Laccase Mediator Research Articles

High-Valence Mn MOF Inspired by Laccase Mediators Enables Versatile Nature-Mimicking Catalysis.

In nature, active Mn3+ -ligand complexes produced by laccase catalyzed oxidation can act as the low-molecular mass, diffusible redox mediators to oxidize the phenolic substrates overcoming the limitations of natural enzymes. Learning from the metal-ligand coordination of natural functional units, high-valence Mn metal-organic framework (Mn MOF) is constructed to simulate the catalysis in natural mediator system. Benefiting from the characteristics of nanoscale size, rich metal coordination unsaturated sites, and mixed valence state dominated by Mn(III), Nano Mn(III)-TP exhibits superior laccase-mimicking activity, whose Vmax (maximal reaction rate) is much higher than that of natural laccase. Referring to natural systems, relevant free radical experiments prove that the material induces the production of active oxygen species with the assistance of carboxylic acid, and active oxygen species further oxidize phenolic substrates. Based on its robust performances, the primary oxidative degradation of an emerging pollutant triclosan (TCS) is creatively applied, an important antiasthmatic medicine terbutaline sulfate (TBT) detection, and the synthesis of non-toxic and black near-natural dyes for dyeing. By simulating the essential mediators of natural enzymatic catalysis, an Mn MOF-based material that demonstrates multiple novel applications is successfully developed, which introduces a new reliable strategy for achieving versatile nature-mimicking catalysis.

High melanin degradation by laccase from a novel isolated white rot fungi Trametes polyzona 023 in the presence of phenolic compounds

The concern to identify a more benign alternative for whitening agents is gaining significant attention in recent investigations. Therefore, this study aimed to investigate the ability of laccase isolated from Trametes polyzona to degrade melanin and explore potential natural phenolic mediators enhancing degradation process. Among phenolic mediators tested, gallic acid showed the most potential for melanin degradation due to 3-fold enhancement, with the highest rate of 77.8 % during a 24-h reaction. The addition of sodium azide inhibited melanin degradation to approximately 56 %, showing the significant role of laccase. The results of Scanning Electron Microscope analysis after treatment showed that the morphology of melanin was significantly altered, with rough and porous surface, as well as small uniform size. However, the untreated melanin had a smooth surface, a round shape, and a larger size. In vitro and molecular docking analyses mutually supported the occurrence of the laccase mediator system in melanin degradation.

Electro-oxidation of synthetic lignin with different mediators for the laccase mediator system (3-hydroxyanthranilic acid, 4-hydroxybenzoic acid, phenol red)

Three mediators for the laccase mediator system, 3-hydroxyanthranilic acid (3-HAA), 4-hydroxybenzoic acid (HBA), and phenol red (PR) were investigated as mediators in an electrolytic mediator system (EMS) for the degradation of guaiacyl synthetic lignin (G-DHP). All the electron-oxidations of G-DHP with 3-HAA, HBA and PR in the absence of 2,6-lutidine proceeded to give the electrolysis residues in moderate yields. The significant β-β and β-5 linkage loss was found in all the electrolysis residues, especially the residue in the electro-oxidation with PR was significant. The addition of 2,6-lutidine as a base increased the yields of the electrolysis residues and influenced the relative ratio of β-O-4, β-5 and β-β linkages to some extent, that is, increase of β-O-4 linkage loss and decrease of β-β linkages loss (in the electro-oxidation with 3-HAA), increase of β-O-4 linkage loss (in that with 3-HBA), increase of β-5 linkages loss (in that with PR at 0.35 V) and decrease of β-O-4 and β-β linkages loss (in that with PR at 0.70 V). Thus, the base such as 2,6-lutidine was also one of the critical factors for reaction efficiency and reaction selectivity in the EMS. Consequently,3-HAA, HBA, and PR could be used as mediators in EMS for lignin degradation, especially 3-HAA is the most preferable because of the low applied potential.

Co-immobilization of laccase-mediator system to catalyze the synthesis of theaflavins from tea polyphenols

The co-immobilized laccase-mediator system (LMS) has broad development prospects in the fields of biocatalysis, biochemistry, and environmental remediation. In this study, laccase and mediator molecules were co-immobilized in functionally modified calcium alginate microspheres to improve their catalytic activity and stability and to systematically characterize the physicochemical properties and evaluate the catalytic performance of the co-immobilized laccase-mediator system. The co-immobilized LMS was used to catalyze the synthesis of theaflavins (TFs) from tea polyphenols, improve the catalytic efficiency, realize the recycling of the system, and explore the safety of the catalytic products. The results showed that the catalytic efficiency of the co-immobilized LMS was significantly improved and the relative activity remained above 50% after 10 cycles. The in vitro safety of the catalytic product was verified by Human hepatocellular carcinoma (HepG2) cell-related indexes. The results show that the co-immobilized laccase mediator system has great development potential in the industrial application of theaflavins.

A novel laccase from Trametes polyzona with high performance in the decolorization of textile dyes

Laccases are multicopper oxidases able to oxidize several phenolic compounds and find application in numerous industrial applications. Among laccase producers, white-rot fungi represent a valuable source of multiple isoforms and isoenzymes of these multicopper oxidases. Here we describe the identification, biochemical characterization, and application of laccase 2 from Trametes polyzona (TP-Lac2), a basidiomycete fungus emerged among others that have been screened by plate assay. This enzyme has an optimal temperature of 50 °C and in acidic conditions it is able to oxidize both phenolic and non-phenolic compounds. The ability of TP-Lac2 to decolorize textile dyes was tested in the presence of natural and synthetic mediators at 30 °C and 50 °C. Our results indicate that TP-Lac2 most efficiently decolorizes (decolorization rate > 75%) malachite green oxalate, orange G, amido black10B and bromocresol purple in the presence of acetosyringone and 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonate)—ABTS. Overall, the laccase mediator system consisting of TP-Lac2 and the natural mediator acetosyringone has potential as an environmentally friendly alternative for wastewater treatment in the textile industry.

Gaining insight into the effect of laccase expression on humic substance formation during lignocellulosic biomass composting

The aim is to enhance lignin humification by promoting laccase activities which can promote lignin depolymerization and reaggregation during composting. 1-Hydroxybenzotriazole (HBT) is employed to conduct laccase mediator system (LMS), application of oxidized graphene (GO) in combination to strengthen LMS. Compared with control, the addition of GO, HBT, and GH (GO coupled with HBT) significantly improved laccase expression and activities (P < 0.05), with lignin humification efficiency also increased by 68.6 %, 36.7 %, and 107.8 %. GH treatment induces microbial expression of laccase by increasing the abundance and synergy of core microbes. The unsupervised learning model, vector autoregressive model and Mantel test function were combined to elucidate the mechanism of action of exogenous materials. The results showed that GO stabilized the composting environment on the one hand, and acted as a support vector to stabilize the LMS and promote the function of laccase on the other. In GH treatment, degradation of macromolecules and humification of small molecules were promoted simultaneously by activating the dual function of laccase. Additionally, it also reveals the GH enhances the humification of lignocellulosic compost by converting phenolic pollutants into aggregates. These findings provide a new way to enhance the dual function of laccase and promote lignin humification during composting. It could effectively achieve the resource utilization of organic solid waste and reduce composting pollution.

Laccases as Effective Tools in the Removal of Pharmaceutical Products from Aquatic Systems.

This review aims to provide a comprehensive overview of the application of bacterial and fungal laccases for the removal of pharmaceuticals from the environment. Laccases were evaluated for their efficacy in degrading pharmaceutical substances across various categories, including analgesics, antibiotics, antiepileptics, antirheumatic drugs, cytostatics, hormones, anxiolytics, and sympatholytics. The capability of laccases to degrade or biotransform these drugs was found to be dependent on their structural characteristics. The formation of di-, oligo- and polymers of the parent compound has been observed using the laccase mediator system (LMS), which is advantageous in terms of their removal via commonly used processes in wastewater treatment plants (WWTPs). Notably, certain pharmaceuticals such as tetracycline antibiotics or estrogen hormones exhibited degradation or even mineralization when subjected to laccase treatment. Employing enzyme pretreatment mitigated the toxic effects of degradation products compared to the parent drug. However, when utilizing the LMS, careful mediator selection is essential to prevent potential increases in environment toxicity. Laccases demonstrate efficiency in pharmaceutical removal within WWTPs, operating efficiently under WWTP conditions without necessitating isolation.

Laccase mediator system as a potential technology for decolorization of textile dyes — Overview and perspectives

Laccase mediator system as a potential technology for decolorization of textile dyes — Overview and perspectives

Spectrophotometric Assay for the Detection of 2,5-Diformylfuran and Its Validation through Laccase-Mediated Oxidation of 5-Hydroxymethylfurfural

Modern biocatalysis requires fast, sensitive, and efficient high-throughput screening methods to screen enzyme libraries in order to seek out novel biocatalysts or enhanced variants for the production of chemicals. For instance, the synthesis of bio-based furan compounds like 2,5-diformylfuran (DFF) from 5-hydroxymethylfurfural (HMF) via aerobic oxidation is a crucial process in industrial chemistry. Laccases, known for their mild operating conditions, independence from cofactors, and versatility with various substrates, thanks to the use of chemical mediators, are appealing candidates for catalyzing HMF oxidation. Herein, Schiff-based polymers based on the coupling of DFF and 1,4-phenylenediamine (PPD) have been used in the set-up of a novel colorimetric assay for detecting the presence of DFF in different reaction mixtures. This method may be employed for the fast screening of enzymes (Z’ values ranging from 0.68 to 0.72). The sensitivity of the method has been proved, and detection (8.4 μM) and quantification (25.5 μM) limits have been calculated. Notably, the assay displayed selectivity for DFF and enabled the measurement of kinetics in DFF production from HMF using three distinct laccase–mediator systems.

Potential of Laccase as a Tool for Biodegradation of Wastewater Micropollutants

In the 21st century, humans are facing a high risk of exposure to emerging pollutants. Water contamination has become a major threat due to its devastating impacts on the entire ecosystem. Relatively great danger for aquatic microorganisms is posed by organic micropollutants, which are a consequence of progressing urbanization and industrialization. This review focuses on laccase of mainly fungal and bacterial origin, which provides an eco-friendly strategy for the transformation of these harmful pollutants to less or non-toxic compounds, as it acts oxidatively on the aromatic ring of a wide range of compounds, releasing water as the only by-product. Laccase alone or with the use of mediators has been used successfully to remove micropollutants from wastewater, including pharmaceuticals and personal care products, biocides, endocrine disrupting agents, steroid hormones, and microplastics. Even though the potential of an LMS (laccase–mediator system) is tremendous, the selection of an appropriate mediator and the persistent monitoring of toxicity after treatment are critical and should be performed routinely. Hence, further research is still needed for the optimization of degradation processes to improve our understanding of the different interactions of laccase with the substrate and to develop sustainable advanced water treatment systems.

Facile co-immobilization of laccase and mediator by polyethyleneimine-induced biomimetic mineralization for dye removal

Laccase, a green biocatalyst, has been widely used in the field of biodegradation with the assistance of electron mediators. Herein, a novel strategy, polyethyleneimine (PEI)-induced biomimetic mineralization, was explored and applied to co-immobilize laccase and 2,2′-azino bis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS). With the PEI doping, calcium phosphate (CaP) could facilely mineralize within 20 min to form PEI@CaP; ABTS could be co-mineralized with PEI to form (PEI + ABTS)@CaP. Laccase, a negatively charged protein, was then immobilized via electrostatic adsorption onto the embedded PEI in the carriers. Both of the immobilized laccase preparations, denoted as Lac-PEI@CaP and Lac-(PEI + ABTS)@CaP, showed high activity recoveries (96.4 % and 88.0 %, respectively), and increased pH tolerance and storage stability as compared to free laccase. ABTS and laccase loading densities of Lac-(PEI + ABTS)@CaP reached 0.83 mmol g−1 and 100 mg g−1, respectively. Lac-(PEI + ABTS)@CaP presented higher malachite green (MG) degradation capability than free laccase and Lac-PEI@CaP with free ABTS, possibly due to the proximity effect of the co-immobilized laccase and ABTS. Moreover, Lac-(PEI + ABTS)@CaP exhibited reusability and kept 83.2 % of MG degradation activity after three recycles and 37.9 % in the sixth. This work provided a new strategy for the co-immobilization of laccase and electron mediator, and demonstrated the potential of the biocatalyst for organic pollutant removal.

Peculiar Properties of Template-Assisted Aniline Polymerization in a Buffer Solution Using Laccase and a Laccase-Mediator System as Compared with Chemical Polymerization.

The conventional chemical polymerization of aniline has been described in multiple publications, while enzymatic polymerization has been poorly explored. A comparative study of the template-assisted enzymatic and chemical polymerization of aniline in a buffer solution of sodium dodecylbenzenesulfonate micelles was performed for the first time. The high-redox potential laccase from the fungus Trametes hirsuta was used as a catalyst and air oxygen served as an oxidant. Potentiometric and spectral methods have shown that oligomeric/polymeric products of the enzymatic polymerization of aniline are synthesized in the conducting emeraldine salt form immediately after the reaction is initiated by the enzyme. The use of the laccase-mediator system enabled a higher rate of enzymatic polymerization and a higher yield of final products. Potassium octocyanomolybdate (IV) served as a redox mediator. The products of the enzymatic polymerization of aniline were studied by the ATR-FTIR, MALDI-TOF and atomic force microscopy methods. The chemical oxidative polymerization of aniline under the same conditions resulted in forming a non-conducting dark brown product.

1-ethyl-3-methyl imidazolium acetate, hemicellulolytic enzymes and laccase-mediator system: Toward an integrated co-valorization of polysaccharides and lignin from Miscanthus

The pretreatment of Miscanthus x giganteus with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]), followed by enzymatic hydrolysis and oxidative depolymerization, resulted in the efficient production of monomeric sugars and selective production of phenolic intermediates. The liquid fractions containing monomeric sugars were obtained with high yields (>80%), while a solid fraction enriched with lignin (>50% w.w−1) was isolated. The phenolic monomers released during [Emim][OAc]-pretreatment were identified and quantified by HPLC. A decrease in the content of vanillin, vanillic acid, and p-hydroxybenzaldehyde was observed during pretreatment, suggesting a selective solubilization of guaicyl and p-hydroxyphenyl units from the lignin polymer in [Emim][OAc]. The decrease in p-coumaric and ferulic acids content suggested a cleavage of lignin-carbohydrate complexes during pretreatment. Both pretreatment and enzymatic hydrolysis affected the content of ferulic acid ether and ester bonds. The NMR structural study (2D HSQC) of the isolated lignin showed the preservation of β-O-4′ interunit bonds, while 31P NMR analysis revealed a high content of aliphatic hydroxyl groups. The oxidative depolymerization of the isolated lignin, catalyzed by the laccase mediator system in the presence of 5% v.v−1 recycled [Emim][OAc] in buffer, mainly led to the production of free p-hydroxybenzaldehyde as a promising platform molecule. The mass balance of glucan, xylan and lignin from Miscanthus was validated throughout this strategy.

An Artifact of Perfluoroalkyl Acid (PFAA) Removal Attributed to Sorption Processes in a Laccase Mediator System.

Fungi and laccase mediator systems (LMSs) have a proven track record of oxidizing recalcitrant organic compounds. There has been considerable interest in applying LMSs to the treatment of perfluoroalkyl acids (PFAAs), a class of ubiquitous and persistent environmental contaminants. Some laboratory experiments have indicated modest losses of PFAAs over extended periods, but there have been no clear demonstrations of a transformation mechanism or the kinetics that would be needed for remediation applications. We set out to determine if this was a question of identifying and optimizing a rate-limiting step but discovered that observed losses of PFAAs were experimental artifacts. While unable to replicate the oxidation of PFAAs, we show that interactions of the PFAA compounds with laccase and laccase mediator mixtures could cause an artifact that mimics transformation (≲60%) of PFAAs. Furthermore, we employed a surrogate compound, carbamazepine (CBZ), and electron paramagnetic resonance spectroscopy to probe the formation of the radical species that had been proposed to be responsible for contaminant oxidation. We confirmed that under conditions where sufficient radical concentrations were produced to oxidize CBZ, no PFAA removal took place.

A green process for flax fiber extraction by white rot fungus (Laccase mediators system) in a less-water environment

Degumming is required for all bast fibers before further processing. In this study, white rot fungi(WRF) were combined with solid state fermentation progressive bio-degumming(SPB) by introducing the electron mediator syringaldehyde(SA) to explore the feasibility of WRF bio-degumming in a less-water environment. The results indicated that the electron mediator (SA) could effectively enhance the oxidation effect of WRF and enhance the diffusion of WRF on the flax surface to improve its action range, thus enhancing the degumming effect. After degumming by this method, the fibers had a high degree of dispersion and a smooth surface. The removal of non-cellulose components was similar to that of the chemical method and meets the requirements for degumming. In addition, the fibers treated by this method had low molecular weight loss and high tensile strength. Thus, the SPB-L method offers a new strategy for the sustainable processing of bast fibers.

Efficient Azo Dye Biodecolorization System Using Lignin-Co-Cultured White-Rot Fungus.

The extensive use of azo dyes by the global textile industry induces significant environmental and human health hazards, which makes efficient remediation crucial but also challenging. Improving dye removal efficiency will benefit the development of bioremediation techniques for textile effluents. In this study, an efficient system for azo dye (Direct Red 5B, DR5B) biodecolorization is reported, which uses the white-rot fungus Ganoderma lucidum EN2 and alkali lignin. This study suggests that the decolorization of DR5B could be effectively enhanced (from 40.34% to 95.16%) within 48 h in the presence of alkali lignin. The dye adsorption test further confirmed that the alkali-lignin-enhanced decolorization of DR5B was essentially due to biodegradation rather than physical adsorption, evaluating the role of alkali lignin in the dye biodegradation system. Moreover, the gas chromatography/mass spectrometry analysis and DR5B decolorization experiments also indicated that alkali lignin carried an excellent potential for promoting dye decolorization and displayed a significant role in improving the activity of lignin-modifying enzymes. This was mainly because of the laccase-mediator system, which was established by the induced laccase activity and lignin-derived small aromatic compounds.

Laccase–mediator co-immobilized doped-HKUST-1 cellulose composite beads and their application for the biodegradation of carbazole

The co-immobilization of laccase and a mediator is essential for water treatment due to the fact that the free laccase and mediator cannot be recovered and reused simultaneously.

Alginate Encapsulation Stabilizes Xylanase Toward the Laccase Mediator System.

Xylanase, a hydrolytic enzyme, is susceptible to inactivation by the oxidative conditions generated by the laccase mediator system (LMS). Given the impetus to develop a mixed enzyme system for application in biomass processing industries, xylanase was encapsulated with either Cu2+- or Ca2+-alginate and then exposed to the LMS with variations such as mediator type, mediator concentration, and treatment pH. Results demonstrate that alginate-encapsulated xylanase retains substantial activity (> 80%) when exposed to the LMS relative to non-encapsulated xylanase. Cu2+-alginate generally provided better protection than Ca2+-alginate for all mediators, and protection was observed even at a low pH, where the LMS is most potent. Despite encapsulation, xylanase was still capable of hydrolyzing its polymeric substrate xylan, given kcat/Km values within an order of magnitude of that for non-encapsulated xylanase. The alginate matrix does not impede the function of the oxidized mediator, since comparable Vmax values were observed for the conversion of veratryl alcohol to veratraldehyde by free and Cu2+-alginateencapsulated laccase. Overall, these results support development of a mixed enzyme system for biomass delignification and, more broadly, show potential for protecting protein function in an oxidative environment.

Laccase-Carrying Polylactic Acid Electrospun Fibers, Advantages and Limitations in Bio-Oxidation of Amines and Alcohols.

Laccases are oxidative enzymes that could be good candidates for the functionalization of biopolymers with several applications as biosensors for the determination of bioactive amine and alcohols, for bioremediation of industrial wastewater, and for greener catalysts in oxidation reactions in organic synthesis, especially used for non-phenolic compounds in combination with redox mediators in the so-called Laccase Mediator System (LMS). In this work, we describe the immobilization of Laccase from Trametes versicolor (LTv) in poly-L-lactic acid (PLLA) nanofibers and its application in LMS oxidation reactions. The PLLA-LTv catalysts were successfully produced by electrospinning of a water-in-oil emulsion with an optimized method. Different enzyme loadings (1.6, 3.2, and 5.1% w/w) were explored, and the obtained mats were thoroughly characterized. The actual amount of the enzyme in the fibers and the eventual enzyme leaching in different solvents were evaluated. Finally, the PLLA-LTv mats were successfully applied as such in the oxidation reaction of catechol, and in the LMS method with TEMPO as mediator in the oxidation of amines with the advantage of easier work-up procedures by the immobilized enzyme. However, the PLLA-LTv failed the oxidation of alcohols with respect to the free enzyme. A tentative explanation was provided.

Biodegradation of Indigo Carmine Dye by Laccase from Bacillus licheniformis NS2324

Indigo carmine dye is used in many industries like textile, paper, cosmetics, etc. It is specially used in textile industries for dyeing denim. The untreated water discharged from these industries, especially the textile industry, poses a significant environmental threat. The conventional physicochemical methods used in treating industrial effluent are very expensive and cause secondary pollution. In the present study, an extracellular laccase-producing Bacillus licheniformis NS2324 was isolated, and a crude enzyme was used to degrade indigo carmine dye. The conditions needed for decolorisation were standardised. Optimisation studies for dye decolorisation by NS2324 laccase revealed that the enzyme was able to decolorize dye efficiently at 50 oC temperature, pH 8, with an enzyme dose of 10 IU/ml after 6 hours. Up to 96.79±1.06 per cent decolorisation was achieved under optimised conditions without the use of any mediator of laccase. Complete degradation of indigo dye by laccase in the absence of mediator makes the present study very useful for treating textile effluent from the denim industry.