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Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase-luminol system, determine its kinetic parameters, and analyze the effects of protein and OH- concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase-luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 μM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase-luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification., Translational Omics and Bioengineering and Regenerative Medicine strategic focus groups of Tecnologico de Monterrey; CONACyT, Grant/Award Number: 492276, With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).

Carbon nanotube (CNT) and gelatin (Gela) molecules are effective substrates in promoting engineered cardiac tissue functions. This study developed a microfluidic-based encapsulation process for biomimetic hydrogel microcapsule fabrication. The hydrogel microcapsule was produced through a coaxial double orifice microfluidic technique and a water-in-oil emulsion system in two sequential processes. The phenol (Ph) substituted Gela (Gela-Ph) and CNT (CNT-Ph), respectively as cell-adhesive and electrically conductive substrates were incorporated in hyaluronic acid (HA)-based hydrogel through laccase-mediated crosslinking. The Cardiomyocyte-enclosing microcapsule fabricated and cellular survival, function, and possible difference in the biological activity of encapsulated cells within micro vehicles were investigated. The coaxial microfluidic method and Lac-mediated crosslinking reaction resulted in spherical vehicle production in 183 μm diameter at 500 capsules/min speed. The encapsulation process did not affect cellular viability and harvested cells from microcapsule proliferated well likewise subcultured cells in tissue culture plate. The biophysical properties of the designed hydrogel, including mechanical strength, swelling, biodegradability and electroconductivity upregulated significantly for hydrogels decorated covalently with Gela-Ph and CNT-Ph. The tendency of the microcapsule for the spheroid formation of cardiomyocytes inside the proposed microcapsule occurred 3 days after encapsulation. Interestingly, immobilized Gela-Ph and CNT-Ph promote cellular growth and specific cardiac markers. Overall, the microfluidic-based encapsulation technology and synthesized biomimetic substrates with electroconductive properties demonstrate desirable cellular adhesion, proliferation, and cardiac functions for engineering cardiac tissue.

Makale (Early Access), WOS:000909732300001, Herein, a novel D-A-D type monomer that contains hexylthiophene as the donor and benzo[c][1,2,5]thiadiazole as the acceptor has been electropolymerized successfully to yield P(DTFBT) and its electrochromic properties and performance in amperometric biosensors have been investigated. The polymer shows great electrochemical features with low band gap and multichromic properties. Additionally, after polymer modification, laccase was immobilized on a modified electrode surfaces for catechol sensing. Biosensor optimization values, cycle number, enzyme amount, pH and also interfering effect of some possible compounds were investigated. With optimum values, analytical characterization of the sensor was determined. Kmapp, LOD and sensitivity values were estimated as 0.11 mM, 0.014 mM and 166.74 mu A/(mM cm(2)), respectively. The experimental results revealed that the sensor shows satisfactory accuracy and confirms the proposed sensor has a potential candidate for catechol quantification., Scientific and Technological Research Council of Türkiye (TUBITAK)

The purpose of this study is to develop a green and safe chitosan-based preservative which can be applied in strawberry preservation. Chitosan (CS) was treated by 2,2,6,6-tetramethylpiperidine oxygen radical/laccase oxidation system (TEMPO/laccase oxidation system), which was mainly used to prepare TEMPO/laccase chitosan (TLCS). Furthermore, on this basis, the structure and performance of TLCS were also studied. The results showed that compared with CS, the solubility of TLCS improved, and the kinetic viscosity reduced significantly. Next, a cinnamaldehyde-TEMPO/laccase chitosan (CIN-TLCS) antibacterial agent was prepared by covalently combining the aldehyde group in cinnamaldehyde (CIN) and the amino group in CS. It was found that CIN combined with TLCS through covalent bonds, which changed the structure and crystallinity of TLCS. In addition, the total antioxidant capacity of CIN-TLCS also improved, which was necessary for the application of CIN-TLCS in extending shelf life. Cytotoxicity experiments showed that CIN-TLCS had no cytotoxicity. Furthermore, strawberries were used to explore the actual bacteriostatic and fresh-keeping effects of CIN-TLCS. The experiment found that CIN-TLCS could maintain the freshness of strawberries at room temperature (23 ± 1°C) for 5 days and had positive effects on strawberry color, loss-weight rate, hardness and pH. These results showed that CIN-TLCS could be used as a potential preserving agent for fruit storage. PRACTICAL APPLICATION: To obtain a green, safe and effective food preservative, chitosan (CS) was modified by a 2,2,6,6-tetramethylpiperidine oxygen radical/laccase oxidation system (TEMPO/laccase oxidation system) to get TEMPO/laccase chitosan (TLCS) and cinnamic aldehyde-TEMPO/laccase chitosan (CIN-TLCS). At the same time, the structure and antibacterial properties of TLCS and CIN-TLCS were analyzed, and their possibility as a new green and safe strawberry preservative was studied. Compared with oxazolidine, imidazole and triazole commercial drugs, CIN-TLCS has the advantages of low price, no pollution, no cytotoxicity and no drug resistance.

Lignin is an abundant natural feedstock that offers great potential as a renewable substitute for fossil‐based resources. Its polyaromatic structure and unique properties have attracted significant research efforts. The advantages of an enzymatic over chemical or thermal approach to construct or deconstruct lignins are that it operates in mild conditions, requires less energy, and usually uses non‐toxic chemicals. Laccase is a widely investigated oxidative enzyme that can catalyze the polymerization and depolymerization of lignin. Its dual nature causes a challenge in controlling the overall direction of lignin‐laccase catalysis. In this Review, the factors that affect laccase‐catalyzed lignin polymerization were summarized, evaluated, and compared to identify key features that favor lignin polymerization. In addition, a critical assessment of the conditions that enable production of novel lignin hybrids via laccase‐catalyzed grafting was presented. To assess the industrial relevance of laccase‐assisted lignin valorization, patented applications were surveyed and industrial challenges and opportunities were analyzed. Finally, our perspective in realizing the full potential of laccase in building lignin‐based materials for advanced applications was deduced from analysis of the limitations governing laccase‐assisted lignin polymerization and grafting., Laccases as builders: Similar to their role in the formation of lignin in nature, laccases are capable of building advanced lignin‐based materials, given the optimum laboratory conditions. This Review evaluates the factors that affect laccase‐catalyzed lignin polymerization and grafting reactions. Patented applications of lignin‐based materials produced via laccase catalysis are highlighted and potential routes towards unlocking the full capacity of laccase in lignin valorization are proposed.

Bisphenol A (BPA) is one of the toxic chemicals, which is widely used for manufacturing epoxy, polyester resin, and polycarbonates. These materials are extensively used in manufacturing of reusable bottles, baby bottles, dental sealants, various medical devices, and so forth. Moreover, canned and packaged foods are sources of bisphenol A, which is unknowingly consumed by many people worldwide. Its endocrine disrupting and teratogenic properties impose potential risk to the wildlife and human health. BPA has been linked to reproductive, metabolic, and immunity disorders in humans. Regardless of BPA ban in reusable and baby bottles, annually, 15 billion pounds of BPA still being produced. BPA pollution and its cleanup are major challenges. Therefore, it is essential to develop a suitable strategy to bioremediate BPA. The Trametes versicolor (TV) laccase-based nanoemulsion calcium alginate bead organogel was able to transform 94% of BPA within 2 h of treatment. Organogel showed 60% of BPA removal from actual industrial wastewater in packed bed batch reactor and 67% of BPA removal in continuous flow packed bed reactor. The biological oxygen demand (BOD) of treated industrial effluent was 14 mg/L, which is very much less than untreated effluent's BOD, which was 48 mg/L. The chemical oxygen demand of industrial effluent was 1240 mg/ml, and treated effluent was 248 mg/L, respectively. Hence, application of nanoemulsion-based organogel in packed bed reactor found to be a potential candidate for the bioremediation of industrial effluent containing BPA. PRACTITIONER POINTS: The TV laccase-based nanoemulsion calcium alginate bead organogel was able to transform 94% of BPA. Organogel showed 67% of BPA removal from industrial wastewater in continuous flow packed bed reactor. The nanoemulsion-based organogel in packed bed reactor found to be potential candidate for the bioremediation of industrial effluent containing BPA.

Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation of a long-carbohydrate-chain alkyl glycoside and cyclodextrin glucanotransferase (CGTase)-catalyzed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl β- d-maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase-catalyzed coupling/disproportionation reactions with α-cyclodextrin and dodecyl β- d-maltoside diuronic acid (DDM-2COOH) or octyl β- d-glucuronic acid (OG-COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non-ionic substrates (dodecyl β- d-maltoside and octyl β- d-glucoside), Toruzyme exhibited high catalytic activity at pH 5-6, but for the acidic substrates (DDM-2COOH and OG-COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM-2COOH and OG-COOH, which exist at lower pH (pKa about 3).

Summary Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well‐studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties., Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P . acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well‐studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.

This study investigated the dye decolorization capacity of three yeast strains. Cyberlindnera fabianii was shortlisted for its high decolorization capacity and was further tested on various azo dyes. Based on the color of the biomass, and the UV-Vis analysis, Acid Red 14 was selected as a model dye, to examine the enzymatic biodegradation. The results showed significant increase in the intracellular and extracellular activities of laccase, tyrosinase, manganese peroxidase, and azoreductase. Phytotoxicity assessment indicated that the AR14 biodegradation by-products were not phytotoxic compared to the original dye molecules. Regarding the decolorization optimization, the screening of factors using the Plackett-Burman design showed that pH, dye concentration, and shaking speed had significant effects. These factors and their combined effect were evaluated using response surface methodology with the Box-Behnken model. The pH was the most significant factor, followed by dye concentration. The analysis of the contour plot and the 3D response surface diagram showed that the decolorization was inversely proportional to the increase in the initial dye concentration, but proportional to the initial pH and shaking speed. At optimal conditions (pH = 5.154, AR14 = 50 mg/L), C. fabianii could decolorize more than 97% of AR14 within 12 hr. PRACTITIONER POINTS: Cyberlindnera fabianii is a successful candidate for dye mycoremediation. Oxidase and reductase are the key enzymes involved in the biodegradation of azo dyes. By-products of Acid red 14 biodegradation are not phytoxic compared to the original dye. Design of experience tools enables to determine optimum conditions for efficient decolorization.

Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana, an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response-generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph-derived in vivo fungal hyphal bodies or be secreted. Targeted gene-knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen-secreted laccase 2 in evasion of insect immune defenses.

Laccases belong to a family of multicopper enzymes able to oxidize a broad spectrum of organic compounds. Despite the well-known property of laccases to carry out bleaching and degradation of industrial dyes and polyphenolic compounds, their industrial use is often limited by the high cost, low efficiency, or instability of these enzymes. To look for new microorganisms which produce laccases that are potentially suitable for industrial applications, we have isolated several fungal strains from a cave in northern Spain. Their phenotypic analysis on agar plates supplemented with ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) disclosed two laccase-positive strains. Further genotyping revealed that they belonged to the Gliomastix murorum and Conidiobolus thromboides species. The secretion of G. murorum and C. thromboides laccase-like enzymes was then confirmed by zymography. Further identification of these polypeptides by mass-spectroscopy revealed the nature of the laccases and made it possible to predict their functional domains and other features. In addition, plate assays revealed that the laccases secreted by both G. murorum and C. thromboides were capable of degrading industrial dyes (Congo Red, Indigo, and Eriochrome Black T). Homology modeling and substrate docking predicted the putative structure of the currently uncrystallized G. murorum enzyme as well as its amino acid residues potentially involved in interactions with these dyes. In summary, new biochemical and structural insights into decolorization mediated by G. murorum laccase as well as identification of laccase-like oxidase in C. thromboides point to a promising future for these enzymes in biotechnology., The work was supported by the Basque Government pre-doctoral grant PRE-2013-1-901, SAIOTEK grant SPE12UN84, and IKERBASQUE (Basque Foundation for Science).

Nowadays natural biopolymers have a wide variety of uses in various industrial applications, such as food, adhesives and composite materials. Among them, cellulose has attracted the interest of researchers due to its properties: high strength and flexibility, biocompatibility and nontoxicity. Despite that, in many cases its practical use is limited because of poor solubility and/or an unsuitable hydrophilic/hydrophobic balance. In this context, enzymatic modification appears as a powerful strategy to overcome these problems through selective, green and environmentally friendly processes. This minireview discusses the different methods developed for the enzymatic modification of cellulose, emphasizing the type of reaction, the enzymes used (laccases, esterases, lipases, hexokinases, etc.), and the properties and applications of the cellulose derivatives obtained. Considering that cellulose is the most abundant natural polymer on Earth and can be derived from residual lignocellulosic biomass, the impact of its use in bio-based process following the logic of the circular economy is relevant.

A homogeneous monomeric laccase (ASL) from Agaricus sinodeliciosus, with a molecular mass of 65 kDa, was isolated using ion-exchange chromatography (CM-cellulose and Q-Sepharose) and gel-filtration chromatography (Superdex 75). This laccase exhibited maximum activity at 50 °C and pH 5.0. Hg2+ and Cd2+ significantly inhibited its activity. The laccase displayed a Km value of 0.9 mM toward 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS). In addition to ABTS, ASL exhibited higher affinity toward o-toluidine and benzidine than other substrates. ASL is able to decolorize malachite green and Eriochrome black T.

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Here, molecular docking simulation was used to predict and compare interactions between a recombinant Trametes sp. C30 laccase from Saccharomyces cerevisiae and four aflatoxins (AFB1 , AFB2 , AFG1 , and AFG2 ) as well as their degradation at a molecular level. The computational result of docking simulation indicates that each of the aflatoxins tested can interact with laccase with a binding ability of AFB1 >AFG2 >AFG1 >AFB2 . Simultaneously, it also demonstrated that aflatoxin B1 ， B2 ， G1 ， G2 may interact near the T1 copper center of the enzyme through H-bonds and hydrophobic interactions with amino acid residues His481 and Asn288; His481； Asn288, and Asp230; His481 and Asn288. Biological degradation test was performed in vitro in the presence of a recombinant laccase. Degradation increased as incubation time increased from 12 to 60 hr and the maximum degradation obtained for AFB1 , AFB2 , AFG1 , and AFG2 was 90.33%, 74.23%, 85.24%, and 87.58%, respectively. Maximum degradation of aflatoxins was determined with a total activity 3 U laccase at 30 °C in 0.1 M phosphate buffer, pH 5.7 after 48-hr incubation. The experimental results are consistent with that of docking calculation on the biological degradation test of four aflatoxins by laccase. PRACTICAL APPLICATION: In this study, the degradation efficiencies of laccase for B and G series of aflatoxins were determined by computer simulation and verified by performing in vitro experiments. It can provide reference for rapid screening of aflatoxin degradation-related enzymes.

International audience; Lignin is the second most abundant polymer after cellulose in lignocellulosic biomass. Its aromatic composition and recalcitrant nature make its valorization a major challenge for obtaining low molecular weight aromatics compounds with high value-added from the enzymatic depolymerization of industrial lignins. The oxidation reaction of lignin polymer using laccases alone remains inefficient. Therefore, researches are focused on the use of a laccase-mediator system (LMS) to facilitate enzymatic depolymerization. Until today, the LMS system was studied using water-soluble lignin only (commercial lignins, modified lignins, or lignin model compounds). This work reports a study of three LMS systems to depolymerize the three major industrial lignins (organosolv lignin, Kraft lignin, and sodium lignosulfonate). We show that an enzymatic depolymerization of these lignins can be achieved by LMS using laccase from Trametes versicolor, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt as mediator and a cosolvent (25% of 1,4-dioxane) to enhance the solubilization of lignins.

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.

Background Ginkgo biloba leaf extract contains many active ingredients that are beneficial for health. However, ginkgolic acid, one of the major components found in G. biloba extract, may cause serious allergic and toxic side effects. The purpose of this study is to immobilize the laccase system on the electrospun nylon fiber mat (NFM) to hydrolyze the ginkgolic acid in G. biloba leaf extract efficiently. Results Novel electrospinning technology successfully produced high-quality nanoscopic fiber mats made of a mixture of multi-walled carbon nanotube and nylon 6,6. Laccase that was immobilized onto the NFM exhibited much higher efficiency in the catalyzation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) than nylon 6,6 pellets. After being immobilized onto the NFM, the pH and temperature stability of laccase were significantly improved. The NFM-immobilized laccase could maintain more than 50% of its original activity even after 40 days of storage or 10 operational cycles. The kinetic parameters, including rate constant (K), the time (τ50) in which 50% of ginkgolic acid hydrolysis was reached, the time (τcomplete) required to achieve complete ginkgolic acid hydrolysis, Km and Vmax were determined, and were 0.07 ± 0.01 min-1 , 8.97 ± 0.55 min, 45.45 ± 2.79 min, 0.51 ± 0.09 mM and 0.49 ± 0.03 mM min-1 mg-1 , respectively. Conclusion The result successfully demonstrated the strong potential of using novel electrospun nanofiber mats as enzyme immobilization platforms, which could significantly enhance enzyme activity and stability. © 2020 Society of Chemical Industry.

Wastewater from the textile industry contaminated with azo dyes affects the environment negatively, causes pollution, and threatens environmental balance. Among various methods for wastewater treatment, bioremediation emerges as an environmentally friendly, economical, and sustainable solution. In this study, white-rot fungus Sporotrichum sp. was employed to decolorize reactive blue 13 (RB13). The long-term decolorization capacity of the fungus was investigated by a sequential batch experiment under optimized conditions. The fungus showed high decolorization efficiency upon repeating usage, and its decolorization efficiency decreased from 97.4% to 87.09% after transferring to a freshly prepared medium seven times. The MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay) method using Chinese Hamster Lung V79 379A was performed to assess the cytotoxicity of treated water samples. This study revealed that Sporotrichum sp. has short-term enzymatic and long-term biosorption capacity on reactive blue 13 and the decolorization potential of the alive and dead cells is impressively high. PRACTITIONER POINTS: White-rot fungus Sporotrichum sp. is able to decolorize sulfonated azo-dye reactive blue 13 upon sequential incubation in freshly prepared dye solution. The decolorization mechanism of the fungus is estimated to be bioadsorption. Sporotrichum sp. can be considered for long-term usage and immobilization applications.

Diethylstilbestrol (DES) is a synthetic form of oestrogen that does not easily degrade in the environment and can be harmful to human health. Herein, the mechanism of the interaction between laccase and DES was investigated by various spectroscopic means and high-performance liquid chromatography (HPLC). The results of fluorescence experiments showed that the quenching of intrinsic fluorescence of laccase by DES was due to a static quenching, forming a binding site. According to the Förster non-radiative energy transfer theory (FRET), the action distance R

Tea, originating from China, is an important part of Chinese traditional culture. There are different qualities of and producing areas for tea on the market, therefore it is necessary to discriminate between teas in a fast and accurate way. In this study, a chemical sensor array based on nanozymes was developed to discriminate between different metal ions and teas. The indicators for the sensor array are three kinds of nanozymes mimicking laccase (Cu-ATP, Cu-ADP, Cu-AMP). The as-developed sensor array successfully discriminated 12 metal ions and the detection limit was as low as 0.01 μM. The as-developed sensor array was also able to discriminate tea samples. Different kinds of tea samples appeared in different areas in the canonical score plot with different response patterns. Furthermore, in a blind experiment, we successfully discriminated 12 samples with a 100% accuracy. This sensor array integrates chemistry and food science together, realizing the simultaneous detection of several kinds of teas using a sensitive method. The as-developed sensor array would have an application in the tea market and provide a fast and easy method to discriminate between teas.

Poly(2‐oxazoline)s (POxs) with 2,2′‐iminodiacetate (IDA) end groups were investigated as inhibitors for laccase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC50 values were found to be in a range of 1–3 mm. Compared with IDA alone, the activity was increased by a factor of more than 30; thus indicating that attaching a polymer chain to an inhibitor can already improve the activity of the former. The enzyme activity drops to practically zero upon increasing the concentration of the most active telechelic inhibitor, IDA‐PEtOx30‐IDA (PEtOx: poly(2‐ethyl‐2‐oxazoline)), from 5 to 8 mm. This unusual behavior was investigated by means of dynamic light scattering, which showed specific aggregation above 5 mm. Furthermore, the laccase could be stabilized in the presence of POx‐IDA, upon addition at a concentration of 20 mm and higher. Whereas laccase becomes completely inactive at room temperature after one week, the stabilized laccase is fully active for at least a month in aqueous solution., Bulking up: Poly(2‐oxazoline)s (POx) with iminodiacetate (IDA) end groups competitively inhibit the enzyme laccase. A telechelic IDA‐POx‐IDA derivative shifts the inhibition mechanism at higher concentrations to become a dead‐end inhibitor. Simultaneously, the polymers stabilize the enzyme to preserve its activity, even for 28 days at room temperature.

The surface display laccase (SDL) biocatalyst, where the enzyme laccase is displayed on the surface of biological cells through synthetic biology, provides a new opportunity to develop sustainable technologies for removal of emerging contaminants from wastewater. This study vigorously characterized biocatalytic properties of the SDL in comparison to free laccase in removing emerging contaminant acetaminophen (APAP), with the aim to understand the effect of surface display on enzyme functionality and identify the strategy to overcome the potential limitation. The SDL could effectively remove APAP. Adding redox mediators substantially improved the removal efficiency. The Michaelis-Menten kinetic analysis showed that the redox mediator 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonate could overcome the limitation of APAP accessing the active site of laccase in the SDL biocatalyst. The APAP removal rate catalyzed by the SDL in real secondary wastewater effluent was higher than that in acetate buffer; comprehensive enzyme kinetic analysis provided clear evidence that there were redox mediating compounds in the wastewater. Analysis of transformation products revealed that surface display did not change laccase functionality in terms of APAP transformation mechanism. In addition, the SDL retained 88% of the initial activity after six repeated APAP biotransformation reactions. Results from this study provide a scientific basis for developing and implementing SDL as an innovative biocatalytic material for contaminant treatment applications.

Upon injury, psychotropic psilocybin‐producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two‐step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4‐O‐phosphate group to yield psilocin, while PsiL oxidizes its 4‐hydroxy group. The PsiL reaction was monitored by in situ 13C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C‐5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3‐ to 13‐mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function., Magic mushroom blues: Hallucinogenic Psilocybe mushrooms turn blue when injured. We report that the phosphatase PsiP and the laccase PsiL degrade psilocybin and initiate blueing. MALDI‐MS and in situ NMR spectroscopy show that the blue color is due to a mixture of quinoid psilocyl oligomers, primarily coupled via carbon 5.

The use of laccases applied in bioremediation processes has been increasingly studied, given the urgent need to overcome the environmental problems caused by emerging contaminants. It is known that immobilized enzymes have better operational stability under reaction conditions, allowing for greater applicability. However, given the lack of commercially available immobilized laccases, the search for immobilization materials and methods continues to gain effort. The use of polyacrylonitrile (PAN) to immobilize enzymes has been investigated since it is a low-cost material and can be modified and functionalized to well interact with the enzyme. This polymer can be used with different morphologies such as fibers, beads, and core-shell, presenting as an easily applicable alternative. This review presents the missing link between polymer and enzyme through an overview of PAN's current use as support for laccase immobilization and polymer functionalization methods, considering the importance of immobilized laccases in several industrial sectors.

Tailoring the hydrophilicity of solid surfaces with a strong affinity to water has been extensively explored in the last 20 years, but studies have been limited to the single function of wettability. Here, the multifunctional properties of tailored surface films are extended from exhibiting superwettability to facilitating biological activities. It is shown that amyloid fibrils can be universally coated onto various substrates, such as fabrics (non-woven organic masks), metal meshes, polyethersulfone (PES), glass, and more, endowing the resulting surfaces with excellent performance in oil/water mixture and emulsion separation, antifouling, and antifogging. Moreover, the biocompatible crosslinked amyloid fibril coatings can serve as a platform for biocatalytic activities by immobilizing enzymes, as shown in the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) oxidation and Reactive Black 5 (RB5) degradation by laccase from Trametes versicolor. The study provides a universal approach to modifying surface morphology and chemical properties via fibrous protein templates, opening the way to unexplored bio-based applications and functionalities., Advanced Materials, 34 (51), ISSN:0935-9648, ISSN:1521-4095

Novel liquid supports for enzyme immobilization and reuse based on aqueous biphasic systems (ABS) constituted by cholinium-based ionic liquids (ILs) and polymers for the degradation of dyes are here proposed. The biocatalytic reaction for dye decolorization using laccase occured in the biphasic medium, with the enzyme being "supported" in the IL-rich phase and the dye and degradation products being enriched in the polymer-rich phase. An initial screening of the laccase activity in aqueous solutions of ABS constituents, namely cholinium dihydrogen citrate ([Ch][DHC]), cholinium dihydrogen phosphate ([Ch][DHP]), cholinium acetate ([Ch][Acet]), polypropylene glycol 400 (PPG 400), polyethylene glycol 400 (PEG 400) and K2 HPO4 was carried out. Compared to the buffered control, a relative laccase activity of up to 170%, 257%, and 530% was observed with PEG 400, [Ch][DHP], and [Ch][DHC], respectively. These ABS constituents were then investigated for the in situ enzymatic biodegradation of the Remazol Brilliant Blue R (RBBR) dye. At the optimized conditions, the ABS constituted by PPG 400 at 46 wt% and [Ch][DHC] at 16 wt% leads to the complete degradation of the RBBR dye, further maintaining the enzyme activity. This ABS also allows an easy immobilization, recovery, and reuse of the biocatalyst for six consecutive reaction cycles, achieving a degradation yield of the dye of 96% in the last cycle. In summary, if properly designed, high enzymatic activities and reaction yields are obtained with ABS as liquid supports, while simultaneously overcoming the safety and environmental concerns of conventional organic solvents used in liquid-liquid heterogeneous reactions, thus representing more sustainable biocatalytic processes.