Engineered tyrosinases with broadened bio-catalysis scope: immobilization using nanocarriers and applications

Abstract

Enzyme immobilization is a widely used technology for creating more stable, active, and reusable biocatalysts. The immobilization process also improves the enzyme's operating efficiency in industrial applications. Various support matrices have been designed and developed to enhance the biocatalytic efficiency of immobilized enzymes. Given their unique physicochemical attributes, including substantial surface area, rigidity, semi-conductivity, high enzyme loading, hyper catalytic activity, and size-assisted optical properties, nanomaterials have emerged as fascinating matrices for enzyme immobilization. Tyrosinase is a copper-containing monooxygenase that catalyzes the o-hydroxylation of monophenols to catechols and o-quinones. This enzyme possesses a wide range of uses in the medical, biotechnological, and food sectors. This article summarizes an array of nanostructured materials as carrier matrices for tyrosinase immobilization. Following a detailed background overview, various nanomaterials, as immobilization support matrices, including carbon nanotubes (CNTs), carbon dots (CDs), carbon black (CB), nanofibers, Graphene nanocomposite, platinum nanoparticles, nano-sized magnetic particles, lignin nanoparticles, layered double hydroxide (LDH) nanomaterials, gold nanoparticles (AuNPs), and zinc oxide nanoparticles have been discussed. Next, applied perspectives have been spotlights with particular reference to environmental pollutant sensing, phenolic compounds detection, pharmaceutical, and food industry (e.g., cereal processing, dairy processing, and meat processing), along with other miscellaneous applications.

Introduction

Enzymes are effective biocatalysts that provide competitive processes than traditional (chemical) catalysts and significantly increase reaction rates in various chemical and biological processes (Choi et al. 2015). There is a need for reliable biocatalysts as enzymatic bioremediation is recently gaining high research interest as a "green" solution (Das et al. 2018). Owing to the unique characteristics, including stability, refinement, and specificity, several forms of enzymes and related materials with high catalytic potentials are commonly used in several industries, including dairy, fabric, medicines, water management, etc. (Du et al. 2017; Al-Rawi et al. 2020; Cunha et al. 2020). Biocatalyst stability is an essential constraint for their manufacturing applications since they must withstand intense reaction circumstances, such as high temperature and excessive pH (Madhavan et al. 2017). However, due to certain limitations of free enzymes, such as susceptibility to harsh environment, poor activity and stability, no recoverability, limited reusability and so on, the use of solvable enzymes in the chemical and biotechnology industries is restricted (Liu et al. 2017).

Tyrosinase (EC 1.14.18.1) is a monooxygenase that contains a Cu molecule in its catalytic site (Fig. 1) (Zaidi et al. 2014). This enzyme is involved in the o-hydroxylation and oxidation of monophenols resulting in catechols and o-quinones, respectively (Fujieda et al. 2020). It catalyzes the ortho-hydroxylation of tyrosine (monophenol) to 3,4-dihydroxyphenylalanine or DOPA (o-diphenol) and the oxidation of DOPA to dopaquinone. It is also active in the biosynthetic pathways of melanin (o-quinone) that can be converted into melanin pigments (Zaidi et al. 2014). In addition to the biochemical substrates tyrosine and L-DOPA, tyrosinase converts a variety of phenols and diphenols to the related diphenols and quinones (Matoba et al. 2018). CuO₂ (II) or a bis (µ-oxo) dicopper (III) is thought to be involved in the transition of an oxygen atom to the phenol substrate (Fig. 2) (Pillaiyar et al. 2018). The resonance Raman spectra of these intermediates are distinctive. Versions of individual intermediate complexes and confirmation for prompt stability among the two modes are provided by synthetic studies (Rolff et al. 2011).

Fig. 1.

Structure of tyrosinase. Reprinted from Zaidi et al. (2014) with permission under the terms of the creative commons attribution license

Fig. 2.

An overview of catalytic cycles of tyrosinase. Reprinted from Pillaiyar et al. (2018) with permission from American chemical society

Tyrosinase activities (monophenol hydroxylase and diphenoloxidase) are the foundation of many industrial applications involving the detoxification of phenol containing wastewater and soil (dos Santosa et al. 2013). In therapeutic sectors, it is also used to manufacture o-diphenols as an indicator for melanoma patients (Gradilone et al. 2010), prodrugs activator (Jawaid et al. 2009), and food manufacturing for amendment of diet proteins (Monogioudi et al. 2011). There is a lot of evidence that this enzyme has a lot of promise in the foodstuff, medication, agronomic sectors, diagnostic and ecological activities (Wang et al. 2005). However, these widespread industrial applications are often hindered by a lack of operational stability, long-term shelf life, and cumbersome recovery and re-utilization. Immobilization of enzymes might solve these drawbacks by developing robust, stable, and insoluble biocatalysts (Das et al. 2017).

Enzymes can be quickly isolated and extracted from the reaction substrate, reducing the chance of free enzyme contamination of the finished product. Furthermore, using immobilized enzymes, it is possible to reduce the expense of the related processes. An effective immobilization can significantly improve enzyme's characteristics, such as stability at higher temperature and pH and increased catalytic efficiency (Tacias-Pascacio et al. 2017). It appears that new functionalized supports with low biodegradability, nontoxicity, and robust mechanical properties are needed for convenient and effective immobilization (Wahba 2017). As a result, great emphasis has been given to the immobilization of enzymes, including tyrosinase on various carriers. The synthesis of an appropriate and robust support matrices is crucial for effective immobilization. Traditional immobilization techniques, which are divided into chemical and physical processes, may be used to accomplish the said purpose (Khan et al. 2017). The weak attachment force between the enzyme and the support substance is one of the most detrimental physical approaches. This is why chemical immobilization and covalent bonding, which improve the stability of modified surfaces, are often suggested (Chung et al. 2017).

Strong support structures usually stabilize the enzymes' configuration, and thus, their functions are maintained (Husain 2017). Immobilized enzymes are thus more stable and resistant to environmental modifications than free enzymes (Wang et al. 2020). Furthermore, heterogeneous immobilized enzyme systems allow fast recovery of enzymes, multiple enzyme reuse, hyper-activity and stability, rapid reaction termination, and a wider range of bioreactor designs (Bernal et al. 2018). In recent years, there has been a lot of curiosity and focus on the ability of immobilized enzymes (Bommarius and Riebel-Bommarius 2004). Immobilized enzymes usually are more stable and simpler to manage than their free versions. Furthermore, the reaction products are not polluted with the enzyme (which is particularly beneficial in the food and pharmaceutical industries). In proteases, the rate of autolysis can be significantly slowed while they are immobilized (Massolini and Calleri 2005). When it comes to the efficiency of the immobilized enzyme mechanism, the matrix properties are crucial. Physical tolerance to compression, hydrophilicity, inertness to enzymes, ease of derivatization, biocompatibility, resistance to microbial attack, and low-cost are all desirable properties (Barry and O'Riordan 2016).

Various methodologies have been established for the immobilization of tyrosinase. Some of which include silica sol–gel composite films (Wang et al. 2000), tyrosinase–modified boron-doped diamond electrodes (Notsu et al. 2002; Zehani et al. 2015), tyrosinase entrapped in an agarose-guar gum (Tembe et al. 2007), glassy carbon electrode modified by gold nanoparticles (Sanz et al. 2005), tyrosinase-FeO₃ nanoparticles-chitosan (Wang et al. 2008a, b), use of polyaniline-carbon nanofibers or nanotubes (Zhang et al. 2009; Branzoi et al. 2010; Yin et al. 2010; Rodríguez Méndez et al. 2013; Apetrei and Apetrei 2015), diazonium salt (Cortina-Puig et al. 2010) or titania sol–gel matrix (Kochana et al. 2015).

This work summarizes recent advancements in the use of a variety of nanostructured materials as carrier matrices for tyrosinase immobilization. The review also spotlights the biocatalytic capabilities and applications of the immobilized tyrosinase-based engineered nano-biocatalytic systems in the medical, nutritional, and biotechnological sectors.

Nanomaterials for enzyme immobilization

Because of the exponential advancement in nanotechnology, nanomaterials and enzymes are increasingly being combined for use in biomedicine, biochemical engineering, ecological control, and biosensors (Chen et al. 2017a, b). Since 2014, there has been significant development in the field of enzyme nanomaterial interactions, including enzymatic alteration or deprivation of nanomaterials, and enzyme immobilization (Chen et al. 2017a, b).

Nanoparticles typically have a diameter of 1–100 nm and are made up of hundreds of atoms. Clusters of particles are described as particles with a diameter of less than 1 nm. Nanoparticles with less than 10 nm diameter are especially intriguing because they can be called almost entirely surface. After all, all atoms are on or near the surface (Shi et al. 2015). Nanostructures have been used to promote enzyme immobilization via various mechanisms, including adsorption, covalent binding, encapsulation, cross-linking and others (Yang et al. 2016). It should be noted that controlling enzyme–support interactions is critical for understanding the various implementations and improvements that can be made, and maintaining the orientation of the enzyme can be particularly important (large substrates, immobilization area) (Barbosa et al. 2013).

Nanomaterials have proven to be one of the most effective immobilization supports owing to their distinctive features, like their large surface area (Sharifi et al. 2020; Liang et al. 2020). Although the high surface-to-volume ratio is advantageous, Brownian motion will affect the action of enzymes immobilized on these matrices (Gupta et al. 2011). It can overcome the diffusional limitations of traditional reaction systems and enhance enzyme stacking per unit mass of support (Zhang et al. 2016). Micron-sized particles have been used as carriers or matrices for enzyme immobilization. The size employed by the carriers/matrices is much greater than that required for the enzyme molecules, which has been a source of concern. One of the main motivations for prompt effort on enzyme aggregates (without carriers) was the need to reduce bioreactor sizes (Bilal et al. 2019). Crosslinked enzyme clusters are a more modern variant of such enzyme aggregates (CLEA). The growing nanotechnology-based research interests and the focus on miniaturization have further strengthened the enzyme immobilization using different nanomaterials in various shapes and sizes (Orfanakis et al. 2018). The implication is that nanomaterials can aid in the reduction of bioreactor size. This is because a greater amount of biocatalyst should be loaded per unit weight of the carrier (Cho et al. 2020). This article summarizes recent progress on using an array of nanostructured materials as carrier matrices for tyrosinase immobilization, biocatalytic properties, and applications of the resulting immobilized tyrosinase-based engineered nano-biocatalytic systems in the medical, nutritional, and biotechnological sectors. The nanomaterials used for tyrosine immobilization are listed in Table 1.

Table 1.

Nanomaterials for immobilization of tyrosinase

Nanomaterial	Application	References
Carbon dots	Biosensor	Samdani et al. (2016) Mohammadi et al. (2011) Baluta et al. (2020)
Platinum nanoparticles along with graphene	Biosensor	Liu et al. (2011)
Carbon black	Biosensor	Nadifiyine et al. (2013) Ibáñez-Redín et al. (2018)
Carbon nanotubes	Biosensor	Kim et al. (2010)
Lignin nanoparticles/nanocapsules		Capecchi et al. (2018, 2019)
Magnetic nanoparticles	Degradation of phenolic compounds	Liu et al. (2018) Abdollahi et al. (2019)
Layered Double Hydroxide (LDH) nanomaterials	Biosensor	Soussou et al. (2017)
Nanofibers	Biosensor	Mercante et al. (2021)
Graphene nanocomposite	Biosensor	Yang et al. (2015) Tang et al. (2013) Reza et al. (2015)
Poly(diallyldimethylammonium Chloride) Capped Gold Nanoparticles	Biosensor	Chaimuangyong et al. (2019)
Zinc oxide	Biosensor	Liu et al. (2006) Gu et al. (2009)

Carbon nanotubes

Because of their unique electronic and mechanical qualities, carbon nanotubes (CNTs) have been intensively researched as electrode materials in recent years (Basiuk et al. 2019). CNTs are supposed to have intrinsic electrochemical qualities comparable to other carbon electrodes commonly applied in numerous electrochemical uses from a chemical standpoint. Compared to other carbon-based nanomaterials like C60 and C70, CNTs have somewhat distinct electrochemical characteristics (Schroeder et al. 2018). When used as an electrode supporter, CNTs' sensitive electrical properties enable them to arbitrate electron transfer reactions with electroactive species in solution. As a result, CNT-based electrodes have become commonplace in electrochemical sensing (Gupta et al. 2018). Electrodes modified with carbon nanotubes have been designed for biosensors (Tang et al. 2017). The CNT-coated electrodes are the most straightforward to create, as long as stable CNT suspensions are preserved. For the fabrication of the CNT/surfactant-modified electrode, suitable surfactants were selected to achieve high catalytic efficiency and strong selectivity against the analytes. However, there is little information about making polymer-modified CNT electrodes extremely catalytic and selective for analytes (Yin et al. 2017). Kim et al. (2010) used PAAc-g-MWNT and PMAn-g-MWNT to make tyrosinase-immobilized biosensors. The detection limits of the tyrosinase-immobilized biosensor dependent on PAAc-g-MWNT and PMAn in the phosphate-buffered solution are 0.2–0.9 mM concentration and 0.1–0.5 mM for phenol.

Carbon dots

Despite their unique qualities, such as hydrophilic nature, less toxic, comparatively high strength, and biochemically stability over time, carbon dots (CDs) are a promising sensor medium (Bonet-San-Emeterio et al. 2020; Ding et al. 2020). The use of the CDs medium to evaluate different compounds, emphasizing their fluorescent qualities, is recorded in the literature, for example, hydrazine in water (Hiremath et al. 2020). CDs are being used in electrochemical sensors for a diversity of uses, including the assessment of ascorbic acid (Zhou et al. 2020), riboflavin (Priyadarshini et al. 2020) and neurotransmitter (Mphuthi et al. 2016). Using a glassy carbon electrode (GCE) updated with FeMoO₄ nanorods, Samdani et al. (2016) anticipated an electrochemical approach for norepinephrine (NE) determination. Adopting cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques, synthesized nanorods were used to oxidase NE. The spectrophotometric response of NE revealed an immediate increase in current. Mohammadi et al. (2011) tested a different approach for detecting NE. SWCNTs and restrained tyrosinase were added to a glassy carbon electrode. For NE assessment, the DPV technique was used. The changed electrode's detection limit for NE was determined to be 0.1 M.

Baluta et al. (2020) used nontoxic reagents to develop a modern biosensor-based method to measure neurotransmitters as easily as possible. The Au-E biosensor was developed and built using tyrosinase immobilized on an electroactive coating of cysteamine and carbon nanoparticles covering the gold electrode. This sensing system used voltammetric techniques, such as CV and DPV, to calculate the catalytic oxidation of norepinephrine (NE) to NE quinone. The organized bio-system had excellent constraints, including a comprehensive direct range (1–200 M), a 196 nM limit of detection, a 312 nM limit of quantification, and high discrimination and sensitivity abilities. It's worth noting that the mentioned approach was effective in determining NE in real samples.

Carbon black

Owing to its lower price and exceptional physicochemical qualities, Carbon Black (CB) has risen to prominence as a carbon nanomaterial to apply electrochemical biosensors (Talarico et al. 2015). Since these attributes are heavily reliant on external interaction, the impact of functionalization has been extensively researched for a variety of uses. In the interim, the impact of CB fabrication on its characteristics in electroanalytical uses is still understudied (Silva et al. 2017). Carbon paste electrodes are common in electroanalysis because of their low surface current, impending high range, biochemical resistance, and ease of preparation from low-cost materials. Carbon has been used in various ways, from graphite, glassy carbon, carbon nanotubes, and more recently, carbon black (Arduini et al. 2010a, b; Deng et al. 2011). Carbon black was commonly used as a carbon substantial to manufacture fuel cells, lithium batteries, sodium batteries, and sensors for identifying gaseous phases (Martinez-Alvarez and Miranda-Hernandez 2008). Conversely, only a few carbon black-based biosensors have been identified (Razumiene et al. 2005). Arduini et al. (2011, 2012) conducted research that showed the appropriateness of carbon black as an electrochemical transducer.

A carbon black paste electrode was developed. This device showed significantly reduced noise compared to enzymatic sensors based on traditional graphite paste electrodes. Using catechol as a substrate, the response was linear in the range 0.013 to 150 µmol L⁻¹ with a detection limit of 6 nmol L⁻¹ (based on three times the S/N ratio). Peroxidase and laccase biosensors were also prepared. The responses of these three enzymatic biosensors to twenty different phenolic compounds were investigated, taking into account their molecular structure and their specific enzyme activity relationship. In addition, another sensor was reported based on semipurified tyrosinase. There was a good statistical correlation between the results obtained with tyrosinase biosensor and Folin–Ciocalteu spectrophotometric methods for phenol determination in olive oils.

Nadifiyine et al. designed a prototype biosensor established by the halt of commercially available tyrosinase onto a carbon black paste electrode (2013). As compared to enzymatic sensors using conventional graphite paste electrodes, this instrument produced significantly less noise. The output was linear in the range of 0.013 to 150 mol/1 using catechol as the substrate, with a diagnosis limit of 6 nmol/L. Biosensors for peroxidase and laccase were also created. The contributions of biosensors to 20 distinct phenolic substances were studied, with their structure and activity association taken into account. Ibáez-Redn et al. (2018) identified the production of responsive electrochemical biosensors. The chemical pre-treatment improved the hydrophilicity of the substance by increasing the amount of exterior oxygenated functional groups, as measured by elemental analysis and FTIR. Compared to unfunctionalized CB, the electron transfer rate constant (k0) was observed to be nearly 100-fold higher, indicating a significant increase in electrocatalytic properties. They developed a tyrosinase biosensor for catechol determination in water samples using functionalized CB and di-hexadecyl phosphate (DHP). Their findings show that functionalized CB is an ideal medium to produce biosensors.

Nanofibers

Nanofibers are effective platforms for immobilizing biomolecules for further use in biosensing (Andre et al. 2021). Electrospinning is a novel tool that blends low price, easiness of processing, and implementation for a wide range of materials, among other techniques for manufacturing such nanofibers (Mercante et al. 2017). Electrospinning is an electrohydrodynamic method that produces fibers with diameters ranging from nm to m by uniaxially stretching and elongating a viscoelastic solution. The nanofibrous membranes have a high surface-to-volume ratio, a porous integrated configuration, a low diffusion barrier, and amendable flexibility (Li et al. 2019). These characteristics allow for the development of hybrid and composite nanofibrous films with high sensitivity and selectivity for various analytes (Yang et al. 2020). Furthermore, the nanofibrous structure allows for high enzyme loadings, which is advantageous for biosensing efficiency. Regardless of the appropriateness of electrospun nanofibers, adjusting the configuration of the nanofibrous film to attain high performance for an assumed analyte requires significant effort (Jankowska et al. 2020).

Mercante et al. (2021) identified a polymeric electrospun nanofiber of polyamide 6 (PA6) and poly(allylamine hydrochloride) (PAH) ornamented with gold nanoparticles (AuNPs), referred to as PA6/PAH@AuNPs, that was dumped onto a fluorine-doped tin oxide (FTO) substrate for perceiving low levels of Bisphenol A (BPA). The immobilization of tyrosinase by the hybrid layer was fantastic, allowing for amperometric identification of BPA with a maximum recognition of 0.011 M in the concentration of 0.05 to 20 M. The collective outcome of the bulky surface area and permeability of PA6/PAH nanofibers, the catalytic action of AuNPs, and Tyr's oxidoreductase capability is credited with the improved sensing efficiency. Such discoveries exposed the way to new biosensing architectures for monitoring BPA and other endocrine-disrupting chemicals (EDCs) in water supplies.

Graphene nanocomposite

Graphene has perfect and unusual properties, such as high charge transport agility, wide surface area, strong chemical stability, and mechanical strength (Du et al. 2011). As a result, it has sparked considerable interest in various technical areas, including nanoelectronics, nanocomposite, biosensing, and bioelectronics (Sun et al. 2011). To satisfy the desired condition in each application, graphene must be chemically functionalized, which can affect considerable reductions in the electrical conductivity of the functionalized graphene (Kuilla et al. 2010). This could be attributed to an increase in blemish quantity and drugging of shielding resources on the graphene surface, all of which would impair electron transport and affect electrical conductivity. As a result, research into combining graphene with other nanomaterials to attain graphene exterior amendment to improve conductivity is also required.

Several environmentally safe processes, such as electrodeposition, an association of metal nanoparticles on functionalized graphene top, and microwave therapy, have been employed to anchor noble metal nanoparticles on graphene nanosheets (Yin et al. 2015). For instance, Yang et al. (2015) used a green approach to anchor AuNPs onto carboxylic graphene nanosheets without adding any reductant or shielding particle. Tang et al. (2013) demonstrated the electrochemical deposition of reduced graphene oxide and gold nanoparticles onto a glassy carbon electrode (GCE) surface without using a harmful reductant. AuNPs have a suitable atmosphere for enzyme immobilization and promote electron transmission between the immobilized enzyme and the electrode, which is why they were chosen as the nanomaterial (Makhotkina and Kilmartin, 2010). Furthermore, graphene is an auspicious support matrix for metal nanoparticles owing to its wide surface area, electronic properties, and mechanical strength. Since these special requirements reduce AuNP agglomeration and improve their stability in the graphene nanosheet matrix, future use in biosensing has been investigated (Li et al. 2010).

Applications of graphene nanocomposites have recently piqued researchers' curiosity in various fields, especially in the construction of biosensors. Reza et al. (2015) developed a tyrosinase biosensor based on rGO/Chit to detect bisphenol A electrochemically. The reduced GO-AuNPs film for tyrosinase halt for phenol discovery has been investigated. Graphene–polyaniline and tyrosinase-modified GCE were also used to study electrochemical biosensors for phenolic derivatives. Additionally, graphene/Co3O4 was successfully employed as a medium for tyrosinase immobilization and catechol detection in fruit samples (Liang et al. 2016).

Platinum nanoparticles

Despite its peculiar qualities (Li et al. 2008; Wang et al. 2008a, b), like strong density, versatile electrical conductivity, and extraordinary heat resistance and physical attributes, graphene has piqued interest as a good resource (Shan et al. 2010). Furthermore, graphene's plane structure provides a large area for charging nanoparticles ideal for electrode amendment (Wang and Hu 2009). Because the incorporation of carbon-based substances and metal nanoparticles, such as CNT–Pt composites, has been shown to have a synergic effect in electrocatalytic applications, (Yao et al. 2006) reason to believe that graphene and platinum nanoparticles would have the same impact. Besides, when the pH is greater than 5, tyrosinase is negatively charged. As a result, electrostatic interference among a positively charged self-assembled mono-layer (SAM) and the negatively charged enzyme particles under an appropriate pH range is a viable way to immobilize tyrosinase (Love et al. 2005). For the identification of organophosphorus pesticides, Liu et al. (2011) created an amperometric biosensor. A glassy carbon electrode adapted with graphene and the enzyme tyrosinase restrained on platinum nanoparticles were used. Different pesticides can be identified in catechol as a substrate since they suppress the enzyme that mediates the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene improve the performance of the electrochemical removal of o-quinone, resulting in increased sensitivity.

Magnetic nanoparticles

The development of an effective method for recovering biocatalyst from reaction mixtures has received a lot of attention. Nano-sized magnetic particles have recently become popular as an excellent help for immobilizing biomolecules since they can be quickly retrieved from the reaction medium by inserting a powerful magnet close to the mixture (Xie and Zang 2017). Magnetic nanoparticles (MNPs) are a viable carrier for biomolecule immobilization artifacts due to their ease of separation with the aid of a magnetic field and their low toxicity (Khoshnevisan et al. 2017; Wang et al. 2016). MNPs may also be isolated from the media with an additional magnetic field (Bezerra et al. 2017).

Because of their excellent advantages of fast synthesis and isolation, magnetic nanoparticles are commonly employed as carriers for enzyme arrest (Feng et al. 2016). For more modifications, they are normally amino-functionalized. Amino-functionalization agents include 3-aminopropyl) triethoxysilane (APTES), polyethyleneimine (PEI), and dopamine (Li et al. 2015). However, these amino-functionalization processes are time-consuming and necessitate the use of assignable agents. The one-pot solvothermal synthesis of aminated magnetic nanoparticles (Fe3O4-NH2) appears to be a promising approach for avoiding the time-consuming amino-functionalization process (Martn et al. 2014).

To overcome the limitations of both the noncovalent and covalent processes, Liu et al. (2018) developed a procedure that fused the immobilization technique of physical adsorption with covalent crosslinking. Abdollahi et al. (2019) immobilized tyrosinase onto functionalized nanoparticles. Superparamagnetic nanoparticles were synthesized, and silica-coated using a simple process, and their ability for tyrosinase immobilization was demonstrated. A combination of tyrosinase and cyanuric chloride functionalized magnetic nanoparticles (Cyc/EMNPs) was shaken at room temperature to immobilize the enzyme.

Lignin nanoparticles

Because of their unique environmentally friendly qualities, green resources are thought to play a positive role in the Circular Economy (Velte and Steinhilper 2016). Paper and biorefinery industries produce more than 50 million tons of lignin per year, making it the most abundant organic polyphenol in nature (Ragauskas et al. 2014). Lignin derivatives are low-price ingredients that work like consequent petroleum equivalents, exhibiting antioxidant activity (Kaur and Uppal 2015), UV-shielding safety, anticorrosion possessions, and electrochemical sensitivity in all charge storing compounds and electrochemical sensors due to their aromatic character (Ding et al. 2016). As a result of the direct consequence of the "size-effect" phenomenon, lignin is incipient as an innovative and affordable base substance for synthesizing nanocapsules with improved physicochemical characteristics (Xiong et al. 2017). In theory, these nanoparticles may be effective polyphenol-based platforms for enzyme immobilization. The supramolecular connection between lignin and proteins has been investigated in-depth to recognize affinity lignin-binding peptide structures and determine the part of polymer structural variations in the molecular classification stage (Leskinen et al. 2017). Protein folding composition and Coulombic forces in the non-ionic medium have occurred as critical constraints in protein self-assembly on the lignin surface (Strobel et al. 2015; Westereng et al. 2015). Lignin and tyrosinase work together to achieve effective oxidative processes. Tyrosinase has been extensively researched due to its applications in skin health, cosmetics, and agriculture industries. When used in the immobilized state, it performs well in industrial applications due to its reusability, increased flexibility, and simplified purification procedures (Dinçer et al. 2012).

Guazzaroni et al. (2012) identified the processing of heterogeneous catalysts for the production of bioactive catechol derivatives by immobilizing tyrosinase on microcapsules of the epoxy resin Eupergit, or MWCNTs. The production of neuroactive 3,4-dihydroxyphenylalanine (DOPA)-containing peptides and lipophilic catechols with extensive spectrum antiviral activity against DNA and RNA viruses was aided by these catalysts, which had a high catalytic ability (Bizzarri et al. 2017; Botta et al. 2015). In general, nanostructured catalysts outperformed microcapsule counterparts in terms of activity, storage life, and reusability (Botta et al. 2017).

Capecchi et al. (2018) identified different methods for immobilizing tyrosinase in organosolv lignin (OL) nanoparticles. OL has a low molecular weight and a discrete solubility in organic solvents compared to native lignin and other scientific lignins. The novel catalysts have been characterized in terms of structural and kinetic properties, and they have been used to convert selected phenols to bioactive catechols in a series of experiments. Notably, behavior and kinetic parameters for lignin-dependent tyrosinase catalysts prepared by layer-by-layer assembly were on par with or better than those previously recorded for catalysts based on conventional inorganic supports.

Layered double hydroxide nanomaterials

Layered Double Hydroxide (LDH) nanomaterials are desirable candidates for electrochemical investigation in broad-spectrum and for enzyme arrest (Tonelli et al. 2013) between host materials previously used for the immobility of enzymes distinguishing polyphenols (Zehani et al. 2015). Subsequently, they display several strategic qualities in the production of hybrid biomaterials. LDHs are a unique type of substance made up of positively charged layers with charge-balancing anions sandwiched. Like their strong ion exchange potential or encapsulation efficiency, special characteristics are due to their unique nature. Finally, these substances have excellent biocompatibility, which is significant in the sense of enzyme immobilization (Shan et al. 2003). In addition, the immobilization of various types of biomolecules, such as amino acids or DNA, onto LDH films has been successfully tested. LDH's properties cause an enzyme to be immobilized while maintaining its automatic configuration without disrupting catalytic sites, minimizing any inhibition consequence (Baccar et al. 2012). Thus, they are also well suited to the production of electrochemical biosensors. Antibodies and enzymes have also been suggested as applications for this kind of substance (Temani et al. 2014). Hybrid matrices composed of chitosan and LDHs were identified in the case of tyrosinase (Han et al. 2007). The use of tyrosinase-MgAlCO3 LDH to functionalize glassy carbon electrodes has also been documented (Han et al. 2012). Furthermore, the chemical composition of LDH material can affect enzymatic behavior. Previous experiments found that tyrosinase is triggered in the existence of Co or Zn and inhibited in the presence of Ni, so they intended to immobilize it using an LDH content containing cobalt (Co1.57Al (OH) xSO4, shortened hereafter as CoAl) (Hidouri et al. 2011). Finally, the electrochemical activity of cobalt-based LDHs has been well studied, as cobalt has excellent electrochemical properties (Han et al. 2011).

For the construction of an electrochemical tyrosinase-based biosensor used for the identification of a hybrid combination of polyphenols isolated from green tea, Soussou et al. (2017) investigated gold screen-printed electrodes glazed with thin films of Layered Double Hydroxides (LDHs) containing Co and Al (Co1.57Al (OH) xSO4, shortened as CoAl). According to physicochemical studies, the resulting biosensor has excellent accuracy, a wide vibrant range, and very small detection confines, 0.33 pg/mL and 0.03 pg/mL for oxidation and reduction.

Gold nanoparticles

Gold nanoparticles (AuNPs) are an excellent option for biosensing electrodes because of their conductivity, durability, biocompatibility, and broad surface region. Gold nanoparticles (AuNPs) and carbon nanotubes (CNTs) were used for their outstanding physio-chemical qualities. AuNPs were discovered to have excellent conductivity, surface area, and catalytic properties (Chen et al. 2018). Rolling graphite sheets create carbon nanotubes (CNTs), which have quasi-one-dimensional (1D) shapes. It has good chemical stability, high surface area, excellent adsorption potential, excellent biocompatibility and can be used to promote electron transfer between electroactive species and electrodes (Amatatongchai et al. 2015). Furthermore, some polymer materials are used in conjunction with other materials. The polymer poly-(diallyl dimethylammonium chloride) (PDDA) was electrochemically dumped on the electrode exterior to create PDDA-capped AuNPs (PDDA@AuNPs), a polymer with high electrical conductivity and ionic power. These materials are being used to produce dopamine biosensors that are accurate, clear, inexpensive, and quick to analyze (Liu et al. 2006).

Chaimuangyong et al. (2019) developed dopamine biosensors with tyrosinase (Tyr)-deposited poly-(diallyldimethylammonium chloride)-capped gold nanoparticles (PDDA@AuNPs) composite and carbon nanotubes (CNTs)-adapted glassy carbon electrode (GCE) (Tyr/PDDA@AuNPs-CNTs/GCE). Dopamine was detected using cyclic voltammetric and amperometric techniques. The parameters of the applied potential, Tyr loading, PDDA@AuNPs number, and carbon nanotubes amount were all optimized. Under mild conditions, the modified electrode had high immunity with no intervention from uric acid, ascorbic acid, cholesterol, ethanol, or aspirin. It demonstrated high reliability for more than 30 years of use. With a maximum sense of 8.50 nM (S/N = 3), the dopamine biosensor showed two linear ranges of 10 nM to 100 nM (R2 = 0.9997) and 100 nM to 1 mM (R2 = 0.9979).

Zinc oxide nanoparticles

Apart from optoelectronic properties, nanostructural ZnO has several benefits for biosensors, including a high aspect ratio, a polar surface along the c-axis, strong electron contact, and nontoxicity (Wei et al. 2006). Notably, ZnO has a high isoelectric point (IEP) of around 9.5, making it ideal for immobilizing biomolecules with low IEP, such as enzymes and proteins, using electrostatic attraction at the suitable pH (Ahmad et al. 2010). Biosensors made of ZnO nanoparticles, porous film, nanocombs, and nanorods have been used to sense cytochrome c, protein, uric acid, glucose, and phenolic acid correspondingly (Wang et al. 2006; Taratula et al. 2006). The semiconductor ZnO is a very appealing material. Rubber, ceramics, paints, pharmaceuticals, and sensors all use bulk zinc oxide (Liu and Zeng, 2003). Topoglidis and colleagues described the immobilization and bioelectrochemistry of proteins on nanoporous TiO₂ and ZnO films, claiming ZnO with a high isoelectric point (9.5) was ideal for the adsorption of proteins with low isoelectric points (Topoglidis et al. 2001). As a result, ZnO nanoparticles (nano-ZnO) merit auxiliary study as a potential candidate for support material in biosensor fabrication.

Liu et al. (2006) established a phenol biosensor that is free of mediators. Tyrosinase with a low isoelectric point was adsorbed on the surface of ZnO nanoparticles (nano-ZnO) with a high isoelectric point, aided by electrostatic connections, and then incapacitated on the glassy carbon electrode by chitosan film formation. The nano-ZnO matrix was found to have a favorable microenvironment for tyrosinase loading due to its desirable isoelectric point and the immobilized tyrosinase maintaining its operation to a large degree. Furthermore, no other electron mediators are needed. The undeviating reduction of biocatalytically produced quinone species at 200 mV was used to evaluate phenolic compounds (vs. saturated calomel electrode). The enzyme electrode's fabrication parameters, as well as the different experimental variables, were augmented. The subsequent biosensor has a sensitivity of 182 lA mmol⁻¹ L and can attain 95% of steady-state current in 10 s. The phenol sensing range was 1.5 * 10–7 to 6.5 * 10–5 mol L⁻¹, with a sensing limit of 5.0 * 10–8 mol L⁻¹ at a signal/noise ratio of 3. The obvious Michaelis–Menten constant (K_M^app) and the enzyme electrode's stability were also calculated. The established biosensor's efficiency was likened to that of biosensors based on different immobilization matrices.

Gu et al. (2009) used zinc powders as raw material and hydrothermally fabricated ZnO nanorods on a gold wire without using some additional surfactant or alleviating agent. The gold wire was expertly handled to increase nucleation for ZnO nanostructure progress and boost the biosensor's efficiency, which was developed by immobilizing tyrosinase (Tyr) on ZnO nanorods for phenol discovery. According to electrochemical measurements, FTIR and SEM studies, the Tyr was stably adsorbed on the ZnO nanorod surface with bioactivity for phenol oxidation. The biosensor achieved a 95% steady-state current of less than 5 s, with a sensitivity of 103.08 A/mM at Cphenol > 20 M and 40.76 A/mM Cphenol 20 M. At a signal/noise ratio of 3, the sensing limit of 0.623 M was obtained.

Applications of tyrosinases

Environmental pollutant sensing

Because of their high sensitivity, quick reaction, and ease of use, enzyme-based biosensors are a promising option for detecting pesticide residues (Amine et al. 2006). Biosensors that use potentiometric or amperometric transducers to inhibit the cholinesterase (ChE) enzyme are the most often used for recognizing and quantifying organophosphates (Arduini et al. 2010a, b). In recent times, biosensors focused on the restriction of tyrosinase action by a compound (monophenol or o-diphenol) have been established to detect contaminants (Anh et al. 2004). Tyrosinase is a catechol oxidase with a dinuclear copper core that is found in a wide range of microorganisms, plants, and animals (Chen and Jin 2010). In the presence of oxygen as an electron acceptor, tyrosinase can catalyze the conversion of monophenols to o-diphenols, which can then be oxidized to o-quinones (Cortina-Puig et al. 2010). On the electrode surface, the produced o-quinones can be electrochemically reduced to o-diphenols again. The analysis of improvements in electrochemical reduction signals of o-quinones resulting from the inhibition of tyrosinase production can be used to detect contaminants. As a result, one of the research's main goals is to create a biosensor with high sensitivity (Liu et al. 2011).

Numerous amperometric biosensors dependent on the inhibition of tyrosinase enzyme action have been employed to determine the presence of triazine and phenylurea herbicides in the atmosphere in recent years (Martinazzo et al. 2018). Herbicides are commonly used despite the risk they pose to the ecosystem since they have less ecological perseverance, leading to severe toxicity. Since their existence might be found in external and earth water, they pose a severe hazard to human health. Vaz et al. (2021) developed a carbon nanosphere-based sensor for herbicide detection (Fig. 3). For the identification and quantifiable assessment of lethal intensities, a variety of techniques are used. Usually, LC or GC are employed, but owing to their simpler sample preparation, electrochemical enzyme sensors are also considered a substitute to traditional spectrometric approaches for pollutant detection (Bucur et al. 2018). Fe₃O₄ nanoparticles have been inserted into a sol–gel/chitosan biosensor membrane to improve biosensor sensitivity and reaction, along with immobilized alkaline phosphates. These nanoparticles, which are used in electrochemical biosensors, may be responsible for a desirable microenvironment for biomolecules like proteins to directly interchange electrons with an electrode (Abdel-Aziz and Heikal 2021). Electrochemical biosensors have also been constructed using enzyme-based biosensors, such as laccase, tyrosinase, glucose oxidase, horseradish peroxidase, and Fe₃O₄ nanoparticles. The tyrosinase electrode is often used to track phenolic and catecholic substances, and it is one of the most powerful transducers for phenol or catechol discovery (Tucci et al. 2019).

Fig. 3.

Carbon nanosphere-based sensor used for herbicide detection. Reprinted from Vaz et al. (2021) with permission from Elsevier

Pharmaceutical application

L-Dihydroxy phenylalanine is a naturally befalling nutritional complement and psychoactive agent present in many foods and herbs and is synthesized in the mammalian body and brain from the amino acid L-tyrosine. L-DOPA is a predecessor of the central nervous system's release of dopamine. As a result, L-DOPA is used as a potent medication to cure Parkinson's disease and regulate neurogenic myocardium damage (Min et al. 2019). The enzyme tyrosinase was used as a biocatalyst to manufacture L-DOPA using L-tyrosine as a substrate and L-ascorbate as a reducing agent (Fig. 4) (Zaidi et al. 2014). The amount of L-DOPA generated in batch reactors using microbial tyrosinases varies from 1.44 to 54 mg (Tan et al. 2019). However, efficiency appears to be significantly lower due to two factors. First, the addition of L-tyrosine seems to be insufficient, with less than 30% of it absorbed throughout the progression. Second, dopaquinone, leukodopachrome, and dopachrome are formed with melanin due to lateral reactions and reversible progression intermediates, which are eliminated by adding L-ascorbate in a concentration equivalent tyrosine (Ilesanmi and Adewale 2020). In continuous and batch processes, Ates et al. (2007) used microbial tyrosinase in Cu-alginate gels. L-DOPA has a global sales share of $101 billion per year, and therefore, new manufacturing processes are also being investigated. The global demand for L-DOPA is shown by an increase of 250 tons per year (Etemadi et al. 2018).

Fig. 4.

L-DOPA production by tyrosinase using L-tyrosine as a substrate and L-ascorbate as a reducing agent. Reprinted from Zaidi et al. (2014) with permission under the terms of the Creative Commons Attribution License

Phenolic compounds detection

Because of the significant concerns about toxicity, the accurate quantification of phenols in composite environmental matrices has received a lot of interest. Wastewater from various industries, including coal conversion, resin and plastics, petroleum refineries, textiles, dyes, iron and steel, and pulp and paper, contains phenolic compounds (Mishra and Chiang 2020). Phenols are harmful pollutants found in industrial waste that pose several health risks, with some of the potential carcinogens. Furthermore, phenol induces coloration of the receiving streams, making it necessary to decontaminate the compound (Fig. 5) (Wee et al. 2019). A number of researchers have investigated the use of enzymes in wastewater management. Elimination of phenol and its byproducts using a polyphenol oxidase enzyme such as tyrosinase has become a very important and efficient process. S. antibioticus tyrosinase, for example, was active against industrial toxins, including 3-and 4-chlorophenols and 3-and 4-fluorophenols (Camargo et al. 2018). The use of bacterial tyrosinase to treat polluted waste has recently been appraised, and it could be achieved using tyrosinase generating stains or the enzyme in an immobilized state as the protagonist. Tyrosinase benefits over other enzyme mechanisms employed for phenol elimination in that it uses molecular oxygen as the oxidant rather than H₂O₂, thereby lowering the cost of implementation (Laranjo et al. 2019). Electrochemical biosensors focused on immobilized tyrosinase have gained the most interest among the various diagnostic approaches employed to quickly check these phenolic compounds. For the identification of phenolic compounds, spectrophotometric or chromatographic methods have traditionally been used. However, new techniques have been introduced that may provide higher precision, lower costs, and quicker and easier sample handling (Mishra and Chiang 2020).

Fig. 5.

Schematic illustrations of (A) EAPC protocol, consisting of enzyme adsorption, precipitation, and crosslinking, and (B) electrochemical reactions occurring in the EAPC electrode on glassy carbon electrode. Reprinted from Wee et al. (2019) with permission from Elsevier

Tyrosinases catalyze the transfer of phenolic substrate to quinine species, electrochemically reduced for phenolic analyte detection at low potentials. Adsorption, crosslinking, on the surface of electrodes, entrapment in silicone films and hydrogels, carbon paste matrix, and graphite-epoxy composite electrodes are all used to immobilize tyrosinase an electrochemical transducer (Yin et al. 2019). Immobilization of tyrosinase to identify phenolic compounds has also been accomplished using electropolymerization, self-assembled monolayers, silica sol–gel, alumina sol–gel, and nanoparticles (Cerrato-Alvarez et al. 2019).

Applications in the food industry

Montereali et al. (2010) used a biosensor based on tyrosinase and laccase to identify polyphenols found in musts and wines. Both enzymes were halted on ferrocene-modified graphite screen-printed electrodes. These biosensors had strong specimen performance associated with spectrophotometric analysis, but due to SO₂, enzymatic activity was inhibited. Tyrosinases are also extracted as a toxin byproduct in industrial fermentation processes (Osma et al. 2010).

Cereal processing

Tyrosinases have been extensively explored for their capability to catalyze the oxidation of phenolic compounds found in breakfast cereal proteins and polysaccharides by forming connections in or from polysaccharides, proteins and polysaccharides, or proteins themselves (Glusac et al. 2018). Tyrosinase (mushroom extract rich in polyphenol oxidase) has been used to study the role of tyrosinase in the wheat dough and the development of 2-S-cysteinyl-DOPA 2,5-di-S-cysteinyl-DOPA, 6-S-cysteinyl-3, 4-DOPA, and di-DOPA crosslinks in gluten proteins (Nawaz et al. 2017).

Dairy processing

To create a smooth consistency securer, crosslinking may be used in dairy products to avoid syneresis. Hetero-cross-linking cereal, milk, and meat biopolymers could create innovative diet yields with specific qualities and properties. Tyrosinase enhances the functionalization of milk-based products by modifying the antioxidation attributes. Crosslinking of casein proteins and oxidation of tyrosyl residues in dairy proteins have previously been identified. Tyrosinases have also been shown to cause partial crosslinking of whey proteins; for example, A. bisporus tyrosinase has been shown to cross-link-lactalbumin (Nawaz et al. 2017).

Meat processing

Crosslinking enzymes are crucial in customizing the gelation qualities of meat, as the ability to shape gels and and the textural and binding properties of meat are all crucial in the production of meat products. Pork and chicken proteins have recently been evaluated for tyrosinase processing. Tyrosinase increases the gel shape quality of a 4 percent chicken breast myofibrillar protein suspension in the presence of 0.35 M NaCl and the insistence of homogenate gels comprising a lower level of phosphate-free beef (Tedeschi et al. 2021; Nawaz et al. 2017).

Other applications

The widespread acceptance of tyrosinase's catalytic property has been attributed to its use in cell culture, which aids nerve cell development. The tyrosinase enzyme is stamped onto plastic sheets, resulting in thin films of melanin in situ. Melanin has a bacteriostatic benefit, so it may help prevent bacterial infection. Entrapment of tyrosinase in polymer, a natural polymer, or transformed polystyrene, or adsorption on nylon zeolite, glass beads, fuller soil, and chitin stimulated with hexamethylenediamine are all used to change L-tyrosine to L-DOPA (Carvalho et al. 2000). Several studies have described the embedding of silk proteins onto chitosan via tyrosinase reactions, indicating that polymers can be tailored. Similarly, L-DOPA has been successfully grafted onto the proteins of wool fibers. It is also used to make hydrogels for skin replacements, medicine transport matrices, and tissue engineering (Mattinen et al. 2008). Tyrosinase was used by Aberg et al. (2004) for biosynthetic splicing of phenolic moieties or protein onto chitosan. The morphology and reusability of the two unique heterogeneous biocatalysts have been studied. Under moderate and environmentally safe laboratory conditions, these biocatalysts have been used to efficiently and selectively synthesize bioactive catechols. Lipophilic catechols are also made with tyrosinase and layer-by-layer assisted tyrosinases. These derivatives with antioxidant activity and long carbon alkyl side chains have shown substantial antiviral activity, indicating the probability of an innovative inhibition contrivance dependent on both redox and lipophilic qualities (Bozzini et al. 2013).

Chen et al. (2002) defined a new method for conjugating gelatin to the polysaccharide chitosan in vitro using tyrosinase. Hydroxytyrosol, a powerful antioxidant found in olives, was also made from tyrosol using tyrosinase. Tyrosinase has been employed as a possible prodrug in treating melanoma patients who have responded well to tyrosinase action. Anghileri et al. (2007) use microbial tyrosinase to make clusters from sericin, a peptide presented in silk textile industry wastewater. EMPA, multidisciplinary research and support organization, recently developed approaches for the recombinant synthesis of bacterial tyrosinase, making biomaterials including crosslinked proteins and melanin, such as recombinant Verrucomicrobium. The protein Spinosum tyrosinase is used to make custom-made melanin and other polyphenolic materials from various phenols and catechols as primary resources. These materials have an extensive range of uses, comprising the manufacture of organic semiconductors and photovoltaics. The tyrosinase enzyme can also be used to make crosslinked proteins, making it possible to recycle enzyme biocatalysts, including lipase (Wang et al. 2021).

Reactive lignin nanocapsules accelerate a pigmenting response to provide a durable, versatile bioink of a new kind. The creation of several pigments/dyes by natural or synthetic method is catalyzed by tyrosinase and laccase. Lignin nanocapsules that work with oxidation enzymes may be a biological catalyst for pigment formation and a newly manufactured pigment as carrier, adhesive, and additive (Polak and Jarosz‐Wilkolazka 2012). Capecchi et al. (2019) identified numerous roles of lignin nanocapsules in the support and enzyme activation needed for pigment production. In addition, lignin nanocapsules were discovered to preserve the melanin pigment from alkaline and UV degradation.

Conclusion and perspectives

Since the first industrial use of immobilized enzymes in the 1960s, enzyme immobilization harmony has advanced rapidly. Currently, the strategy of immobilized enzymes that fit various precise implementations has abandoned the traditional trial-and-error methodology and gradually migrated to a coherent scheme. This has also been distinguished by the fact that enzyme immobilization is now used to anticipate the reuse of expensive enzymes. The presence of a stable immobilized enzyme is now widely recognized as providing prompt perception into method progress and saving costs in process development and manufacturing. One of the most important classes of industrial enzymes is tyrosinase enzymes. These enzymes are widely used in manufacturing processes, including pharmaceuticals, cosmetics, and food. Several studies suggest that this enzyme has a lot of promise in medicine, agriculture, and analytical and environmental applications. The immobilized tyrosinase had a wider pH and temperature operating range and improved reusability and storage flexibility. Furthermore, since the immobilized tyrosinase was stabilized in its active conformation by electrostatic interaction before covalent crosslinking, it had a higher affinity for the substrate. Therefore, sufficient immobilization matrices for tyrosinase immobilization are needed to improve its usability.

Declarations

Conflict of interests

The authors declare no conflicting interests.