Investigation of laccase activity in cholinium-based ionic liquids using experimental and molecular dynamics techniques.

Laccases hold great potential for biotechnological applications, particularly in environmental pollutant remediation. Laccase activity is governed by the solvent environment, and ionic liquids (ILs) emerge as a versatile solvent for activation or stabilization of enzymes. Herein, effects of cholinium-based ILs formulated with carboxylic acids, inorganic acid, and amino acids as anionic species, on the catalytic activity of laccase from Trametes versicolor were investigated by experimental and computational approaches. Experimental results showed that laccase activity was enhanced by 21.39 % in 0.5 M cholinium dihydrogen citrate ([Cho][DHC]), in relation to the laccase activity in phosphate buffer medium. However, cholinium aminoate ILs negatively affected laccase activity, as evidenced by the partial deactivation of laccase in both cholinium glycinate and cholinium phenylalaninate, at concentrations of 0.1 M and 0.5 M, respectively. Molecular dynamics studies revealed that the enhancement of laccase activity in [Cho][DHC] might be attributed to the highly stabilized and compact structure of laccase, facilitating a better internal electron transfer during the laccase-substrate interactions. Enhanced catalytic performance of laccase in [Cho][DHC] was postulated to be driven by the high accumulation level of dihydrogen citrate anions around laccase's surface. [Cho][DHC] holds great promise as a cosolvent in laccase-catalyzed biochemical reactions.

Impact of surface-active ionic solutions on the structure and function of laccase from trametes versicolor: Insights from molecular dynamics simulations.

Many protein-ionic liquid investigations have examined laccase interactions. Laccases are a class of poly-copper oxidoreductases that retain significant biotechnological relevance owing to their notable oxidative capabilities and their application in the elimination of synthetic dyes, phenolic compounds, insecticides, and various other substances. This study investigates the impact of surface active ionic liquids (SAILs), namely, decyltrimethylammonium bromide [N10111][Br] and 1-decyl-3-methylimidazolium chloride [C10mim][Cl] as cationic surfactant ionic liquids and cholinium decanoate [Chl][Dec], an anionic surfactant ionic liquid, on the structure and function of laccase from the fungus Trametes versicolor (TvL) by the molecular dynamics (MD) simulation method. In summary, this study showed that laccase solvent-accessible surface area increased in the ionic liquid [Chl][Dec] while it decreased in the other two ionic liquids. Interestingly, [Chl][Dec] ionic liquid components formed hydrogen bonds with laccase, while [N10111][Br] and [C10mim][Cl] components were unable to form hydrogen bonds with laccase. The quantity of hydrogen bonds formed between water molecules and the enzyme was also diminished in the presence of [Chl][Dec] in comparison to the other two ionic liquids. especially at a concentration of 250 mM. In 250 mM concentrations of [N10111][Br] and [C10mim][Cl], clusters of long-chain cations are likely to form near the copper T1 site. However, even at low [Chl][Dec] concentrations, long [Dec]- chains were observed to penetrate the enzyme near the copper T1 site, and at 250 mM [Chl][Dec], a large cluster of anions occupied the opening of the active site. The results of the analysis also show that the interaction between the [Dec]- anion and the enzyme is stronger than the interaction between [N10111]+ and [C10mim]+ with laccase; in addition, the [Dec]- anion, compared to [Br]- and [Cl]- has a much greater tendency to bind with the enzyme residues.

Mechanism of non-phenolic substrate oxidation by the fungal laccase Type 1 copper site from Trametes versicolor: the case of benzo[a]pyrene and anthracene.

Laccases (EC 1.10.3.2) are multicopper oxidases with the capability to oxidize diverse phenolic and non-phenolic substrates. While the molecular mechanism of their activity towards phenolic substrates is well-established, their reactivity towards non-phenolic substrates, such as polycyclic aromatic hydrocarbons (PAHs), remains unclear. To elucidate the oxidation mechanism of PAHs, particularly the activation mechanism of the sp2 aromatic C-H bond, we conducted a density functional theory investigation on the oxidation of two PAHs (anthracene and benzo[a]pyrene) using an extensive model of the T1 copper catalytic site of the fungal laccase from Trametes versicolor.

Ligninolytic enzyme potential of Trametes spp. associated with leaf litter in riparian forest of the Amazônia region.

The present study explored the potential of leaf litter as a source of fungi able to produce ligninolytic enzymes for the biodegradation of anthraquinone dyes. Within the colonies isolated from the leaf litter, only three colonies of two species Trametes were selected based on the detection of oxidation and decolorization halos in Petri dishes with PDA (potato-dextrose-agar) + Guaicol and PDA + RBBR (Remazol Brilliant Blue R). The identification of the colonies was done through sequencing of the ITS region. The enzymatic activity of Lac (lacase), MnP (manganês peroxidase) and LiP (lignina peroxidase) was analyzed by spectrophotometry during fermentation in PD+RBBR imedium. Isolates A1SSI01 and A1SSI02 were identified as Trametes flavida, while A5SS01 was identified as Trametes sp. Laccase showed the highest enzymatic activity, reaching 452.13 IU.L-1 (A1SSI01, 0.05% RBBR) after 96h. Isolate A1SSI02 reached the highest percentage of decolorization, achieving 89.28% in seven days. The results imply that these Trametes isolates can be highly effective in waste treatment systems containing toxic anthraquinone dyes. Keywords: laccase, peroxidases, basidiomycete, litter and biodecolorization.

Two Zn2Cys6-type transcription factors respond to aromatic compounds and regulate the expression of laccases in the white-rot fungus Trametes hirsuta.

White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o-toluidine exposure, in Trametes hirsuta AH28-2. Two Zn2Cys6-type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o-toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o-toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs.IMPORTANCEWhite-rot fungi differentially express laccase isoenzymes when exposed to aromatic compounds. Clarification of the molecular mechanisms underlying differential laccase expression is essential to elucidate how white-rot fungi respond to the environment. Our study shows that two Zn2Cys6-type transcription factors form heterodimers, interact with the promoters of laccase genes, and positively regulate laccase transcription in Trametes hirsuta AH28-2. Aromatic monomer addition induces faster heterodimer formation and rate of activity. These findings not only identify two new transcription factors involved in fungal laccase transcription but also deepen our understanding of the mechanisms underlying the response to aromatics exposure in white-rot fungi.

Biofoams with untapped enzymatic potential produced from beer bagasse by indigenous fungal strains.

White rot fungi are promising organisms for the production of mycelial-based biofoams, providing a sustainable means of valorizing lignocellulosic wastes. This study explores the utilization of two indigenous fungal species, isolated from Argentina and belonging to the genera Trametes, for producing biofoams from brewery waste. The resulting biofoams exhibited an average density of 0.30 g cm-3, a Young's modulus of approximately 1 MPa, and a compressive stress of around 19 MPa. Additionally, the variation of laccase activity throughout the biofoam production process was evaluated. Surprisingly, residual laccase activity was detected in the biofoams following oven drying at temperatures of 60, 80, and 100 °C. This detection highlights the untapped enzymatic potential of the biofoams and positions them as promising green catalysts for various biotechnological applications.

Improved productivity and dye removal performance of Trametes versicolor pellets using rice husk as a co-substrate.

Pellet production represents a critical step for several processes requiring fungal biomass, nevertheless, its optimization is seldom reported. The use of finely ground rice husk as a microcarrier and co-substrate permitted a marked increase (≈ 2.7×) in the productivity of fungal pellet production using Trametes versicolor compared to traditional production methods. The pellets show similar structure and smaller size compared to typical sole-mycelium pellets, as well as comparable laccase activity. The efficiency of the pellets for biodegradation was confirmed by the removal of the crystal violet dye, achieving significantly faster decolorization rates compared to the traditionally produced pellets. The use of these pellets during the continuous treatment of the dye in a stirred tank bioreactor resulted in 97% decolorization operating at a hydraulic residence time of 4.5 d.

Using low-shear aerated and agitated bioreactor for producing two specific laccases by trametes versicolor cultures induced by 2,5-xylidine: Process development and economic analysis.

Laccase isoforms from basidiomycetes exhibit a superior redox potential compared to commercially available laccases obtained from ascomycete fungi, rendering them more reactive toward mono-substituted phenols and polyphenolic compounds. However, basidiomycetes present limitations for large-scale culture in liquid media, restraining the current availability of laccases from this fungal class. To advance laccase production from basidiomycetes, a newly designed 14-L low-shear aerated and agitated bioreactor provided enzyme titers up to 23.5 IU/mL from Trametes versicolor cultures. Produced enzymes underwent ultrafiltration and LC/MS-MS characterization, revealing the predominant production of only two out of the ten laccases predicted in the T. versicolor genome. Process simulation and economic analysis using SuperPro designer® suggested that T. versicolor laccase could be produced at US$ 3.60/kIU in a 200-L/batch enterprise with attractive economic parameters and a payback period of 1.7 years. The study indicates that new bioreactors with plain design help to produce low-cost enzymes from basidiomycetes.

Unambiguous NMR Structural Determination of (+)-Catechin-Laccase Dimeric Reaction Products as Potential Markers of Grape and Wine Oxidation.

(+)-Catechin-laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone-d6 led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.

Immobilization of Trametes versicolor laccase on chitosan/halloysite as a biocatalyst in the Remazol Red RR dye.

In this study, the laccase obtained from Trametes versicolor was immobilized onto the chitosan(CTS)/halloysite (HNT) beads. In the immobilization step, the effects of chitosan (1-3% w/v), halloysite (0-2% w/v), glutaraldehyde (0.5-1.5% v/v) and enzyme concentrations (1-3%) on loading and immobilization efficiency were investigated. SEM, FT-IR, XRD, TGA and XPS analyses were performed to examine the structure of beads. In addition, the effects of parameters such as pH (4-10), temperature (25-55 °C), storage life on the activity of free and immobilized laccase were also investigated. The activities of free and immobilized laccase preserved 23% and 56% of its initial activity at the end of 59 days of storage. The effects of mediators such as 2.2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1-Hydroxybenzotriazole hydrate (HBT), 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) and violuric acid (VLA) on the dye removal efficiency were investigated. Reusability of the CTS/HNT/Lac in the presence of HBT and VLA mediators, which enable the highest dye removal, was tested. After 15 cycles, 42% and 54% dye removal were achieved with the CTS/HNT/Lac in the medium containing HBT and VLA, and 42% and 49% of the activity is preserved, respectively. This study showed that CTS/HNT/Lac can be used repeatedly for Remazol Red RR dye removal.

Facile synthesis of recyclable laccase-mineral hybrid complexes with enhanced activity and stability for biodegradation of Evans Blue dye.

Two morphologies of laccase-mineral hybrid complexes, i.e., laccase-mineral hybrid nanoflowers (La-HNF) and nanopetals (La-HNP), were synthesized via biomineralization using Cu3 (PO4)2·3H2O as the mineral for Evans Blue (EB) dye biodegradation. XRD patterns and FT-IR spectra results revealed the successful immobilization of laccase via in-situ formed Cu3(PO4)2·3H2O crystals. Compared with free laccase, laccase-mineral hybrid complexes showed higher enzymatic activity due to the activation effect induced by copper ions of Cu3(PO4)2·3H2O, further, the improved kinetic parameters of laccase-mineral hybrid complexes could be ascribed to nanoscale-dispersed laccase molecules within hybrid complexes. For EB dye biodegradation, the reason why the biodegradation efficiency (94.9%) of La-HNF was higher than that (86.8%) of La-HNP could be synergistic effect of immobilized laccase within 3D hierarchical structure of La-HNF. In addition, the optimized biodegradation conditions (pH 4.6 and 40 °C) of La-HNF were obtained, moreover, 93.2% and 48.1% of EB dye were biodegraded by La-HNF after stored for 30 days and reused for 10 cycles, respectively, demonstrating La-HNF have good practicability.

A novel process for the covalent immobilization of laccases on silica gel and its application for the elimination of pharmaceutical micropollutants.

In the present work, pharmaceutical micropollutant degradation by laccase immobilized on silica through an innovative process is proposed. The influence of different parameters on the immobilization conditions was evaluated by a 23 full factorial design, and parameters leading to the highest activity were identified. Under these conditions, laccase activity reached 14 ± 2 U g-1 of silica with a protein immobilization yield of 35%. The biocatalyst characterization did not show any change in pH and thermal stabilities but enhanced the long-term storage of laccases. Immobilized T. versicolor laccases were then tested to remove four pharmaceutical micropollutants (amoxicillin, ciprofloxacin, carbamazepine, and sulfamethoxazole) in the presence of redox mediators (syringaldehyde, p-coumaric acid, and ABTS). High removal yields (50-100% according to the pollutant) were obtained within 4 h of treatment due to the synergistic effect of laccase-mediator biotransformation and adsorption on the support. Overall, the pharmaceuticals' removal efficiency was highly influenced by their physicochemical properties; however, the presence of redox mediators impacted not only the oxidation mechanism but also the interactions between the biocatalyst and micropollutants. Finally, the reusability of the biocatalyst was proved during 7 degradation cycles.

Immobilization of laccase via cross-linked enzyme aggregates prepared using genipin as a natural cross-linker.

Genipin is a nontoxic natural cross-linker that was successfully used to prepare cross-linked enzyme aggregates (CLEAs) of Trametes versicolor laccase. The recovered activity of CLEAs was influenced by the co-solvent type, genipin concentration, cross-linking time, preparation pH, and bovine serum albumin (BSA; amino group feeder) concentration. The characteristics of CLEAs prepared using genipin under optimal conditions (genipin-BSA-CLEAs) were compared with those of typical CLEAs prepared using glutaraldehyde or dextran polyaldehyde. Genipin-BSA-CLEAs were nano-sized (average diameter, approximately 700 nm), had a ball-like shape, showed a narrow size distribution, and exhibited the highest substrate affinity among the prepared CLEAs. The thermal stability of genipin-BSA-CLEAs was 6.8-fold higher than that of free laccase, and their pH stability was also much higher than that of free laccase in the tested range. Additionally, genipin-BSA-CLEAs retained 85% of their initial activity after 10 cycles of reuse. Particularly, genipin-BSA-CLEAs showed higher thermal and pH stability than CLEAs that were cross-linked using glutaraldehyde. Therefore, genipin represents an alternative to toxic compounds such as glutaraldehyde during cross-linking to prepare CLEAs.

Kinetic characterization of purified laccase from Trametes hirsuta: a study on laccase catalyzed biotransformation of 1,4-dioxane.

OBJECTIVE: Laccase is one of the best known biocatalysts which degrade wide varieties of complex molecules that are both non-cyclic and cyclic in structure. The study focused on enzyme kinetics of a purified laccase from Trametes hirsuta L. fungus and its application on biotransformation of a carcinogenic molecule 1,4-dioxane. RESULTS: Laccase was purified from white-rot fungus T. hirsuta L. which showed specific activity of 978.34 U/mg after the purification fold of 54.08. The stable laccase activity (up to 16 h) is shown at 4-6 pH and 20-40 °C temperature range. The purified enzyme exhibited significant stability for 10 metal ions up to 10 mM concentration, except for Fe2+ and Hg2+. The Cu2+ ion induced laccase activity up to 142% higher than the control at 10 mM concentration. The laccase enzyme kinetic parameters Km was 20 ± 5 µM and 400 ± 60 µM, whereas Kcat was 198.29 ± 0.18/s and 80.20 ± 1.59/s for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol respectively. The cyclic ether 1,4-dioxane (100 ppm) was completely degraded in presence of purified laccase within 2 h of incubation and it was confirmed by HPLC and GC analysis. The oxidation reaction was accelerated by 25, 22, 6 and 19% in presence of 1 mM syringaldehyde, vanillin, ABTS and guaiacol mediators respectively. CONCLUSIONS: In this study, fungal laccase (a natural biocatalyst) based degradation of synthetic chemical 1,4-dioxane was reported for the first time. This method has added advantages over the multiple methods reported earlier being a natural remedy.

Imidazole-rich copper peptides as catalysts in xenobiotic degradation.

Laccases, oxidative copper-enzymes found in fungi and bacteria were used as the basis in the design of nona- and tetrapeptides. Laccases are known to be excellent catalysts for the degradation of phenolic xenobiotic waste. However, since solvent extraction of laccases is environmentally-unfriendly and yields obtained are low, they are less preferred compared to synthetic catalysts. The histidine rich peptides were designed based on the active site of laccase extracted from Trametes versicolor through RCSB Protein Data Bank, LOMETS and PyMol software. The peptides were synthesized using Fmoc-solid phase peptide synthesis (SPPS) with 30-40% yield. These peptides were purified and characterized using LC-MS (purities >75%), FTIR and NMR spectroscopy. Synthesized copper(II)-peptides were crystallized and then analyzed spectroscopically. Their structures were elucidated using 1D and 2D NMR. Standards (o,m,p-cresol, 2,4-dichlorophenol) catalysed using laccase from Trametes versicolor (0.66 U/mg) were screened under different temperatures and stirring rate conditions. After optimizing the degradation of the standards with the best reaction conditions reported herein, medications with phenolic and aromatic structures such as ibuprofen, paracetamol (acetaminophen), salbutamol, erythromycin and insulin were screened using laccase (positive control), apo-peptides and copper-peptides. Their activities evaluated using GC-MS, were compared with those of peptide and copper-peptide catalysts. The tetrapeptide was found to have the higher degradation activity towards salbutamol (96.8%) compared with laccase at 42.8%. Ibuprofen (35.1%), salbutamol (52.9%) and erythromycin (49.7%) were reported to have the highest degradation activities using Cu-tetrapeptide as catalyst when compared with the other medications. Consequently, o-cresol (84%) was oxidized by Tp-Cu while the apo-peptides failed to oxidize the cresols. Copper(II)-peptides were observed to have higher catalytic activity compared to their parent peptides and the enzyme laccase for xenobiotic degradation.

Influence of humic acids on fungal laccase-initiated 17α-ethynylestradiol oligomerization: Transformation kinetics and products distribution.

Fungal laccase has aroused great concern in rapidly removing estrogens because of its ability to accelerate humification and oligomerization. Here, the effect of two humic acids (HAs) on the reaction kinetics and products distribution of 17α-ethynylestradiol (EE2) in laccase-initiated humification and coupling was systematically elucidated. Laccase from Trametes versicolor exhibited over 98.3% removal rate for EE2 at pH 5.0 within 120 min, while HAs invariably restrained EE2 transformation by competing with target-substrate for the enzymatic catalytic center. EE2 removal followed pseudo-first-order kinetics, and the rate constant was decreased markedly with increasing concentration of two HAs (0-60 mg L-1). Additionally, laccase heightened the aromaticity and humification degrees (A250 nm/A365 nm ratio) of HAs probably due to the formation of new humic polymers such as (HA)m and/or (HA)m-(EE2)n (m and n represent the number of HA and EE2 units, respectively). Three major EE2 oligomers were identified, in accordance with a mechanism involving the phenoxy radical-driven polymerization to yield a wide variety of self-coupling products. Notably, HAs diminished the extent of EE2 self-coupling but aggrandized the cross-coupling between EE2 and HAs, and the inhibition degree of EE2 self-coupling increased with the concentration of HAs. One major reason is EE2 could be covalently incorporated into humic molecules to produce (HA)m-(EE2)n cross-coupling products via radical-caused C-C, C-O-C, and/or C-O-C bonds, thereby reducing EE2 self-oligomerization. These findings highlight that HAs play a vital role in the fungal laccase-induced humification and oligomerization of EE2, which obviously alter the geochemical fate and transport of EE2 in natural aquatic ecosystems.

Comparative catalytic degradation of a metabolite 3,5-dichloroaniline derived from dicarboximide fungicide by laccase and MnO2 mediators.

A ternary catalysis system was investigated to evaluate the comparative degradation of toxic fungicide metabolite 3,5-dichloroaniline (3,5-DCA) by laccase and MnO2 with mediators. In this study, copper based fungal enzyme laccase (Trametes versicolor origin) and metal catalyst MnO2 with various combinations of phenolic mediators (catechol, syringaldehyde, syringic acid, caffeic acid and gallic acid) were monitored to optimize and screen the better one for 3,5-DCA degradation assay. Catechol showed better potentiality in reduction of 3,5-DCA among the studied mediators. Catechol (2mM) showed the highest reduction rate (99-100%) followed by syringaldehyde (40.51%) with 2U/mL of laccase at 25 °C within 24 h reaction time. Similarly, complete degradation of 3,5-DCA was obtained by catechol (2mM) with 2 mg/mL of MnO2 in MnO2-mediator assay. The notable finding of current study indicated the triggering of catechol for better 3,5-DCA degradation at higher pH condition but inertness in laccase-mediator assay due to laccase destabilization. The reaction pathways of optimized mediator-based catalysis for laccase and MnO2 were proposed. Finally, the optimized laccase-catechol based degradation was considered as a pioneer green catalysis approach to reduce the toxic metabolite 3,5-DCA concentrations in aqueous medium as compared to MnO2-catechol catalysis.

Enzymatic hydrolysis of barley straw for biofuel industry using a novel strain of Trametes villosa from Paranaense rainforest.

Agricultural practices generate lignocellulosic waste that can be bioconverted by fungi to generate value-added products such as biofuels. In this context, fungal enzymes are presented as an alternative for their use in the hydrolysis of cellulose to sugars that can be fermented to ethanol. The aim of this work was to characterize LBM 033 strain and to analyze its efficiency in the hydrolysis of cellulosic substrates, including barley straw. LBM 033 strain was identified as Trametes villosa by molecular techniques, through the use of the ITS and rbp2 markers and the construction of phylogenetic trees. The cell-free supernatant of T. villosa LBM 033 showed high titers of hydrolytic enzymatic activities, necessary for the hydrolysis of the holocellulosic substrates, hydrolyzing pure cellulose to cellobiose and glucose and also degraded the polysaccharides contained in barley straw to short soluble oligosaccharides. These results indicate that macro fungi from tropical soil environments, such as T. villosa LBM 033 can be a valuable resource for in-house, cost effective production of enzymes that can be applied in the hydrolysis stage, which could reduce the total cost of bioethanol production.

Ecotoxicological test based on inhibition of fungal laccase activity: Application to agrochemicals and the monitoring of pesticide degradation processes.

Ecotoxicological evaluations require the use of assays with several bioindicators from different trophic levels. Only a few ecotoxicological tests using fungi have been developed, reason why, detection of adverse effects from compounds that exert fungicide action may be overlooked. This work developed a toxicity test based on the inhibition of laccase enzymatic activity in the fungus Trametes versicolor. The test was applied to several fungicides and succeeded to determine inhibition values (half maximum effective concentration, EC50) for most of them (flusilazole, imazalil, pyrimethanil, tetraconazole), though a clear dose-response was not evident for others (thiabendazole, metalaxyl). The application on atrazine (herbicide), imidacloprid (insecticide) and oxytetracycline (antibiotic), proved the proposed test is suitable towards other agrochemicals. The test was also used to estimate the detoxification resulting from two different approaches employed in the removal of agrochemicals. (a) First, in the liquid-phase elimination by fungal biomass simultaneously removing atrazine, imazalil, tebuconazole and triadimenol, the test showed a significant decrease in toxicity by biodegradation (adsorption contribution to detoxification was negligible). (b) Second, a solid-phase biomixture (used for pesticide degradation from agricultural wastewater) partially removed atrazine, imazalil, metalaxyl and pyrimethanil after 33 d; nonetheless, this system could not reduce the toxicity of the matrix, and higher laccase inhibition was detected after the treatment. The design test increases the battery of available bioassays to determine the toxicity of agrochemicals, and provides an interesting tool to monitor biodegradation processes.

Amperometric biosensor based on coupling aminated laccase to functionalized carbon nanotubes for phenolics detection.

A biosensor for phenolic compounds based on a chemically modified laccase from Coriolus hirsuta immobilized on functionalized screen-printed carbon electrodes (SPCEs) was achieved. Different enzyme modifications and immobilization strategies were analyzed. The electrochemical response of the immobilized laccase on SPCEs modified with carboxyl functionalized multi-walled carbon nanotubes (COOH-MWCNT) was the highest when laccase was aminated prior to the adsorption onto the working electrode. The developed laccase biosensor sensitivity toward different phenolic compounds was assessed to determine the biosensor response with several phenolic compounds. The highest response was obtained for ABTS with a saturation value of Imax = 27.94 µA. The electrocatalytic efficiency (Imax/Kappm) was the highest for ABTS (5588 µA µM-1) followed by syringaldazine (3014 µA.µM-1). The sensors were considerably stable, whereby 99.5, 82 and 77% of the catalytic response using catechol as substrate was retained after 4, 8 and 10 successive cycles of reuse respectively, with response time average of 5 s for 12 cycles. No loss of activity was observed after 20 days of storage.