of phenolic mediators through radicals or their redox recycling has been shown to be useful in increasing the diversity of laccase substrates available. Laccase enzymes are split into two kinds depending on their redox potential: low redox potential and high redox potential enzymes. Low redox potential enzymes are those that have a low redox potential. Bacteria, plants, and insects all contain enzymes with modest redox potential, but fungi have laccases with a high redox potential [153]. Because laccases speed up the conversion of one molecule to another, they are beneficial in both anabolic and catabolic processes. Fungal laccases degrade lignin and humus, resulting in a catabolic reaction in the plant. These anabolic processes are important during morphogenesis; for example, they catalyze cuticle sclerotization [59], pigment synthesis [111], polyflavonoid synthesis [22], soil organic matter humidification (Chefetz et al. 1998), humidification [163]. Because of the redox potential of laccases, which may be found in plants, bacteria, and insects, radical coupling reactions in anabolic processes are thermodynamically viable without using extra chemicals in anabolic processes (Mikolasch et al. 2009). This kind of reaction, which results in the formation of pigments, lignins, polyflavonoids, and humus, often uses low molecular weight phenolic compounds as substrates. Purified laccases from plant tissues have been shown to polymerize lignin and polyflavonoids in the presence of radicals as has been demonstrated for Eucalyptus lignin (Harakava et al. 2005) and Arabidopsis thaliana (seed coat formation) [179]. The use of flavonoids and monolignols for radicalmediated lignin polymerization by purified laccases isolated from plant tissues has been shown. For example, in the plant Populus trichocarpa [182], oligomeric molecules generated by such processes are randomly covalently linked to cell wall components of plant fibers, resulting in the formation of oligomeric molecules. According to the research [216], laccases related to the insect cuticle matrix have a comparable radical-based relationship to one another. In Manduca sexta and Tribolium castaneum, cross-linking activities mediated by the laccase were identified to produce cuticle sclerotization, which was previously thought to be caused by a virus [13]. In the process of cuticle sclerotization, catechol, N-acetyldopamine, and N-alanyldopamine decompose into quinones, which are subsequently radical bound to histidyl residues in cuticular proteins [113].
Laccases must create free radicals to carry out their catabolic functions. These free radicals oxidize the lignin and humus, which are then decomposed. The first generation of free radicals is produced by fungal laccases created from natural methoxyhydroquinones. As a result of these free radicals, the Fenton reaction is activated, resulting in forming a range of reactive oxygen species (ROS). Second, when low molecular weight redox mediators that occur naturally are oxidized, radicals are generated as a byproduct. In most cases, the oxidized target substrates are the source of these mediators. When phenolic lignin units undergo preferential oxidation, as occurs in the presence of phenolic acid, minute phenolic residues with oxidized side-chains are released into the environment. The basidiomycetes, which are very effective lignocellulosic decomposers, are the most often oxidized species. However, when applied alone, laccases have been shown not to affect the depolymerization of native lignin but modify its surface characteristics. Due to limited analytical tools, it is presently unable to determine the specific attack mechanisms and enzymatic properties of laccases in connection to lignin modification [153]. When applied to model phenolic compounds of lignin, laccase has been shown to break the bonds between the carbons C1 and C2 (C-C cleavage) as well as the bonds between the carbons C1 and the aryl group (alkyl-aryl cleavage). According to Kawai et al. [106] and Rochefort et al. [189], laccase needs a mediator for breaking the connections between non-phenolic lignin subunits. Natural laccase redox mediators have been identified in lignin degradation products, phenolic secondary metabolites (p-coumaric acid and acetosyringone), and extracellular fungal metabolites (p-cinnamic acid, 4-hydroxybezylic alcohol, syringaldehyde, sinapic acid, and 3-hydroxyantharnillic acid [233]. The fungal laccase enzyme oxidizes all these phenolic compounds to phenoxyl radicals which can oxidize non-phenolic lignin residues by a number of mechanisms, including hydrogen abstraction [52]. In addition, phenolic compounds compared to those found in lignin-decomposing bacteria have been detected in the laboratory environment. Many bacteria, including Bacillus species, Pseudonomas putida, Aneurinibacillus aneurinilyticus, Streptomyces species, and Paucimobilis species, generate benzaldehyde with methoxy, hydroxyl, or trimethoxy substitutions. Bacillus sp and P. putida cultures have been shown to create cinnamic acid with methoxy and hydroxy substitutions and cinnamic acid with hydroxy and methoxy substitutions [136].
When numerous phenolic compounds associated with lignin degradation were investigated in vitro, they have mediation qualities; however, the existence of natural laccase mediators during in vivo wood decay has not yet been proven. Natural mediators, which occur naturally in the form of phenolic chemicals released from a lignin polymer and may interfere with laccase's solitary activity, making it difficult to rule out interference while studying laccase's solitary activity.

Bacterial Laccase
Recent years have seen an increase in the popularity of bacteria-derived laccases, owing to the huge industrial benefits they offer over fungal laccases. These benefits include the capacity to operate across a broad temperature and pH range while demonstrating outstanding resistance to a wide range of inhibitors. Apart from that, bacterial laccases offer a number of benefits, including wide substrate specificity, rapid enzyme production, and ease of cloning and expression in the host, especially when the enzyme is modified. It may also be used for biobleaching pulp and paper, decolorizing and degrading textile colors and wastewater, and producing biosensors, among other applications [37,38]. A wide variety of microorganisms may be used to extract and produce bacterial laccases, and the activity can subsequently be measured at the cellular level.
Prokaryotes have been found to include MCOs that are capable of producing laccase [41]. These enzymes are collectively called "polyphenol oxidases," "multicopper oxidases," or "laccase-like enzymes" depending on the method by which they catalyze a reaction. They catalyze the same oxidation of traditional laccase substrates, which makes them interchangeable. Azospirillum lipoferum isolation from plant roots contained the first known bacterial protein having polyphenol oxidase activity, which was identified as LMCO [82]. In addition to its activity is associated with the formation of a dark brown colour, laccase is a multimeric enzyme [58].
In soil and water, prokaryotic laccase is produced by bacteria from the phyla Aquificae, Deinococcus-Thermus, Firmicutes, and Cyanobacteria, as well as members of the Archaea phylum, which includes members of the Aquificae and Deinococcus-Thermus phylas. [184], Bacillus pumilus, Bacillus subtilis, B. licheniformis [112], S. lavendulae [217], S. griseus [66], Escherichia coli (Alexandre et al. 2000), Porphyromonas syringae [210], Thermus thermophilus [145], Oscillatoria boryana [165], Halofeax volcanii (Uthandi et al. 2010), and Marinomonas mediterranea [196] including species existing in extreme environments [74]. When it comes to prokaryotes, the placement of laccase inside the cell differs significantly from species to species. It seems to be closely related to the physiological activity of the enzyme and controlled by the enzyme's developmental stage as well as the presence of stimulating substrates in the environment. Bacillus subtilis [134], S. meliloti, Thermophilus thermophilus [145], and Mycobacterium mediterranea (Sanchez-Amat et al. 1997) are the bacteria that make the vast bulk of the laccases that are produced in the United States. Because the presence of laccase intracellularly has the potential to create harmful repercussions, bacteria need a means of dealing with the enzyme. Endogenous reactive quinones created by laccases are considered to cause electron transport system reorganization in laccase-positive cells in response to the presence of these reactive quinones. Bacilli and filamentous actinomycetes produce laccase outside of their cell walls [212]. Other human disorders have been linked to the discovery of laccase-like genes in organisms such as E. coli, Bordetella pertussis, P. aeruginosa, Yersinia pestis, Campylobacter jejuni, and Mycobacterium leprae, to name a few [8]. According to some researchers, the production of melanin and the activity of the laccase enzyme are both important factors in the pathogenicity of these organisms [42].
CotA, located on the outer coat of Bacillus subtilis and other Bacillus species endospores (Hullo et al. 2001), has received the greatest attention in the scientific community. The EPR spectra of the 65 kDa CotA protein produced by an overproducing E. coli strain are characteristic of the blue multicopper oxidase family, which is represented by the EPR spectra of the CotA protein. The use of comparative modeling estimated the structure of the CotA. This laccase has an exposed copper core (T1) and two buried copper centers (T2 and T3), which are all hallmarks of a fungal laccase [134]. This laccase also has two hidden copper centers (T2 and T3). The enzyme's crystal structure revealed a larger potential substrate-binding cavity compared to fungal or plant laccases [64]. When the ligands binding to the T1 site of CotA are modified, the biochemical properties of CotA are significantly affected. Copper coordination has been harmed in the wake of the T1 site modifications. At 80 °C, it has a halflife of about two hours and is very thermostable. The CotA recombinant laccase is notably active in the oxidation of ABTS and syringaldazine at pH ranges between 3.0 and 7.0 [134]. For example, the genome of filamentous S. coelicolor includes a protein known as SLAC [126], which is more active than the CotA recombinant laccase. A representative of the laccases with two domains, this protein has a different protein architecture from the others and seems quite stable in acidic circumstances. The oxidation of 2,6-dimethoxyphenol (DMP) by the SLAC displayed a previously unidentified high pH optimum (pH 9.4), yet the recombinantly generated enzyme exhibited conventional laccase paramagnetic properties [126]. According to the literature, actinomycetes are the second most prolific laccase producers in nature after fungi, and their SLACs are regarded to represent significant evolutionary intermediates in creating three-domain MCOs. Unlike a typical laccase, they are not as active as they might be since their crystal structure is more similar to that of nitrite reductase or human ceruloplasmin rather than a conventional laccase [86].
Laccases have a molecular weight ranging from 32 to 180 kDa and may be found as monomers, trimers, or tetramers, depending on where they came from and how they were assembled. Even though there is unambiguous evidence for glycosylation of prokaryotic proteins (Nothaft et al. 2010), very few studies have been done on the glycosylation of bacterial MCOs, and only a few studies have been done on the carbohydrate content of bacterial laccases [174]. Prokaryotic MCOs are similarly poorly known in electrochemistry, which is a scenario comparable to that of bacteria in this regard. Low-redox potential enzymes, such as bacterial (and plant) laccases, are present in the T1 site (E 0′ T1 ), which has a redox potential of 460 mV when compared to the NHE [86]. In contrast to fungal laccases, bacterial polyphenol oxidases are significantly less active at high pH. The pH optimum of many Streptomyces laccases (Gunne et al. 2012) was found to be high (8.5 and 9 for ABTS and DMP, respectively), although McoP isolated from Pyrobaculum aerophilum [73] and T. thermophilus laccase (92 °C) [145] exhibited the highest temperature optimum of all (85 °C). As a result of their extraordinary metabolic capabilities, bacterial laccases have the potential to be a useful source of biotechnological catalysts in a range of applications.
MCOs with laccase activity have been found to have moonlighting proteins [multifunctional enzymes that perform a range of jobs depending on where they are located in the cell] [133] that have been identified in bacterial MCOs [204]. Different hypotheses have been improved regarding the mechanism of multifunctional enzyme switching. It has previously been shown that the putative actions of prokaryotic laccases, but their biological significance in vivo remains unclear. The vast majority of the bacterial proteins that have been found so far have nothing to do with lignin degradation [55]. Despite the fact that the use of bacterial laccases in the degradation of lignin has never been shown, their involvement in the process is presently being investigated in great depth. The lignin polymer has been modified to allow other enzymes access to cellulose, and hemicellulose has recently been advocated as a possible solution [55]. It has been shown that laccase-related gene products are important in metal homeostasis/oxidation, morphogenesis, sporulation cell, and spore coloring. They are often associated with tolerance to a range of environmental stresses. An increasing number of studies have linked the Azospirillum laccase to a variety of processes such as cell pigmentation [82], the use of naturally occurring phenolic compounds generated during lignin metabolism [71], and/or electron transport. In the rhizosphere, these abilities are most likely connected to Azospirillum sp competitiveness, and they play a critical role in plant root colonization, especially when the oxygen content in the soil environment fluctuates [58]. Aside from these functions, Streptomyces laccase may also play a role in morphogenesis and pigmentation, lignocellulose breakdown, antibiotic production, or bacteria-bacteria interactions. Therefore, the vast majority of the laccases found in Bacillus are present on the spore's outer layer, which protects endospores from a number of stressors and poisons that they may encounter. The cotA gene produces a brown spore pigment, a melanin-like polymer, to protect the organism from ultraviolet radiation. A laccase-positive strain of B. subtilis is more resistant to infection than cotAdeficient strains [134], showing that this method is feasible. CotA has also been shown to protect cotA-deficient strains of B. subtilis, indicating that this strategy is viable. Aside from that, the brown-pigmented Bacillus sp. HR03 spores exhibit exceptional resistance to hydrogen peroxide as well as UV A and UV C. Because of this, Bacillus spore pigments are important for the bacteria's ability to withstand adverse conditions, and laccase is a crucial enzyme in the creation of these pigments [207]. In Bacillus subtilis, laccase is also involved in producing a variety of different colours. According to another idea, laccases were responsible for crosslinking tyrosine to di-tyrosine in spore coat proteins. This function was comparable to the function of laccases in constructing plant cell walls. Crosslinking of tyrosine to di-tyrosine has been seen in B. subtilis spores [194], which lends support to this notion. Due to the fact that B. subtilis does not have CumA, the spore coat protein of Pseudomonas sp., it does not participate in metal oxidation and does not produce MnII oxidation. CumA is an MCO with a structure similar to a laccase [155]. It contributes to the oxidation of MnII, which may aid the cell's ability to survive when exposed to metal ions. In addition to being resistant to CuII toxicity, B. halodurans is also a producer of the enzyme laccase [194]. It has also been identified that bacteria such as E. coli (PcoA and CueO, formerly YacK), P. syringae (PcoA), and others have laccase-like coding genes. The existence of these pseudo-laccases, which are identical structurally to multicopper oxidases, is critical for developing bacterial copper resistance. The yacK gene encodes a putative multicopper oxidase that also functions as a phenol oxidase and a ferroxidase. It has the copper-binding sites predicted in this enzyme [110]. The enzyme is not only efficient against the siderophores that E. coli uses to absorb iron (FeII), but it also protects E. coli against copper exposure. In the CueO protein from E. coli, a Met and His-rich region partly encircle the T1 copper active site's entrance [188]. Because of CuII binding, it is possible that more Cu-binding sites will be created in this region and a modification in the active site structure. In their results, they discovered that Pseudomonas CopA has just a minor but significant degree of sequence similarity with MCO proteins and that it is needed for overall copper resistance in these bacteria [140]. Because of its bioremediation capabilities, laccase's participation in cyanobacteria has been studied [12], with data showing that it may help to protect the cell from harsh environmental circumstances. On the other hand, there has been little study on cyanobacterial laccases [2].

Plant Laccases
As a result of the substrate selectivity and features of the pure enzyme, laccase has historically been isolated from gymnosperms, angiosperms, and other plants. A laccase enzyme isolated from Tetracystis aeria (green algae) has been verified as a laccase in recent years based on the substrate specificity and characteristics of the enzyme [164]. Laccases have been identified in a number of higher plants, including Arabidopsis thaliana, Acer pseudoplatanus, Pinus taeda, P. trichocarpa, Nicotiana tabacum, Lolium perenne, Liridendron tulipifera, Oryza sativa, Zea mays, Brassica napus, and Saccharum officinarum [164].
Yoshida discovered laccase in the Chinese lacquer tree Rhus vernicifera, which was the first time it had been discovered. The enzyme in R. succedanea and other Anacardiaceae species (such as Schinus molle, Mangifera indica, Pleogynium timoriense, and Pistacia palaestina) was found by Gabriel Bertrand after ten years of study [93]. Laccase was identified in high concentrations in the resin ducts of the representative cells of these individuals. It was Bligny and Douce that discovered laccase synthesis and secretion in A. pseudoplatanus cultures, and the enzyme was later found in the tissues of plants such as P. taeda [200], P. euramericana [183], and N. tabaccum [187].
It is possible to find laccase isoforms in a broad variety of plant species. A total of five laccase genes have been identified in P. trichocarpa xylem tissues [183], there have been eight laccase genes discovered in the xylem tissues of P. taeda xylem tissues [200], and up to 17 laccase genes have been identified in the xylem tissues of Arabidopsis thaliana xylem tissues [28]. Laccases in plants have a molecular weight of 60-130 kDa and an amino acid content of 500-600 amino acids [231]. This enzyme is glycosylated to a high degree (22-45%) and performs best when the pH ranges between 5 and 7 are maintained. According to [28,231], the enzymes have an isoelectric point (pI) ranging from 5 to 9.6, and they are extensively glycosylated (22-45%). In the presence of NHE, laccases from plants have a very low redox potential (430 mV), which is especially true for copper type I laccases (T1). Among the characteristics that define plant laccases is their ability to oxidize their substrates without mediators [178].
Laccases in plants have been associated with a variety of different processes. Populus sp. seed coats contain laccase from Arabidopsis thaliana, which is thought to be important in stem lignification. Laccase has also been discovered in various organs and tissues of A. thaliana, including the seed coat [28]. It has been shown that Anacardiaceae plants have the enzyme in their resin ducts, which suggests that they may be able to defend themselves against herbivores, predators, as well as microbial and fungal invasion [138].
Green algal laccases are responsible for detoxifying dietary phenolic chemicals present in both terrestrial and aquatic environments. They are also engaged in the growth of cell walls and the creation of UV-absorbing compounds, to name a few activities. Furthermore, green algal laccases play a part in the metabolism of lignocellulosic substrates, which results in the uptake of nutrients by the organism. It has been suggested that laccases, which are present in algae, may be capable of playing a role in the biotransformation and biodegradation of natural and xenobiotic aromatic pollutants [164].

Fungal Laccases
A large amount of knowledge regarding fungal laccases comes from the orders Basidiomycota and Ascomycota, which are fungi that are found in nature. Lignocellulose polymer breakdown, defense, and protection, virulence and pathogenicity, coloration, and sporulation are all processes in which fungus laccases participate. The biodegradation of lignocellulose by fungal laccase is a basic activity of the fungal laccase, and this action contributes to the carbon cycle in the biosphere. Because of the amazing resilience of the lignin polymer to biological and chemical degradation, wood decay occurs slowly and is difficult to detect physiologically in most cases. A high redox potential, around 800 mV vs. NHE, is shown by laccases from fungal species, particularly white-rot species. This redox potential facilitates the removal of electrons from substrates and may also function as redox mediators during the laccase assault on lignin. Fungal laccase has the ability to break down bonds in phenolic lignin model compounds without the requirement for additional mediators. Specifically, laccase can only destroy covalent connections in non-phenolic lignin subunits by using mediators [153].
In nutrient recycling, litter breakdown is an essential stage, which is mediated by a diverse variety of fungal taxa and characterized by the fast succession of saprotrophic species. Litter breakdown is also important in nutrient recycling [230]. Other fungi, particularly those belonging to the Glomeromycota and Zygomycota, have also been discovered. Despite the fact that Ascomycota and Basidiomycota are the most abundant fungus with extracellular enzymes engaged in this process, other fungi, notably Zygomycota and Glomeromycota, are also found [23,230]. Genetic evidence supporting the presence of laccase genes in litterdegrading fungus has been discovered, with the number of basidiomycete laccase genes being shown to be 5-10 times greater in species found on a high-lignin forest floor compared to species found in a low-lignin environment [153]. It has been known that aquatic fungal species were unable to degrade lignin for a long time. Species mostly belonging to the Ascomycota, Chytridiomycota, and Oomycota are significant litter decomposers and laccase producers [23], but species predominantly belonging to the Ascomycota, Chytridiomycota, and Oomycota are not. Sole et al. [211] detected two laccase-encoding gene segments in pure cultures of Clavariopsis aquatica, which suggests that laccase is a cell-associated enzyme [131]. A variety of aquatic hyphomycetes [1] and aquatic Ascomycota fungi, including Phoma sp. and Coniothyrium [104], have also been reported to have laccase activity.
Humic substances (HS) are the second most important carbon source in nature. Microorganisms, particularly fungi that produce oxidizing enzymes, are essential for the biosynthesis, breakdown, transformation, and mineralization of hexavalent chromium [84]. According to Chefetz (Chefetz et al. 1998), the humification process is aided by the laccase from the fungus Chaetomium thermophilum, which produces water-soluble polymers containing hydrophobic acids as part of the humification process. In contrast, enzymes from the Basidiomycota are responsible for the degradation and transformation of HS. To choose which transformation route to use (humification vs. HS degradation), factors such as availability of the substrate and reaction parameters such as pH, humidity, and the presence of co-substrates must be considered [84]. Because of the vital function that pH plays in the degradation and transformation of HS, the involvement of numerous laccase isoenzymes may be critical for increasing HS degradation and transformation efficiency [84]. Feng et al. [72] established a link between the quantity and variety of fungal and bacterial laccase activity in arable subtropical soil, with fungal laccase activity serving as the dominant source.
A fungal reaction to antagonistic situations such as other microorganisms, xenobiotics, heavy metals, poisons, and physiologically active substances is laccase secretion, one of the most fundamental. Fungal laccases may oxidize a wide range of non-phenolic substrates, including aromatic amines, polycyclic aromatic hydrocarbons, synthetic colors, antibiotics, and other less apparent laccase substrates [177].
They can also oxidize phenolic substances. As an enzyme "tool" for removing natural and manmade toxins from the environment, Laccase contributes to fungal active defense by eliminating poisons from the environment.
It has been shown that laccase secretion is enhanced in the presence of Trichoderma sp. in the cultures of Agaricus bisporus, Porphyromonas ostreatus, and Lentinula edodes [75]. According to Sjaarda et al. [209], the presence of a toxic extract of T. aggressivum in the medium promotes the production of A. bisporus laccase genes, and resistance is associated with laccase activity. Laccase production in the presence of Bacillus sp. and A. ochraceus cells is increased in the presence of T. viride cells. According to Lakshmanan and Sadasivan [116], the laccase inhibition of T. viride renders this fungus incapable of competing with harmful bacteria in the environment.
The BcLCC2 laccase from Botrytis cinerea is a good example of how this enzyme is linked to antibiotic resistance to 2,4-diacetylphloroglucinol, as seen in the figure below (2,4-DAPG). Tannic acid is required to decompose 2,4-DAPG [202], and this is the only environment in which it may occur. Cinnabarinic acid is an antibiotic that has been shown to protect against bacterial infection in this fungus. Laccases play a key role in melanin's metabolism in the body. Many melanin pigments are antimicrobial and function as virulence factors, assisting fungal organisms in their fight for life and reproduction. In addition to being found in bacterial endospores, fungal spores/conidia, and inside cell walls [161], they may also be found in bacterial endospores. The potential of C. neoformans to infect people has been explored in connection to the melanization of the organism. This yeast-like fungus is responsible for a variety of diseases, including childhood infection, cryptococcal pneumonia-like illness, and meningoencephalitis (cryptococcosis) [83]. Increased expression of a single CNLAC1 gene was seen in a C. neoformans culture under the conditions of glucose restriction, acidity increase, and temperature reduction [69]. Several studies have shown that laccase is the most important diphenol oxidase involved in producing a melanin-like pigment by C. neoformans when the organism is exposed to an external substrate [236]. Laccase from Candida neoformans also enhances virulence in macrophages by converting iron III to iron II in macrophages, blocking the oxidative burst, and interfering with the body's natural defenses [120]. It is probable that C. neoformans laccase is involved in the reaction to potentially harmful hydroxyl radicals produced by macrophages since it is strongly linked to the cell wall [248].
AIDS patients have been infected with Talaromyces marneffei, which shows that the laccase enzyme may have a role in the pathogenicity of this opportunistic infection [198]. One probable explanation is that T. marneffei generates a red soluble pigment in both yeast and mold forms, which might explain the phenomenon. T. marneffei pathogenicity was investigated using single, double, triple, and quadruple deletions of the lacA, lacB, lacC, and pbrB laccase genes. The goal was to better understand the function of laccase in the pathogenicity of T. marneffei. One study found that only the triple mutant conidia were particularly sensitive to the effects of antifungal medicines, as well as to phagocytosis and death by the monocyte cell line THP-1 [198]. It seems that the laccase produced by T. marneffei plays a role in the pathogen's resistance to host immune responses [198]. Inhibition of immunological recognition is caused by the pigment or other laccase products, which may affect the signaling pathways of monocytes [235]. Laccase genes have also been discovered in the genome of Fonseca sp., a therapeutically significant fungus that resembles black yeast and is a source of fungicides [149].
Anthracnose in Mangifera indica fruits is caused by the fungus Colletotrichum gloeosporioides, which is the most prevalent cause of the disease [232]. Genetically modified Lac1 mutants (LAC1) had reduced pigmentation, fewer conidia production, lower aerial mycelial mass, and lower radial growth rates than wild-type Lac1 mutants (LAC1). It has also been shown that the lac1 mutants are less harmful in virulence assays on mango leaves and fruits, both damaged and uninjured. As reported by Kuo et al. [115], laccases in Heterobasidion annosum may have the ability to operate as a virulence factor when in contact with Plantago edulis seedlings [115]. Inhibiting the fungal pathogen's ability to synthesize melanin may be useful for treating fungal illnesses in plants and animals and reducing fungal degradation in wood.

Animal Laccase
Laccases in insects have been the subject of some of the most in-depth investigations ever conducted in the animal kingdom. Among the creatures that contain the enzyme are the Anophales, Calliphora, Bombyx, Drosophila, Diploptera, Menduca, Lucilia, Papilio, Monochamus, Rhodnius, Phormia, Schistocerca, Sarcophaga, and Tenebrio [128,14,60]. The Hymenoptera, Diptera, Lepidoptera, and Coleoptera are the four major groups of insects. In addition, mollusks have been revealed to carry numerous different isoforms of the enzyme, showing that it exists in the lowest metazoan taxon possible (i.e., sponges). Recent research has connected a genetic mutation in the human laccase gene to an increased chance of developing leprosy, Crohn's disease, ulcerative colitis, and idiopathic arthritis [108,109], among other diseases. A number of mammalian species have been found to contain putative sequences encoding for this enzyme. It was observed that laccase could be found in the epithelial cells of human and termite intestinal epithelial cells [100]. Laccase is most likely a protective component in the intestines of insects against potentially dangerous lignin derivatives formed from a plant-based diet, which are present in their diet in large amounts. Furthermore, it has been demonstrated to be limited to the intracellular space [129] and its molecular mass has been reported to vary between 70 and 100 KDa in a variety of insect species (including M. sexta) [219]. The pH and pI values range from 5 to 6.5 and 5.1 to 6.3, respectively, while the pH ranges from 5 to 6.5 [106,60]. Several studies have shown that sponges utilise laccase as an antibacterial agent, in addition to detoxifying and eliminating xenobiotics from their filtered food [119]. Among insects, laccase is well-known for its function in cuticle sclerotization in the epidermis of larval, pupal, and adult stages of the fruit fly Drosophila virilis [81,231]. Sclerotization occurs before to or soon after the onset of ecdysis because of the oxidative incorporation of acyldopamines into the cuticular matrix (NADA, IV) and N-alanyldopamine into the cuticular matrix (N-alanyl Dopamine) (post-ecdysial sclerotization). A condition known as sclerotization of the delicate larval cuticle occurs in several Dipteran species during the puparium development [11]. In the intermoult stage of the life cycles of D. virilis, L. cuprina, and B. mori, laccase gene expression and activity are low, but they quickly increase during puparium development and thereafter fall [11]. Laccase is found in two forms in insects: laccase-1 and laccase-2. Laccase-2 is required for cuticle tanning in T. castaneum and M. sexta (e.g., larval, pupal, and adult stages) [13], whereas laccase-1 is found in M. sexta's salivary glands, Malpighian tubules, midgut, epidermis and fat body. The laccase-1 enzyme oxidizes potentially toxic compounds consumed by insects, hence acting as a protective factor in the insect stomach. Another well-known biological purpose of laccase in melanin production in the midgut in response to parasite invasion [56,138]. According to research, the laccase-2 gene was shown to be highly expressed in the epidermis of M. sexta and B. mori before ecdysis. Immediately after ecdysis, the cuticle of newly molted pupae is devoid of laccase activity, which becomes apparent a few hours later when the pupae is exposed to light. As a result of these observations, the notion that cuticle laccase is created as an inactive precursor before being activated during the ecdysis stage has been supported [246]. Laccase-2 is important for the coloring of the pupal cuticle and the sclerotization of the adult cuticle in Monochamus alternatus [157]. To produce covalent cross-links between polypeptides, pigmentation and sclerotization, also known as cuticle tanning, are two processes that entail oxidative and nucleophilic reactions involving catechols and amino acid side-chain groups, as well as oxidative and nucleophilic reactions involving catechols and amino acid side-chain groups. It has been shown that protein conjugation thickens and darkens the exoskeleton of insects [13], as well as the cuticle, the egg capsule, the chorion, the ootheca, and the silk cocoon. Hattori et al. [94] discovered a laccase in the salivary glands of the insect Nephotettix cincticeps, which secretes watery saliva that assists the insect in detoxifying potentially dangerous monolignols while feeding. Laccase from M. sexta has been reported to be involved in the oxidation of potentially toxic compounds contained in food, the metabolism of iron [98,99], and other processes. Figure 2 represents various sources of laccase.

Biotechnological Use of Laccase
There is no doubt that the future is bright in the subject of laccase research for biotechnological applications. It is clear that the issue is popular in the manufacture of laccases and the development of immobilization processes for the use of laccases [16,17]. Laccase find applications in textile industries, dye degradation, environmental pollutant degradation, food processing, biosensors, and delignification, paper and pulp manufacture, and polymer synthesis are examples of applications for these enzymes in biotechnological processes (Fig. 3).

Food Processing Industry
Laccase is used in the food sector to remove unwanted phenolic compounds in wine stabilization, baking, juice processing, and wastewater bioremediation [45]. It increases both the functioning and the sensory qualities of a cell [129]. Laccase is used in the beer business to offer stability as well as extend the shelf life of the product. Chill haze results from the naturally occurring proanthocyanidins polyphenol stimulating the production of haze in beer. Warming beer to room temperature or higher may redissolve the complex. After a given amount of time, the phenolic rings are substituted with the sulphydryl group, resulting in a persistent haze that cannot be removed. Laccase has been employed to prevent polyphenol oxidation since it has the ability to absorb excess oxygen and thereby extends the shelf life of beer. Laccase is widely used to stabilize fruit juice. The color and flavor of the juice are provided by phenol chemicals and their oxidative Fig. 2 Various sources of laccase products, which are naturally present in the fruit juice. Their colour and scent change when phenolic and polyphenolic acids polymerize and oxidize. These alterations are known as enzymatic darkening [186] and are caused by the increased quantity of polyphenol. Laccase treatment eliminates phenol as well as the substrate-enzyme combination via membrane filtering, resulting in color stability despite turbidity. A variety of enzymes are employed to improve bread's texture, volume, taste, and freshness. Laccase boosts the stability of gluten structures in dough and baked goods, resulting in increased product volume, enhanced crumb structure, and improved baked goods softness. Stickiness lowers, strength and stability increase, and machineability improves due to the laccase addition, as shown by using low-quality flour [143]. A significant amount of phenolic and polyphenolic compounds during the crushing and pressing stages plays a key role in wine manufacture. Color and astringency are influenced by the high polyphenol concentration present in the seeds, stems, and skins, which varies depending on the grape varietal and vinification circumstances [144]. Polyphenol oxidation happens in wines and musts due to a complicated mechanism, culminating in a change in color and flavor known as maderization [137].

Pulp and Paper Manufacturing Industry
Laccase is capable of depolymerizing lignin and delignify wood pulps, kraft pulp fibers, and chlorinated water to produce chlorine-free biopolpation and delignify wood pulp and kraft pulp fibers [18,30]. When laccases and mediators are combined to delignify pulp, the delignification of the pulp is improved [103]. Using mediators, it is feasible to oxidize the non-phenolic residues that remain after oxygen delignification is complete. In this scenario, the mediator molecule is oxidized by the laccase enzyme, which oxidizes lignin subunits that would otherwise not be oxidized by the laccase enzyme. Besides removing pitch and dyes from woodbased goods, laccase mediation systems may also be used to remove pitch and colours from other materials. Laccases may be used to bind a wide range of materials, including fibers, particles, and paper, to one another [171]. As a consequence of the production of hazardous chlorinated lignin degradation products (such as chlorophenols, chlorolignins, and chloroaliphatics) by pulp and paper mills, which produce intensely colored black liquors that are harmful to the environment. The acidity of the effluents modifies the pH of the land and water bodies where these extremely alkaline paper mill effluents are discharged, and this changes the pH of the effluents. It has been shown that laccase considerably impacts the color and toxicity of these bio-samples [228]. The pH, dye concentration, and enzyme activity were all measured in this experiment to ascertain if Acid Blue 92 was decolored by laccase [185].
The degradation of residual lignin in kraft pulp by bleaching chemicals creates environmental pollution. Using alkaliand thermo-tolerant bacterial laccases is a prodigious biological substitute for chemical processing. Treatment with a novel alkali-and thermostable laccase from Bacillus tequilensis SN4 induced a significant reduction in kappa number and improvement in the brightness of softwood pulp by 28 and 7.6%, respectively, without using a mediator. In the presence of N-hydroxy benzotriazole as a redox mediator, the kappa number was reduced to 47%, along with a 12% increase in brightness, making SN4 laccase a robust candidate for deployment in pulp biobleaching [213].

Textile Manufacturing Industry
During the wet processing stage of the textile industry, a significant amount of water and chemicals are used. It is possible to come across chemicals ranging from inorganic to organic compositions in a range of shapes and forms. Because of their chemical structure, when dyes are exposed to light, water, and other environmental factors, they do not deteriorate. The fact that laccase destroys dye has led to the development of laccase-based procedures, which combine synthetic colors and are currently in use in the industry [62,102]. These techniques are now being applied in the field. Because of its capacity to break down the colors that are currently used in the textile industry, laccase is being investigated as a potential solution for textile effluent issues [171]. Combined with cellulose, laccase is used in stone washing to minimize damage to the exterior fibers while protecting the inside fibers [147]. Other applications include decolorizing dyed cloth to provide a brighter color and eliminating blot after stone washing [135]. Blanquez et al. [34] discovered using T. versicolor are effective in treating a black liquors discharge in order to detoxify and reduce the color, aromatic compounds, and chemical oxygen demand of the discharge, and they found that they were successful in doing so. Their experiments discovered that the amount of color and aromatic compounds was decreased by up to 70-80%, and the amount of carbon dioxide was reduced by up to 60%. Following the completion of their studies, they came to the conclusion that T. versicolor is capable of producing laccase. T. versicolor completely degraded Tropaeolin O, Amaranth, Congo Red, and Reactive Black 5 in the absence of dye sorption. In contrast, it only partially decolorizes the Brilliant Yellow 3B-A, Brilliant Red 3G-P, and RBBR, which are only partially decolorized in the absence of dye sorption. The Brilliant Red 3G-P, Brilliant Yellow 3B-A, and RBBR, which are only partially.
In their experiments with dyes, they discovered that the toxicity of certain hues remained the same. In contrast, the toxicity of others was reduced or altogether removed [181].
Laccase-based hair dyes are less irritating and simpler to work with than conventional hair dyes [192], which are more difficult to work with than traditional hair dyes due to the use of laccases rather than hydrogen peroxide (H 2 O 2 ) in the color composition. Aside from that, the dechlorination process makes use of the enzyme laccase. Extreme dechlorination activity is facilitated by xylidine, which works as an inducer of laccase activity, resulting in the decline in concentration of dissolved oxygen in the solution [223]. Excessive dechlorination activity is facilitated by the presence of xylidine in the solution. In a study conducted by Romero et al. [191], S. maltophilia was found to have the ability to decolorize numerous synthetic dyes (including methyl blue, toluidine blue, methyl green, Congo red, and pink) and industrial effluents.

Biodegradation and Bioremediation
In addition to being excellent bioremediators, immobilized laccases have the advantage of being able to be used forever, as do many other enzymes. Laccase is immobilized in (Polyvinyl alcohol) PVA-based polymers that have been crosslinked with nitrate or boric acid [40]. Laccases are regarded to be environmentally friendly biodegrading agents to degrade an array of textile dyes and other environmental pollinates because of their ligninolytic activity (Fig. 5) [19,20]. It degrades xenobiotics, polycyclic aromatic hydrocarbons (PAHs) derived from natural oil deposits and fossil fuels, chlorinated and phenolic contaminants, and diesel, and its activity in plant tissues increases as its concentration in the soil increases. It also degrades phenolic and chlorinated phenolic contaminants. Environmental contamination occurs due to fast industrialization and excessive pesticide usage to increase agricultural output. This contamination affects soil, water, and air quality and is a significant environmental concern in today's world. T. versicolor is used to bioremediate atrazine in soils with low moisture and organic matter content, which are common in semiarid and Mediterraneanlike climates [24]. Keum and Li [107] isolated laccase from T. versicolor and Pleurotus ostreatus for the breakdown of PCBs and phenol and observed that the rate of degradation decreased with increasing chlorination. They found that 3-hydroxy biphenyl resisted laccase breakdown better than its 2-or 4-hydroxy counterparts [222].

Nanobiotechnology and Biomedicine
Laccases have been widely explored to develop biosensors and biofuel cells, owing to their ability to catalyze direct electron transfer in a variety of situations [190]. Laccases turn oxygen into water in biosensors, and the biosensor then measures the amount of oxygen used during the oxidation of analytes. Laccase-based biosensors have been widely used in the food industry to detect polyphenols in fruit juices, wine, and teas, as well as to determine the presence of fungal contamination in grape musts among other applications [57].
Nanobiotechnology is presently primarily concerned with developing implantable biofuel cells that harvest energy from renewable sources, such as sunlight. Preliminary results of an enzyme biofuel cell operating in vivo in orange were recently presented at a conference [127]. Anode and cathode catalytic electrodes comprising immobilized fructose dehydrogenase and glucose dehydrogenase on the anode and laccase from T. versicolor on the cathode formed the basis of the biofuel cell design. In this experiment, a cathode and anode pair were implanted in orange pulp, enabling electricity to be recovered from it (the glucose and fructose in the juice). An electronic gadget was powered by the energy taken from the orange after it was charged with the energy from the orange. It is a recent trend in biomaterial and biomedical research to develop bioresponsive polymers that may be used to detect potentially hazardous microorganisms. Lysozyme is a protein found in infected wound fluids and cellulases produced by potentially dangerous microbes [201].

Pharmaceuticals and Cosmetics
Given that laccases are very precise biomolecules, pharmaceutical companies use this enzyme to create a wide range of complex pharmaceutical chemicals, including anesthetic medicines, anti-inflammatory medications, antibiotics, sedatives, and other sedatives [130]. According to preliminary research, laccase-based hair dyes, which have recently been created as cosmetics for skin whitening, may be less irritating and safer than currently available hair colors. Deodorants, toothpaste, mouthwash, detergent, soap, and diapers might all benefit from the use of protein altered laccase, which has a lower allergenicity than regular laccase [171].

Biosensors
Laccase has been widely used in electrocatalytic oxygen reduction and in developing biosensors for phenolic substrates, among other applications [227]. A major difference between laccase biosensors and peroxidase biosensors is that they do not react with phenolic substrates to produce hydrogen peroxide. In addition to having poor enzyme stability and being significantly inhibited by reaction products, biosensors based on tyrosinase have also been less effective [88]. On the other hand, laccase is a promising choice for use in phenolic chemical detection biosensors.
Lignin biosynthesis, oxidative polymerization of monolignols, and a variety of other physiological processes are all facilitated by laccases. Laccases are multifunctional enzymes found in plants involved in cytokine homeostasis, phenolic pollution resistance, flavonoid polymerization in seed coats, anthocyanin degradation, and iron metabolism, When it comes to laccases, they play a wide variety of functions in many different types of cells and organisms at various developmental stages. Given their high oxidation potential, they are significant proteins in a range of industries, including the foodstuff and textile industries, the paper and fuel industries, and the environment. In order to commercialize laccases, it is necessary to overexpress them in plants and yeasts. Laccase-expressing transgenic plants that secrete recombinant laccase, notably by rhizosecretion, are being studied as new phyroremediation options. Figure 6 represents the development of an enzyme-based biosensing device and its mechanistic role.

Concluding Remarks and Perspectives
Laccases have received increasing attention as exceptional biocatalytic tools for a wide range of biotechnological applications owing to their inherent ability to oxidize a wide variety of phenolic and non-phenolic compounds. Therefore, the biotechnological importance of these enzymes has led to a marked increase in their demand. A multimillion-dollar industry for laccases is being developed and commercialized, and it is expected to grow in the coming years. However, despite its broad scope, most of the laccase research is still carried out on a laboratory scale or under controlled laboratory circumstances. More attention should be focused on the research of scaling-up procedures for manufacturing, economic analyses, and the expansion of laccase use in various applications. Furthermore, implicating laccasebased biocatalytic systems in actual or simulated scenarios (e.g., real textile effluents) will effectively evaluate whether these enzymes can be used on a larger scale in the future. These difficulties constitute a significant hurdle for researchers who want to pursue novel approaches to using laccases as sustainable biocatalysts. The high cost of redox mediators and their inhibiting effect on laccase activities are another important issue. In this juncture, implementing a laccasemediator system based on natural mediators seems a promising choice requiring further investigation. In addition, designing laccases with tailor-made characteristics via protein engineering and directed evolution is likely to expand the portfolio of highly efficient enzyme variants for a large range of entirely new areas of application where enzymes have not been employed previously. Laccases are enzymes that have a broad range of roles in a variety of species that express them at various developmental stages. Because of their high oxidation potential, they have become important proteins with many uses in various sectors, including the food, textile, paper, fuel, and environmental sections of the economy. Overexpression of laccases in plants and yeasts is being pursued as part of efforts to commercialize the enzymes. Additionally, laccase-expressing transgenic plants secrete recombinant laccase, particularly through rhizosecretion, are being considered novel candidates for phytoremediation applications. Because of this, laccase is attracting the attention of scientists worldwide.