Keywords

1 Introduction

Around 2.45 billion years ago, oxygen (O2) concentration in the biosphere increased which gradually oxidized water-soluble iron (Fe) II to water-insoluble Fe III. Due to Fe III’s insolubility, iron was not readily available to the living systems for their metabolic processes. Under this evolutionary pressure, living systems such as aerobic organisms were forced to find naturally available iron-like metals with high redox potentials. As a response, they started utilizing copper (Cu II/Cu I) and manganese (Mn III/Mn II) which had a similar function as iron (Fe II/Fe III) (Andrews et al. 2003). Copper-containing proteins are mostly are extracellular (Crichton and Pierre 2001). They help with O2 transport and activation, as well as electron transfer during redox reactions. These proteins are multicopper oxidases (MCOs) and can oxidize huge range of substrates with help of O2 as an electron acceptor and function as electron transfer proteins (Janusz et al. 2020). One such interesting MCO is laccase. Laccase was first described by Yoshida (1883) that he found in exudates of Rhus vernicifera. Laccases along with peroxidases help in the development of plant cell walls. The presence of high levels of laccase-like MCO and its expression in vascular tissues of Liriodendron tulipifera indicated the requirement for the uptake of high-efficiency iron pumps in lignified tissues (Hoopes and Dean 2004). Laccases found in Anarcardiaceae resin ducts are thought to aid in defense against herbivores as well as a bacterial and fungal invasion (Mayer and Staples 2002). Most of the high redox potentials laccases are from fungi havng biotechnological and industrial significance (Nunes and Kunamneni 2018). It has been detected in several fungal strains and its production is most efficient in white-rot fungi (Shraddha et al. 2011). Fungal laccases only need oxygen and produce water as a byproduct. Because of their requirements and broad substrate specificity, they are regarded as green catalysts with biotechnological applications, including direct bio-electrocatalysis. Laccase and laccase-mediator system (LMS) have its applications in delignification (Virk et al. 2012), biocomposites (Nasir et al. 2014), biobleaching of pulp (Boruah et al. 2019), removal of aromatic pollutants (Khambhaty et al. 2015), treatment of industrial wastewater (Viswanath et al. 2014), biofuel cells and biosensors (Le Goff et al. 2015; Ribeiro et al. 2014) and degradation of diclofenac (DCF) and chloramphenicol (CAP) by laccase in presence mediators (Nguyen et al. 2014). Laccase has sparked tremendous interest for prospective biotechnological applications due to its catalytic characteristics (Abdel-Hamid et al. 2013). Laccase TEMPO oxidation treatment has been used used on cotton fibers for grafting octadecylamine grafting that enhanced the hydrophobic nature of the fiber (Ding et al. 2016). Bertrand et al. (2002) found the primary catalytic function of laccase in the lignification process. In the following year, laccase was applied in bioremediation processes. Pozdnyakova et al. (2006), demonstrated that laccase was used as a degradation tool to degrade polycyclic aromatic hydrocarbons (PAHs). Thus, with more knowledge and research, laccase was utilized in various industries such as food processing, textile industries, and wine stabilization. Table 1 gives an outline of the research varied out with laccase since 2000 to present 2021.

Table 1 Timeline and application of laccase
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Thus, the present chapter would discuss the general mechanism of action of laccase, the role of laccase in the synthesis of fibreboard, and the mechanism for the synthesis of biocomposite. Further other reported applications of laccases its limitation and the future prospect have also been elaborated.

2 A Fascinating MCO-Laccase

Laccases (EC 1.10.3.2) bio-catalyze a electron (e−) oxidation of substrates and then passes four (e−) to the catalytic copper (Cu) atoms, that are oxidized without releasing partially reduced O2 called reactive oxygen species (ROS) (Janusz et al. 2020; Mehra et al. 2018).

2.1 Structure of Laccase

The 3D structure of MCOs is mainly constructed of β-sheets and turns. They contain a 10–20 kDa sized cupredoxin- like domain. MCOs are mainly of 3 types—2-domain, 3-domain, and 6-domain enzymes. Laccase consist of Greek key β barrel topology and it is ~500 amino acid residues structured in three successive domains. The first domain consists of 150 amino acids, second domain from 150 to 300 amino acids, and the third domain from 300 to 500 amino acids. The presence of disulfide bonds in-between the domains I and II and between I and III stabilizes the structure of laccase (Bertrand et al. 2002; Plácido and Capareda 2015). The structure of laccase has been studied using crystallography, isolating plant and animal laccase as crystals had been difficult to obtain due to the unavailability of proper purification protocols. Despite their broad taxonomic distribution and variety of substrates, it has been demonstrated that Cu in laccases exists in four different Cu catalytic forms per protein unit. These four catalytic Cu atoms are type 1 Cu (T1 Cu) and tri-nuclear Cu clusters (T2 Cu, T3α Cu, and T3β Cu) at the T2/T3 site across all multicopper oxidases. These four Cu ions are divided into three types of structures: Type 1 (paramagnetic ‘blue’ Cu), Type 2 (paramagnetic “normal/non-blue” Cu) and, Type 3 (diamagnetic spin coupled Cu-Cu pair). The majority of the proteins are represented in Table 2.

Table 2 Sources and application of laccase in various industries
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2.2 General Mode of Action

Catalytic participation of laccase’s in coupling reactions is dependent on C–C, C–N, and C–O molecule linkages. Laccase cleaves phenolic components in three ways: Cα–Cβ cleavage, Cα oxidation, and aryl–alkyl cleavage. In laccase-catalyzed oxidation, reaction the initial e− acceptor is T1 Cu that is situated in the cavity near the enzyme surface. The reduction of T1 Cu is a rate-limiting step and the internal electron then moves from T1 to T2 to T3 Cu. Meanwhile, at T2 and T3 Cu sites, O2 is reduced to H2O. Laccase converts phenolic compounds to phenoxyl radicals, which are then polymerized by radical rearrangement or coupling. However, based on the stability of the phenoxyl radicals, redox reversibility with oxidation of a targeted substrate is observed. By acting as mediators, radical-based coupling/redox recycling of phenolic substrates broadens the spectrum of laccase substrates (Patel et al. 2019; Agrawal et al. 2018; Kunamneni et al. 2007).

3 Laccase in the Synthesis of Biocomposite

The bio/wood composite is made by the use of two components the i.e., the wood fiber and the adhesive. In the case of synthetic adhesive, formaldehyde and phenol formaldehyde are generally used. However, due to its toxic and harsh effects, the shift has occurred towards the biological synthesis of bio/wood composites (González-García et al. 2011; Moubarik et al. 2010). Also, the Government of Korea stated that the emission level above 4.0 mg/m2.h for the total volatile organic compound (TVOC) is prohibited (JIS A 1901, small chamber method) (ASTM-D6007-96 1996; Kim et al. 2007). The lignin component of the plants is the second most abundantly available polymer after cellulose. As lignin has structural similarity to the phenol-formaldehyde it has been regarded as a potential substitute for the already available synthetic adhesive (Zhou et al. 2011; Kumar et al. 2009). However, despite the two advantages i.e., high availability and a potential substitute for synthetic adhesive it has restricted use as most (80–85%) lignin available are either burned or discarded (Vishtal and Kraslawski 2011; Mai et al. 2000; Pizzi 2003). Thus, the use of lignin can be a game-changer for the industry of biocomposites and as it is a renewable resource the fear of its scarcity in the future would not be an issue (Agrawal and Verma 2020d).

4 Mechanism of Action of Laccase on Plant Fiber

The fiber modification has been an integral part of the synthesis of biocomposite and various physical-chemical, methods have been applied and reported in literature e.g., alkaline, microwave, high temperature, and steam treatments (Verma et al. 2005, 2009, 2011; Verma and Mai 2010). However, due to high cost, energy requirement, and less environmental sustainability, the past decades have been diverted towards the biological and enzyme-mediated treatment of fibers. Laccase is ubiquitous and multi-dimensional protein and has been used for the removal of lignin (Agrawal et al. 2019; Agrawal and Verma 2020a). The biological treatment methods are milder, specific, and more sustainable and cause minimal/no damage to the biological structure of the fiber (Kunamneni et al. 2008). Laccase enzyme is large and cannot penetrate the cells of the fiber it only results in surface modification (van de Pas et al. 2011). It acts on phenolic polymers of lignin with the resulting in reduction of O2 to H2O (Witayakran and Ragauskas 2009). It is due to these properties that laccase is an intensively studied oxidoreductase having numerous applications and recently in biocomposite synthesis (Agrawal et al. 2019; Agrawal and Verma 2020a, e). Also, laccase-mediated oxidation of lignin, free radicals of phenol and polyphenols are formed. As these free radicals are highly reactive it results in depolymerization, co-polymerization, and grafting (Saastamoinen et al. 2012). Further, the structure of lignin exhibits similarity to phenol-formaldehyde and can thus be a potential adhesive for the synthesis of biocomposite (Zhou et al. 2011; Kumar et al. 2009). Despite the advantage of lignin, the major drawback is its transformation to insoluble lignin and thus requires additional cross-linking e.g., maleic anhydride (Syukri et al. 2021). The laccase mediated treatment also has numerous advantages such as improvement in crystallinity index (Agrawal et al. 2019; Agrawal and Verma 2020a) removal of amorphous phenolic and non-phenolic components with no effect on the microfibril core that ultimately enhances the crystallinity of the cellulose of the fiber along with surface modification to form an effective biocomposite (Nasir et al. 2015) (Fig. 1).

Fig. 1
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Schematic representation of the laccase-mediated synthesis of biocomposite

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5 Other Applications of Laccases

An overview of the various scientific and industrial applications has been represented in Fig. 2 and has been elaborated in the following section.

Fig. 2
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Applications of laccase in different industrial and scientific sectors

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5.1 Paper and Pulp Industry

Wood is made of small wood fibers that are adhered to by lignin. To separate these wood fibers chemical and physical methods of pulping are used. In chemical pulping fibers are separated by dissolving and degrading lignin using chemical agents whereas in physical pulping fibers are physically ripped apart (Bilal et al. 2019a, b; Singh et al. (2015). Pulping is followed by sheeting which results in the production of paper. Chlorine-based chemicals are used for pulp bleaching; as a result, chlorinated aliphatic and aromatic compounds are formed. The compounds are said to be carcinogenic, mutagenic, and toxic. Extensive research has been undertaken in recent years to develop environmentally sustainable enzymatic bleaching technologies. Pulp bio-bleaching has been demonstrated using laccase-mediated systems, but the lack of proper and cheap mediators has hampered their practicality. Laccases can remove potentially toxic phenols produced during lignin degradation, allowing them to depolymerize lignin and delignify wood pulps. Laccase starts by interacting with small phenolic fragments of lignin, which then react to degrade with the lignin polymer. Moreover, the use of ligninolytic fungi to pretreat wood chips strengthens the pulp while lowering the energy required for mechanical pulping. It is also used to reduce the kappa number of pulp and improve the pulp’s papermaking properties. Thus, the use of laccases in bio-bleaching processes in the pulp and paper industry is an environmentally safe approach (Bilal et al. 2019a, b; Virk et al. 2012).

5.2 Dye Degradation

Massive quantities of wastewaters are released by the textile industries which are contaminated by a large spectrum of chemicals for example azo dyes, which are the primary source of environmental pollution (Paździor et al. 2019). For the environment’s safety, treatment of industrial wastewater has become very important before its safe release into the environment (Salem et al. 2019). These effluents contain recalcitrant dyes (e.g., azo) that pollute the freshwater with their color and carcinogenic intermediates such as the aromatic amines. These chemical reagents are usually complex, synthetic and are unaffected to decolorization in presence of H2O, light, and different chemicals. They are also resistant to various existing dye degradation methods e.g., chemical treatments that are ineffective, and results in the production of intermediate compounds that are mutagenic or carcinogenic (Bilal et al. 2019a, b). As a result, the laccase-assisted dye bioremediation has gained interest due to their diverse potential for the degradation of various dyes via sustainable approach (Couto and Toca-Herrera 2006; Verma 2001; Verma and Madamwar 2002a, b). Since traditional treatment methods based on chemical or physical processes are extremely costly and involve massive quantities of resources, various techniques have recently been investigated as alternatives. Laccase due to its ability to catalyze reactions that can degrade a wide variety of pollutants. For textile wastewater treatment, many aerobic and anaerobic bioprocesses have been developed and extensive research has been done fungal laccases for the production of laccase to improve bioprocesses for the degradation of dyes (Verma and Madamwar 2005). The majority of current dye wastewater treatment processes are inefficient and costly. As a result of their ability to degrade dyes fungal laccase mediated remediation of dyes may provide an appealing solution for a sustainable future.

5.3 Bioremediation

Major issues globally today are polluted air, soil, and water that have disastrous consequences. Industrialization and the widespread use of pesticides in agriculture, contamination of the atmosphere is major problem. Industries have been subjected to stringent regulations to handle their waste effluents before their discharge. Numerous remediation strategies have been reported but only a few have been adopted by the industries. Recently, the ability of fungi to transmute diverse chemicals has sparked the interest of the scientific community (Bollag et al. 2003). Also, low cost, high efficiency, and environmental friendliness it has been considered as a feasible alternative to the pre-established chemical-physical methods (Balcázar-López et al. 2016). Further, enzymatic therapy is now being considered as an substitute strategy for the removal of xenobiotics (Balcázar-López et al. 2016). Laccases can remediate polluted soils via immobilization as they can oxidize toxic organic contaminants including chlorophenols, PAHs, etc. (Zhang et al. 2008; Niu et al. 2013). Farnet et al. (2000) investigated the ability of Marasmius quercophilus laccase to treatalkylphenols. Saparrat et al. (2010) investigated the detoxification of “alpeorujo,” which is a solid by-product from the olive oil extraction industry by Coriolopsis rigida laccase. Laccase has been reported for the removal of dichlorodiphenyltrichloroethane (Yuechun et al. 2010) and 2,4-dichlorophenol (Bhattacharya et al. 2009). The degradation of PAHs by Pleurotus ostreatus laccase has been reported by Pozdnyakova et al. (2006) and high tannin from wastewater by Coriolopsis gallica laccase (Yagüe et al. 2000).

5.4 Food Processing

Laccases have a lot of potential as food additives and manufacturing aids in the food industry (Osma et al. 2010). Laccase-based biocatalysts are energy-efficient and biodegradable, making them ideal for food industries and also to produce low-cost, nutritious foods (Brijwani et al. 2010). Laccases can reduce food processing costs while still being environmentally friendly and to fully realize its ability a detailed understanding of their mode of action is required. Laccase’s versatility in action and widespread presence in many fungi species attest to its ease of use in biotechnological processes. Despite the presence of turbidity, after treatment with laccase and active filtration color consistency improved in fruit juices. Also, the phenolic content of juices reduced after laccase treatment along with stability of color (Ribeiro et al. 2010). Dough enhancement additives are added in bread-making process to improve its taste, texture, volume, and freshness. Thus, laccase addition in dough had an oxidizing effect thereby increasing gluten structures strength in baked goods. Also it improved the crumb structure, softness, increased volume, stability, weight, and, reduced stickiness. It also has to be noted that where laccase decreased extensibility in both flour and gluten dough and increased its resistance. The laccase and proteolytic enzymes when added to oat flour increased loaf specific volume and reduced crumb stiffness, chewiness respectively, and eventually improved its texture. Also, Jurado et al. (2009) stated that the induction of laccase acts as a fermentation inhibitor and increased the output of ethanol from steam-exploded wheat straw and reduced phenolic compounds (Larsson et al. 1999). The polymerization of phenols and polyphenols and the natural co-oxidation reactions have resulted in unwanted fragrance and color changes (Ribeiro et al. 2010). Thus, laccase has been reported and used for the clarification of fruit juices (Narnoliya et al. 2019). Giovanelli and Ravasini (1993) investigated the use of laccase along with filtration for stabilizing apple juice. Phenols were removed more efficiently by laccase treatment over other treatments, such as activated coals (Brijwani et al. 2010). Ribeiro et al. (2010) stated that treatment by laccase significantly decreased the phenolic content of juices while increasing color stability. It has also been found to be more beneficial as compared to traditional treatments e.g., addition of ascorbic acid and sulfites along with the enhancement of its functionality as well as sensory properties. Laccase also contributes in beverage stabilization, role in overall food quality improvement, and use in the baking industry (Manhivi et al. 2018; Di Fusco et al. 2010). Further knowledge of laccase kinetic parameters would be beneficial for functional applications of the enzyme.

5.5 Personal Care Applications

Laccase-generated products contain antimicrobial, detoxifying, or personal care active ingredients and has been used to synthesize anesthetics, anti-inflammatory medicines, etc. (Upadhyay et al. 2016). Couto and Toca-Herrera (2006) stated that the dyeing formula’s hydrogen substitution method based on laccase can resolve the inconvenience of chemical dyes by replacing the hydrogen with oxide. In recent years, skin lightening has been also used for cosmetics and dermatological preparations containing staining proteins. Laccase can be used as fragrant agents in personal care items such as toothpaste, mouthwash, detergent, and soap.

5.6 Pharmaceutical Pollutants

Active pharmaceutical ingredients have been detected in wastewater, and no effective method for the removal of are currently in use at large scale. Also these pollutants when released in water severely damages the aquatic environment or drinking water sources (Sui et al. 2010). This perilous condition necessitated the creation of a system for effectively removing pharmaceutical-based pollutants from wastewater. Researchers have confirmed bioremediation and removal of various pharmaceutically active ingredients using laccase (Rana et al. 2017; Xu et al. 2015). Lonappan et al. (2018) confirmed DCF biodegradation by immobilized laccase and enzyme’s binding improved when biochars were pretreated with citric acid. Remarkably, mature pig biochar immobilized laccase demonstrated a notable ability to fully extract DCF (500 μg L−1) in 2 h. Naghdi et al. (2017) investigated the removal of carbamazepine by immobilized laccase. After three cycles of reusability, the immobilized biocatalytic device retained 70% of its original operation and removed 83% of the carbamazepine from the spiked water. In a study by Taheran et al. (2017) used polyacrylonitrile-biochar composite that was home-prepared for laccase immobilization to degrade chlortetracycline from aqueous solution medium. Furthermore, the composite nanofibrous membrane-immobilized laccase demonstrated notable chlortetracycline removal efficacy (Taheran et al. 2017).

5.7 Biosensor

Oxidation of various organic pollutants, present in wastewater, especially phenolic compounds is catalyzed by laccases. It has a significant effect on the production of biosensors for both environmental and clinically relevant metabolites and it does not need any cofactors for e− transfer reactions. Due to laccase’s wide substrate range in biosensor technology, a large range of phenolics and azides can be detected (Rodríguez-Delgado et al. 2015; Sezgintürk et al. 2005). Laccase coupled multi-walled carbon nano tubes-based biosensors are used to calculate the polyphenol index in wines. A bio-sensor based on laccases coupled with multi-walled carbons nano-tubes measures the index of polyphenols in wine. This biosensor gives a clear and fast amperometric response to gallic acid (Di Fusco et al. 2010). The ultrasensitive amperometric detection of nanomolar catecholamine neurotransmitters (dopamine, epinephrine, and norepinephrine) is achieved by co-immobilization-based enzyme electrodes and laccase on glassy carbon electrodes. The enzyme’s selectivity to different phenolic compounds has been altered by the hybrid material of Nafion/sol-gel silicate used to immobilize laccase (Abdullah et al. 2007).

5.8 Miscellaneous Pollutants

As the population is increasing, agriculture production is being improved. This has led to heavy industrialization and excessive use of pesticides, which has caused a dreadful environmental condition. This has polluted the soil, water and, air with toxic chemicals which can create havoc on human health and climate. Due to these factors, it has become a major concern for the world. Potentially hazardous substances such as fungicides, herbicides, pesticides pharmaceutical compounds, phenolic compounds, PPCPs, and recalcitrant synthetic compounds can be biodegraded by laccase. Bisphenol-A, which has a carcinogenic effect, can be degraded by glutaraldehyde cross-linked chitosan beads. Laccase can degrade a wide range of substances, including polyvinyl chloride (Sumathi et al. 2016), xenobiotics e.g., polynuclear aromatic hydrocarbons (Dias et al. 2003; Cañas et al. 2007), polychlorinated biphenyls (Keum and Li 2004), etc. Laccase catalysis is used to regulate contaminants in the environment where fungal laccases can efficiently degrade and mineralize a variety of environmental contaminants, including BPA (Uchida et al. 2001), chlorophenol (Gaitan et al. 2011), nonylphenol (Tsutsumi et al. 2001), and chlorinated hydroxyl biphenyl (Schultz et al. 2001). It has also been used for the removal of 2,4,6-trinitrotoluene (TNT) (Cheong et al. 2006) and catechol (Tušek et al. 2017).

6 Limitations and Future Aspects

The main limitations of the application aspect using laccase are deactivation factors such as inhibitors, elevated pH, temperature, and non-reusability of free laccase. These drawbacks can be mitigated using new systems such as laccase-mediator or immobilized-laccase catalyzed systems. The lack of capacity to produce large quantities of active enzymes prevents its utility on a large scale. However, these issues can be addressed by recombinant organisms or screening for naturally hypersecretory strains. Thus, strain proficient of producing high titre of a suitable enzyme should be selected followed by optimization of the conditions for laccase production. Recent biotechnological advances, particularly in protein engineering and directed evolution, have enabled essential tools for the efficient development of better enzymes with improved properties with better applicability. Also, the production of new enzymes has been tailored to completely new areas of application where enzymes had not previously been used. Although laccase is still produced in limited quantities, their prospective ability is immense; many of these remain to be revealed. Enzyme immobilization could be used to overcome these limitations while also boosting biodegradation efficiency and enzyme reuse. Since the discovery of laccase, its use has expanded in a variety of sectors and has gained significant interest in the synthesis of biocomposites.

7 Conclusion

Laccase has tremendous potential in the application of biocomposite using plant fibers and the research must now be directed toward less focused aspects of the enzyme to broaden the enzyme’s applications. One of the major limitations of using laccase is the high cost of downstream processes such as laccase purification that raises the overall cost of production, preventing it from being commercialized. As a result, research should concentrate on the development of more efficient and cost-effective methods for large-scale production and commercialization of laccase-based applications. It would facilitate the development of a “greener” approach for a “clean” environment by contributing towards chemical-free treatment in industries, development of a chemical-free biocomposite.