Trametes Versicolor Laccase Research Articles

Biocatalytic degradation of environmental endocrine disruptor chlorobenzene via surfactant-optimized laccase-mediator system.

The emergence of environmental endocrine disruptor chlorobenzene (CB) in surface water and its potential environmental impacts have attracted serious global attention. It is still very difficult to achieve effective degradation of it by catalytic oxidation process under mild conditions. Here, an optimized method for degrading CB in aqueous solution using Trametes versicolor laccase and surfactant-assisted laccase-mediator (SALM) system was investigated. The use of a Tween 80 surfactant enhanced the solubility of CB and promoted its efficient degradation. Under favorable conditions, the SALM system yielded a degradation efficiency of 43.5% and a dechlorination efficiency of 41.55% for CB (25mg/L) within 24h. The possible degradation pathway of CB by this system was speculated by detecting the intermediates produced during the reaction. The outcome of the proliferation assays on MCF-7 human breast cancer cells demonstrated a reduction in the estrogenic activity of the CB solution following treatment with the SALM system. Furthermore, the influence of the quantity and positional variation of chlorine substituents on the degradation process was methodically investigated. Moreover, molecular analyses were employed to study the detailed interaction mechanism between laccase and CB, which revealed that the hydrophobic interaction contributed dominantly to binding process. These findings provide an efficient and environmentally friendly degradation system for the development of purification strategies for halogenated pollutants.

Immobilization of Laccase in β-Cyclodextrin Composite Hydrogel for Efficient Degradation of Dye Pollutants

A stable and efficient biocatalyst was prepared by encapsulating Trametes versicolor laccase using an acrylic acid-grafted β-cyclodextrin hydrogel (Lac-CD-PAA). Scanning electron microscopy and nitrogen adsorption-desorption experiments showed that there were regularly distributed channels in the spongy Lac-CD-PAA. In addition, a large number of mesopores and macropores existed in the wall of the hydrogel lamellae. This network structure reduced the diffusion resistance of the hydrogel to the target substrate. The relative activity of the resulting Lac-CD-PAA could be maintained at 35.8% after six cycles of use. Lac-CD-PAA exhibited higher thermal and chemical stability compared to free laccase. The negative charge on the surface of Lac-CD-PAA gives it the ability to pretreat cationic dyes. In six consecutive methylene blue decolorization tests, Lac-CD-PAA decolorized better than free laccase. The results showed that the prepared β-cyclodextrin-based composite hydrogel was a good carrier for laccase.

Selection of organic solvents as reaction media for laccase from Trametes versicolor

The ability of laccases to work effectively in properly selected aqueous-organic solutions significantly expands the scope of their application, making them commercially attractive and more competitive. However, it must be considered that molecules of organic solvent are very aggressive towards enzymes and destroy weak non-covalent interactions that maintain the spatial structure of the protein. In this work, we quantitatively described the influence of the nature of water-miscible organic solvents and their concentration on the activity and stability of Trametes versicolor laccase during the oxidation of ABTS (2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) through the maximum rates and the Michaelis constants KM. The obtained kinetic parameters make it possible to compare the effect of various organic solvents on laccase and select the most optimal one, which minimizes the decrease in enzyme activity, maintains its stability and allows repeated use. Among the solvents studied, polyhydroxy compounds have the least negative effect on laccase activity; however, in all cases, a high concentration of organic solvent (≥30% (v/v)) leads to suppression of enzymatic activity. It has been shown that the longer the alkyl chain (hydrophobic fragment of the structure), the stronger the negative effect of alcohols on enzymes. The most toxic solvent for Trametes versicolor laccase is isopropanol. In addition, numerous examples of various uses of Trametes versicolor in aquatic-organic environments are discussed here.

Determination of the total phenolic content in Hardaliye by amperometric laccase-based biosensor and comparison of the LC-MS/MS

In this study, a laccase-based amperometric biosensor was fabricated for the quantitative analysis of total phenolic content in Hardaliye. The biosensor was constructed using a carbon paste electrode with simple modifications. The laccase obtained from Trametes versicolor was immobilized into a carbon paste electrode modified with a gelatin matrix. The immobilization parameters for biosensor fabrication, as well as its working conditions were optimized with gallic acid as the phenolic standard. Additionally, molecular docking simulation was used to examine the interaction between Trametes versicolor laccase and gallic acid. The biosensor's analytical parameters were determined under optimal conditions including linearity, repeatability, stability and selectivity. The linear range of gallic acid was calculated as 0.5–100 μM, with the detection limit of 0.1323 ± 0.1 μM. With good selectivity and stability, the biosensor successfully determined the total phenolic contents in Hardaliye, and red-wine samples. Comparison studies showed that the biosensor results agreed with those obtained from the Liquid Chromatography-Tandem Mass Spectrometry method. This work indicates that the proposed biosensor is a promising analytic tool for total phenolic analyses of beverages.

Optimization of 5-hydroxymethylfurfural oxidation via photo-enzymatic cascade process

Trametes versicolor's laccase (LacTV) was efficient catalyst in HMF oxidation to FDCA. Its use in an enzymatic and photocatalytic cascade process permitted a 40-fold increase in FDCA productivity compared to traditional enzymatic batch systems.

Statistical optimization of the yield of the oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid catalyzed by laccase from Trametes versicolor

Lignocellulosic residues are an interesting alternative to produce furans as 5-hydroxymethylfurfural (HMF), which can be oxidized to produce 2,5-furandicarboxylic acid (FDCA). According to the latter, this research aims to optimize the oxidation yield of HMF to FDCA catalyzed by a Trametes versicolor laccase using TEMPO as a mediator. For yield optimization, a Box–Behnken experimental design was developed with three factors and three levels. The model achieved 90 % fit (R2) with a maximum yield of 100 % FDCA, using 30 mM HMF, 55 mM 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO), and 6 mg laccase. The oxidation kinetics (at 30 and 100 mM HMF) evidenced the presence of the intermediate compound 5-formyl-2-furancarboxylic acid (FFCA), being higher when working at a higher concentration of substrate (100 mM HMF). Optimizing the operational conditions resulted in a notable improvement, boosting the yield of FDCA by approximately 10 % compared to the initial experimentation. Finally, the values of the variables predicted by the program were validated experimentally, reaching 100 % performance.

Fabrication of cellulose acetate/chitosan/poly(ethylene oxide) scaffold as an efficient surface area substrate for immobilization of laccase

In this study, the properties of the immobilized laccase process on cellulose acetate (CA)/chitosan (CHI)/poly (ethylene oxide) (PEO) electrospun nanofiber was studied. Trametes versicolor laccase's immobilization on the porous electrospun nanofibers (ENFs) with 89–119 nm diameter by polyethyleneimine (PEI) and glutaraldehyde covalent cross-linking was also investigated. Blending CA nanofibers with high hydroxyl (OH) groups and CHI with high reactive hydroxyl and amino functional groups leads to a strong affinity for various proteins and outstanding mechanical properties. In Fourier-transform infrared spectroscopy (FT-IR) spectra, the vibrations at 1560 and 1640 cm−1 about NH stretching indicated that laccase was immobilized on ENFs. Differential scanning calorimetry (DSC) demonstrated that the thermal stability of CA/CHI/PEO improved with immobilization. The laccase loaded on the membranes was around 803 mg/g at pH 5.5, and the immobilized laccase relative activity was about 76.32%. The oxidation of ABTS determined the performance of immobilized and free laccases. Compared with free laccase, the pH and thermal stability of the immobilized laccase were remarkably improved. Over 50% of the immobilized laccase life was maintained for up to 5 cycles and had greater than 75.8% of initial activity and 64.87% of optimum activity. The immobilized laccase on CA/CHI/PEO ENFs with fine diameters enhanced the loading of the enzyme. Hence, immobilized laccase might be an ideal candidate for industry usage instead of free enzymes.

Trametes versicolor laccase activity modulated by the interaction with gold nanoparticles

In this study, the impact of gold nanoparticles (AuNPs) on the structure and activity of laccase from Trametes versicolor (Lc) was described. Fluorescence experiments revealed that AuNPs efficiently quench Lc's tryptophan fluorescence by a static and dynamic process. By using differential scanning microcalorimetry and circular dichroism spectroscopy, it was determined how the concentration of nanoparticles and the composition of the medium affected the secondary structure of Lc. The data revealed that upon binding with AuNPs, conformational changes take place mainly in presence of high amounts of nanoparticles. The complex kinetic analysis unveiled the Lc activity enhancement at low concentrations of AuNPs as opposed to the concentrated regime, where it can be reduced by up to 55%. The Michaelis-Menten tests highlighted that the activity of the biocatalyst is closely related to the concentration of AuNPs, while the Selwyn analysis demonstrated that even in a concentrated regime of Lc it is not deactivated regardless of the amount of AuNPs added. The thermal parameters improved by twofold in the presence of low AuNPs concentration, whereas the activation energy increased with AuNPs content, implying that not all collisions are beneficial to the enzyme structure. The effect of AuNPs on the decomposition of a recalcitrant dye (naphthol green B, NG) by Lc was also evaluated, and the Michaelis-Menten model revealed that only the high AuNPs content influenced negatively the Lc activity. The isothermal titration calorimetry revealed that hydrogen bonds are the main intermolecular forces between Lc and AuNPs, while electrostatic interactions are responsible for NG adsorption to AuNPs. The results of the docking analysis show the binding of NG near the copper T1 site of Lc with hydrogen bonds, electrostatic and hydrophobic interactions. The findings of this work provide important knowledge for laccase-based bio-nanoconjugates and their use in the field of environmental remediation.

Tuning the Type 1 Reduction Potential of Multicopper Oxidases: Uncoupling the Effects of Electrostatics and H-Bonding to Histidine Ligands.

In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O2 is reduced to H2O. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the ∼350 mV difference in potential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range of spectroscopies performed on the oxidized and reduced T1 sites in these MCOs shows that they have equivalent geometric and electronic structures. However, the two His ligands of the T1 Cu in Fet3p are H-bonded to carboxylate residues, while in TvL they are H-bonded to noncharged groups. Electron spin echo envelope modulation spectroscopy shows that there are significant differences in the second-sphere H-bonding interactions in the two T1 centers. Redox titrations on type 2-depleted derivatives of Fet3p and its D409A and E185A variants reveal that the two carboxylates (D409 and E185) lower the T1 potential by 110 and 255-285 mV, respectively. Density functional theory calculations uncouple the effects of the charge of the carboxylates and their difference in H-bonding interactions with the His ligands on the T1 potential, indicating 90-150 mV for anionic charge and ∼100 mV for a strong H-bond. Finally, this study provides an explanation for the generally low potentials of metallooxidases relative to the wide range of potentials of the organic oxidases in terms of different oxidized states of their TNCs involved in catalytic turnover.

Plasticizer phthalate esters degradation with a laccase from Trametes versicolor: effects of TEMPO used as a mediator and estrogenic activity removal.

Phthalate esters (PAEs) are toxic and persistent chemicals that are ubiquitous in the environment and have attracted worldwide attention due to their threats to the environment and human health. Dimethyl phthalate (DMP) is a relatively simple structure and one of the most observed PAEs in the environment. This study investigated the degradation of the DMP using Trametes versicolor laccase and its laccase-mediator systems. The degradation effect of laccase alone on DMP was poor, while the laccase-mediator systems can effectively enhance the degradation efficiency. Within 24h, 45% of DMP (25mg/L) was degraded in the presence of 0.8 U/mL laccase and 0.053mM 2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO). A certain concentration (1mM) of metal ions Al3+, Cu2+ or Ca2+ can positively promote DMP degradation with the laccase-TEMPO system. Moreover, the structure of PAEs also had a great influence on the degradation efficiency. Higher degradation efficiencies were observed when incubating PAEs with short alkyl side chains by the laccase-TEMPO system compared to that with long alkyl side chains. Additionally, the branched-chain PAEs had a better degradation effect than the straight-chain. The estrogenic activity of the DMP solution after reaction was much smaller than that of the original solution. Finally, transformation products ortho-hydroxylated DMP and phthalic acid were identified by GC-MS and the possible degradation pathway was proposed. This study verifies the feasibility of the laccase-TEMPO system to degrade PAEs and provides a reference for exploring more potential value of laccase.

Laccase‐based amperometric biosensor enhanced by Ni/Au/PPy‐COOH multisegmental nanowires for selective dopamine detection

AbstractOne‐dimensional Ni/Au/PPy‐COOH nanowires with multiple segments were synthesized in this study. Smooth surfaces and magnetic properties of nanowires were investigated by scanning transmission electron microscopy (SEM), Energy‐dispersive X‐ray spectroscopy (EDX), and Electron Spin Resonance (ESR) techniques. The nanowires were used to modify the screen‐printed electrode surface and as a micro‐environment for Trametes versicolor laccase. The ability of this enzyme biosensor to detect dopamine change in human biological samples was demonstrated by a wide linear range (0.01–50 μM) and a low LOD (2.265 nM). In addition, the biosensor exhibited excellent selectivity allowing the detection of dopamine in the presence of ascorbic acid, uric acid, L‐Cys, serotonin, and glucose, with high sensitivity of reduction currents obtained at −0.2 V (vs. Ag/AgCl). The proposed biosensor allowed the detection of dopamine in commercial serum and artificial urine with recovery values close to 100 %. It also demonstrated reproducibility, reusability, and long‐term storage stability. The sensitivity, Kmapp, and Imax values of the biosensor were determined as 2.05 μM and 1.03 μA, respectively. The LAC‐Ni/Au/PPy‐COOH/NAF/SPE biosensor is a reliable design for detecting dopamine with a wide linear range.

Laccase modified GO/TiS2 nanocomposite based amperometric biosensor for (-)-epicatechin detection

In this study, 2-dimensional graphene oxide-titanium disulfide (GO/TiS2) nanolayers were synthesized, and the surface and size properties of nanolayers were investigated by transmission electron microscopy (TEM) technique. The nanolayers were used to modify the screen-printed electrode surface and applied as a micro-medium for Trametes versicolor laccase. The ability of this enzyme biosensor to detect the (-)-epicatechin has been demonstrated with a wide linear range (0.1–250 µM), LOD (0.073 µM), and sensitivity (0.3264 µAµM−cm−2). In addition, the biosensor exhibited excellent selectivity allowing selective detection of (-)-epicatechin in the presence of other polyphenolic compounds and high reducing currents sensitivity (vs Ag/AgCl) examined at −0.6 V. The proposed biosensor showed reproducibility, reusability, long-term storage stability, and applicability in actual food samples with recovery values close to 100 %. Also, Kmapp and Imax values ​​of the biosensor were determined as 2.245 µM and 0.757 µA, respectively.

Biodegradation of chrysene and benzo[a]pyrene and removal of metals from naturally contaminated soil by isolated Trametes versicolor strain and laccase produced thereof

The objective of this study was to assess the degradation rates of chrysene and benzo[a]pyrene, as well as the removal of aluminium and iron from contaminated soil collected in the upper layer (0–30 cm) in Lagos, Southwest Nigeria. Trametes versicolor was isolated from this soil and used in degradation experiments, with plantain peels as support. After 8 weeks, 81.0% of chrysene degradation was achieved by T. versicolor , and by adding support this increased to 91.0%. Benzo[a]pyrene was less degradable, with 38.0% and 49.1% of degradation, respectively. Trametes versicolor was also capable of accumulate 46.1% of aluminium and 57.2% of iron. By adding plantain peels, these amounts increased to 48.2% and 61.8%, respectively. At the same time, laccase was produced by Trametes versicolor on plantain peels, achieving 37.8 U/g of crude laccase during SSF at 30 °C for 3 weeks. Laccase degradation experiments were set up in packed-bed reactor (PBR), with a constant feed of 21.6 mL/day of laccase, with and without mediators. In 35 days, 75.8% degradation of chrysene was achieved by laccase. The highest degradation was observed with ABTS (2,2 ′ -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) as mediator, 87.9%. Benzo[a]pyrene degradation with laccase reached 35.6%, raising to 38.8 % with ferulic acid as mediator. In addition, 99.2% of iron and 99.6% of aluminium was removed by laccase, being the treatment for this last mediated with ABTS. • T. versicolor efficiently degrades chrysene in soil in the presence of metals. • Laccase from T .versicolor efficiently removes metals in heavily contaminated soil. • Influence of metal co-contamination on PAH dissipation is studied in soil matrices.

Functionalisation of organosolv lignin by enzymatic demethylation for bioadhesive formulation

Bio-based adhesives offer a sustainable alternative to formaldehyde-based adhesives, which is the most common product applied in wood-based panel manufacturing. Despite the undeniable interest in lignin as a bulk chemical for the synthesis of bio-based adhesives, the main problem is its low reactivity. Therefore, it is crucial to study techniques to increase their reactivity, such as demethylation. One of the parameters to monitor the success of the demethylation is the methanol concentration measured in the reaction medium and complementing these results with the use of non-quantitative analyses such as FT-IR, MALDI-ToF and SEC. In order to maximize the action of the enzyme, a preliminary screening was performed with laccases with different redox potentials, which were added to organosolv lignin in the form of purified enzymes and cocktails. Trametes versicolor laccase was selected as the best candidate for the demethylation of organosolv lignin, with a methanol released concentration of 107 mg/L. Based on these results, parameters affecting both enzyme action and enzyme-substrate interaction were evaluated. Optimal working conditions were strongly defined by the use of surfactants that enhanced lignin solubility (Tween-80) and mediators that favored enzyme catalysis to participate directly in the enzyme catalytic cycle (HBT). However, it was also observed that factors such as pH, temperature and aeration have a direct impact on the enzymatic demethylation of lignin. Considering the operational variables that allowed better demethylation values, the amount of methanol released increased to 363 mg/L, which means an improvement of 239 %. These results were also supported by the increase in molecular weight observed in the SEC analysis and the reduction of the intensity of the FT-IR bands related to the methyl/methoxyl groups.

Chemoenzymatic deracemization of lisofylline catalyzed by a (laccase/TEMPO)-alcohol dehydrogenase system

This article reports on a novel biocatalytic method for the synthesis of both enantiomers of lisofylline based on Trametes versicolor laccase, TEMPO as a redox mediator and stereocomplementary recombinant alcohol dehydrogenases as biocatalysts.

Immobilization of Trametes Versicolor laccase on Cu-alginate beads for biocatalytic degradation of bisphenol A in water: Optimized immobilization, degradation and toxicity assessment

The present work investigated the immobilization of laccase (Trametes versicolor) on Cu-alginate beads by entrapment method and its utilization to degrade bisphenol A (BPA) in aqueous medium. The immobilized laccase demonstrated higher storage stability as compared to free enzyme. Box-Behnken experimental design was used to optimize the immobilization conditions (sodium alginate concentration, CuSO4 molarity, and hardening time). The optimal conditions were found to be 3% sodium alginate concentration (W/V), 0.141 mM CuSO4 and 90 min hardening time. Experimental data exhibited that immobilized laccase (0.5 g) could degrade 96.12% of BPA (10 mg/L) at pH 5.0, 30°C, 150 rpm and 1 h of reaction. The degradation products of BPA were identified by LC-MS analysis; hence the degradation pathways were suggested. The toxicity of BPA products was judged by phytotoxicity test. Immobilized laccase could also degrade BPA in real water, hence could be considered as an efficient candidate for removing BPA in water.

Bioremediation of phenolic pollutant bisphenol A using optimized reverse micelles system of Trametes versicolor laccase in non-aqueous environment.

The online version contains supplementary material available at 10.1007/s13205-021-02842-4.

Trametes versicolor laccase-assisted oxidative coupling of estrogens: Conversion kinetics, linking mechanisms, and practical applications in water purification

The potential application of fungal laccase-assisted bioremediation for estrogen elimination has caused tremendous attention. Herein, naturally-occurring Trametes versicolor laccase (Tvlac) was capable of significantly removing 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) at pH 5. A lower (or higher) pH caused decline in the velocity constants (kprcs) by obstructing the single-electron oxidation of estrogen at Tvlac T1-Cu site (or intercepting the intramolecular-electron transfer between Tvlac T1-Cu and T2/T3-Cu sites). Humic acid (HA) and natural phenolic compounds (PCs) are omnipresent in water and can influence the conversion kinetics of estrogen in Tvlac-triggered reactions. Compared with HA-free, the kprcs values of E2 and EE2 in presence of HA respectively reduced 58.61%–83.72% and 69.72%–95.62% at different pH levels. Additionally, the kprcs values for estrogen were also hampered obviously by O-dihydroxyphenol structure of model PCs, owning to the created monomeric/polymeric O-quinones inverted estrogen phenoxy radicals. The generated carbon‑carbon/oxygen self-linking aggregates during Tvlac-mediated estrogen oxidative coupling were characterized, and the polymerization mechanisms were speculated. A fluid-bed reactor with cross-linked-entrapped Tvlac in Ca-alginate beads was established to treat E2 and EE2, respectively. Compared with free Tvlac, immobilized Tvlac prominently exhibited a higher pH and temperature stability. Especially, the fluid-bed reactor with immobilized Tvlac could be reused, keeping its high conversion efficiencies of E2 and EE2 in natural water, only changing from 99.2% to 73.6% and from 98.5% to 70.9% after 1 to 5 cycles, respectively. These findings present an alternative immobilized fungal laccase-based clean biotechnology for continuously handling estrogen-contaminated waterbodies at a large-scale.

Laccase immobilized on amino-functionalized magnetic Fe3O4-SiO2 core–shell material for 2,4-dichlorophenol removal

ABSTRACT In this study, an amino-functionalized magnetic silica microsphere material (Fe3O4-SiO2-NH2) was prepared. Using glutaraldehyde as a cross-linking agent, Trametes versicolor laccase was adsorbed-covalently bonded and immobilized on the material to prepare Laccase @ Fe3O4-SiO2. In addition, the materials were characterized and analysed by SEM, TEM, XRD, FT-IR and VSM. Finally, the thermal inactivation dynamics of immobilized laccase in polar/non-polar/toxic systems and the adsorption and degradation of 2,4-DCP were studied. The results showed that Laccase @ Fe3O4-SiO2 under the optimal conditions (pH 6, temperature 65°C, initial concentration of 2,4-DCP 10 mg/L), the removal rate was as high as 81.6%. Moreover, compared with free laccase, immobilized laccase had good tolerance under low pH and high-temperature conditions, and storage stability was also greatly improved. After repeated use for 7 times, Laccase @ Fe3O4-SiO2 can still maintain 59% removal rate of 2,4-DCP, which gives it the potential for industrial applications.

Laccase immobilization on core-shell magnetic metal-organic framework microspheres for alkylphenol ethoxylate compound removal

An amino-functionalized magnetic core-shell structure metal-organic framework (MOF), Fe3O4-NH2@MIL-100 (Fe), has been successfully constructed by a step-by-step method. The composite microspheres exhibited excellent magnetic characteristics, large surface area, and pore size, which were employed for the Trametes versicolor laccase immobilization. The immobilized laccase exhibited high activity recovery (72.05%) and significantly improved stability. The reactive activity remained 75% of its initial activity after storage for 4 weeks. Additionally, when the ambient reached 80 °C, the good stability enabled the immobilization laccase to keep 33% residual activity even after 6 h storage. Application of the immobilized laccase for environmental pollutant alkylphenol polyethoxylate (APEO) removal was also investigated. The results showed the immobilized laccase can remove 100% octylphenol polyethoxylate (OPEO) and 98.16% nonylphenol polyethoxylate (NPEO) in 200 min, and can still achieve 63.42% and 46.17% removal efficiency for OPEO and NPEO after reusing four cycles, respectively. The novel MOF based core-shell magnetic composite microspheres introduced by this study showed promising applications as laccase immobilization support, and both the immobilized laccase and its support had great potential in wastewater treatment.