Search Results (151)

Water quality is essential for safeguarding human health and ensuring the stability of ecosystems. Nonetheless, the rising prevalence of emerging contaminants, particularly pharmaceutical compounds, has raised serious environmental concerns due to their bioactivity, widespread use, persistence, and potential toxicity. Among these, acetaminophen (paracetamol) is one of the most frequently detected pharmaceutical pollutants in aquatic environments. Among the various degradation strategies explored, biological methods, especially those involving white-rot fungi, have shown substantial promise owing to their production of ligninolytic enzymes capable of degrading complex pollutants. This study investigates the use of laccases from Ganoderma parvulum, covalently immobilized on chitosan microspheres, for acetaminophen degradation. The immobilization involved a 10% crosslinking agent, 60-min crosslinking time, and 10,000 U/L enzyme concentration, resulting in an immobilization efficiency of 123%, 203%, and 218%, respectively. The immobilized enzymes displayed enhanced stability across pH 3–8 and temperatures between 20 and 60 °C. Biodegradation assays achieved 97% acetaminophen removal within four hours. Nuclear Magnetic Resonance (¹H NMR and COSY) confirmed structural transformation. The enzymes also retained over 95% catalytic activity after multiple reuse cycles. These findings highlight the novel application of laccases as efficient and reusable biocatalysts for pharmaceutical pollutant removal, providing valuable insights into the mechanisms of enzymatic environmental remediation. Full article

In the present investigation, an eco-friendly biocatalyst was developed using Pleurotus eryngii laccase (PeLac) through a copper (Cu)-based protein–inorganic hybrid system for the degradation of bisphenol A, a representative xenobiotic. After partial purification, the specific activity of crude PeLac was 92.6 U/mg of total protein. Immobilization of PeLac as Cu₃(PO₄)₂–Lac (Cu–PeLac) nanoflowers (NFs) at 4 °C resulted in a relative activity 333% higher than that of the free enzyme. The Cu–PeLac NFs exhibited greater pH and temperature stability and enhanced catalytic activity compared to free laccase. This enhanced activity was validated through improved electrochemical properties. After immobilization, Cu–PeLac NFs retained up to 8.7-fold higher residual activity after storage at 4 °C for 30 days. Free and immobilized laccase degraded bisphenol A by 41.6% and 99.8%, respectively, after 2 h of incubation at 30 °C. After ten cycles, Cu–PeLac NFs retained 91.2% degradation efficiency. In the presence of potent laccase inhibitors, Cu–PeLac NFs exhibited a 47.3-fold improvement in bisphenol A degradation compared to free PeLac. Additionally, the synthesized Cu–PeLac NFs demonstrated lower acute toxicity against Vibrio fischeri than Cu nanoparticles. This study presents the first report of PeLac immobilization through an eco-friendly protein–inorganic hybrid system, with promising potential for degrading bisphenol A in the presence of inhibitors to support sustainable development. Full article

Estradiol is a natural estrogen belonging to the group of natural steroid hormones. This paper presents new electrochemical biosensors—simple and low-cost tools for the determination of β-estradiol. The receptor layer of the sensor is the enzyme laccase, which was immobilized on the substrate surface using the soft plasma polymerization technique. This technique is innovative and environmentally friendly as it allows for the effective deposition of the enzyme onto unmodified and modified electrode substrates. Three types of substrates were used: an unmodified glassy carbon electrode and two electrodes modified with composite layers—multi-walled carbon nanotubes combined with CuO nanoparticles and multi-walled carbon nanotubes combined with carbon nanofibers, respectively. Biosensors modified with such materials have not been described previously. In the course of the study, electrochemical measurement conditions (composition, concentration and pH of the base electrolyte, sensor response time, and interference effects) were optimized, and sensor parameters were determined. It was found that the modification of the substrate electrode increased the sensitivity of the sensor by more than 25 times in both cases and led to a lower detection limit for the sensor modified with the carbon nanotubes/carbon nanofiber composite. The best performance was achieved with the sensor containing the carbon nanotube/carbon nanofiber composite layer, which showed a linearity range of 0.1–5 µM, a sensitivity of 7.32 ± 0.22 µA/µM, and a limit of quantification of 0.078 µM. The analytical utility of this biosensor was confirmed by its successful application in the determination of estradiol in pharmaceutical preparations and river water samples. Full article

The immobilization of a laccase from Pycnoporus sanguineus UEM-20 via the formation of cross-linked enzyme aggregates (CLEAs) was optimized through a central composite design (CCD) of response surface methodology (RSM). Both free and immobilized enzymes were investigated for their physico-chemical characteristics, and their adequacy in removing bisphenol A (BPA) and decolorizing malachite green dye in solution was evaluated. The immobilization caused only minor differences in thermostability. Upon immobilization, the enzyme experienced some changes in its kinetic properties. The V_max decreased by a factor of 1.1, and the K_M increased by a factor of 1.89. These kinetic changes did not modify in any remarkable way the capacity of the immobilized enzyme in degrading BPA and decolorizing malachite green dye. Its sensitivity to NaCl was also minimally affected by immobilization. However, its sensitivity to sodium sulfate was substantially decreased. After 1 month’s conservation, the activity of the free form had suffered a drastic drop. The immobilized form, by contrast, remained 100% active after 6 months. All these findings predict that the immobilized laccase from P. sanguineus UEM-20 may be useful in the enzymatic bioremediation of pollutants such as endocrine disruptors and synthetic dyes. Full article

Amperometric biosensors (ABSs) and enzymatic biofuel cells (BFCs) share several fundamental principles in their functionality, despite serving different primary purposes. Both devices rely on biorecognition, redox reactions, electron transfer (ET), and advanced electrode materials, including innovative nanomaterials (NMs). ABSs and BFCs, utilizing microbial oxidoreductases in combination with electroactive NMs, are both efficient and cost-effective. In the current study, several laboratory prototypes of BFCs have been developed with bioanodes based on yeast flavocytochrome b₂ (Fcb₂) and alcohol oxidase (AO), and a cathode based on fungal laccase. For the first time, BFCs have been developed featuring anodes based on Fcb₂ co-immobilized with redox NMs on a glassy carbon electrode (GCE), and cathode-utilizing laccase combined with gold–cerium–platinum nanoparticles (nAuCePt). The most effective lactate BFC, which contains gold–hexacyanoferrate (AuHCF), exhibited a specific power density of 1.8 µW/cm². A series of BFCs were developed with an AO-containing anode and a laccase/nAuCePt/GCE cathode. The optimal configuration featured a bioanode architecture of AO/nCoPtCu/GCE, achieving a specific power density of 3.2 µW/cm². The constructed BFCs were tested using lactate-containing food product samples as fuels. Full article

Polyethylene terephthalate (PET) is a polyester used in the beverage bottling industry that generates a serious pollution problem. Films were obtained to reuse discarded PET bottles, and their surface was modified to determine their effectiveness in supporting the immobilization of the laccase enzyme applied to a catechol biodegradation assay. Radiofrequency (RF) plasma was used to modify the PET surface using different precursors: (a) with the use of air as precursor, the effect of the etching caused by the air on the greater or lesser immobilization was observed; (b) with the use of ethylenediamine, a mixture of N₂/H_2, or aniline as precursors, it was observed which of these three precursors presented the greater or lesser number of amino groups deposited on the PET surface. After plasma modification, the films were cross-linked with glutaraldehyde to immobilize the laccase enzyme. Finally, the catechol test was performed. It was found that the best etching time using air as a precursor was 90 min, and the precursor that caused a higher insertion of amino groups on the surface was ethylenediamine, which reached a density of amino groups of 3.98 ± 0.10 g·mm⁻². The highest percentage of laccase immobilization achieved on the surface of ethylenediamine-modified PET was 97.30%. In the catechol assay, the highest retention was 86.11%. This research reveals how the effect of plasma increases the surface area on a PET surface and, in conjunction with ethylenediamine as the best precursor of the three precursors evaluated, can immobilize a greater amount of enzyme and oxidize more catechol. There is no scientific evidence from previous studies that used air plasma technology to erode and then used three different precursors to modify a surface to immobilize the laccase enzyme and remove a water contaminant. Full article

The extensive use of pharmaceuticals in human and veterinary medicine has led to their persistent environmental release, posing ecological and public health risks. Major sources include manufacturing effluents, excretion, aquaculture, and improper disposal, contributing to bioaccumulation and ecotoxicity. Mycoremediation is the fungal-mediated biodegradation of pharmaceuticals, offers a promising and sustainable approach to mitigate pharmaceutical pollution. Studies have reported that certain fungal species, including Trametes versicolor and Pleurotus ostreatus, can degrade up to 90% of pharmaceutical contaminants, such as diclofenac, carbamazepine, and ibuprofen, within days to weeks, depending on environmental conditions. Fungi produce a range of extracellular enzymes, such as laccases and peroxidases, alongside intracellular enzymes like cytochrome P450 monooxygenases, which catalyze the transformation of complex pharmaceutical compounds. These enzymes play an essential role in modifying, detoxifying, and mineralizing xenobiotics, thereby reducing their environmental persistence and toxicity. The effectiveness of fungal biotransformation is influenced by factors such as substrate specificity, enzyme stability, and environmental conditions. Optimal degradation typically occurs at pH 4.5–6.0 and temperatures of 20–30 °C. Recent advancements in enzyme engineering, immobilization techniques, and bioreactor design have improved catalytic efficiency and process feasibility. However, scaling up fungal-based remediation systems for large-scale applications remains a challenge. Addressing these limitations with synthetic biology, metabolic engineering, and other biotechnological innovations could further enhance the enzymatic degradation of pharmaceuticals. This review highlights the enzymatic innovations, applications, and challenges of pharmaceutical mycoremediation, emphasizing the potential of fungi as a transformative solution for sustainable pharmaceutical waste management. Full article

In an enzyme-based fuel cell system, glucose oxidase and laccase were immobilized on carbon paper as the anode and cathode electrodes. A conductive polymer (polypyrrole) was added to improve conductivity. The mediator and enzymes were mixed in a phosphate-buffer solution for entrapment. A Nafion 212 membrane separated the two half-cells. Power density measurements were taken at a glucose concentration of 10 mM across different operating voltages. Potassium hexacyanoferrate III was used as a redox mediator in the anode and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) in the cathode to boost power output. The biofuel cells, constructed from acrylic (40 × 50 × 50 mm) with a working volume of 20 × 30 × 40 mm, were assembled using a rubber gasket to secure the Nafion membrane. The use of micropore tape covering the electrodes extended the system’s operational lifespan. Without the micropore tape, the maximum power density was 57.6 μW/cm² at 0.24 V. With the micropore tape, the cell achieved a maximum power density of 324.9 μW/cm² at 0.57 V, sustaining performance for 20 days. Thus, micropore tape effectively enhances enzyme retention and biofuel cell performance. Full article

Glycine is an essential amino acid involved in synthesizing a variety of important biomolecules, and its concentration can influence numerous biochemical processes, including the severity of symptoms in a wide range of conditions in humans, such as cancer, schizophrenia, major depression, and diabetes. While a few costly or labour-intensive methods are currently available, we have developed a new enzymatic biosensor that can accurately measure glycine levels with remarkable simplicity. By employing immobilized laccase enzymes in combination with a surface plasmon resonance (SPR) device, our system achieved a limit of detection (LOD) of 9.95 mM and a limit of quantification (LOQ) of 33.19 mM. In addition, it demonstrated a recovery rate of 97.64 ± 7.71%. Moreover, the biosensor maintained consistent signal intensity over 21 days and supported a total of 60 analyses using the same immobilized enzyme setup, demonstrating excellent reusability. Notably, this study marks the first time glycine has been determined using an enzymatic SPR-based platform. Full article

In this study, Cu²⁺-chelated magnetic silicon dioxide nanoparticles were synthesized as carriers for laccase immobilization. The prepared magnetic immobilized laccase was applied in the clarification of sugarcane juice. The optimal conditions for the clarification of sugarcane juice with magnetic immobilized laccase in a shake flask were determined to be as follows: a temperature of 35 °C, pH of 5.5, rotation speed of 150 r/min, and immobilized laccase dosage of 1.0 mg/mL. The sucrose in the sugarcane juice inhibited both free and immobilized laccase. The inhibitory effect was characterized as mixed inhibition, wherein competitive inhibition played a dominant role. An alternating magnetic field was introduced into the catalysis process using magnetic immobilized laccase, and the catechin degradation rate was improved to 77.2% under a magnetic field intensity of 80 Gs and magnetic field frequency of 400 Hz. Under the optimal alternating magnetic field conditions, the treatment time of sugarcane juice was reduced to 20 min when catalyzed by the magnetic immobilized laccase, wherein a decolorization rate of 54.4%, reduction in turbidity of 89.7%, and total phenol degradation rate of 43.4% were achieved. Compared with the shaking condition, the assistance of alternating magnetic fields can shorten the clarifying time, increase the clarifying effect, and enhance the catalyst reusability. These results reveal useful information about the enzymatic treatment of high-sugar juice and provide a potential strategy for juice clarification with magnetic immobilized enzymes. Full article

Laccase from Trametes versicolor was immobilized via the formation of interlinking enzyme aggregates (CLEA). Its free and immobilized enzymes were characterized, and its efficiency was tested via the removal of bisphenol A (BPA) in aqueous solution. The resistances against thermal denaturation and pH variations were improved upon immobilization. Although the optimal pH of the enzyme was not modified by immobilization, the latter considerably increased its stability in the pH range of 2.0 to 8.0. The immobilized form was still 50% active after 6 months of storage, while the free form took 1 month to suffer a similar drop in activity. Both free and immobilized T. versicolor laccases were efficient in removing 200 µM BPA from aqueous solutions. The free laccase removed 79% and 92.9% of the compound during the first hour of reaction when 0.1 and 0.2 U were used, respectively. The immobilized form, on the other hand, removed 72% and 94.1% of 200 µM BPA during the first hour of reaction when 0.2 and 0.5 U were used, respectively. The immobilized enzyme allowed seven reuse cycles in the oxidation of ABTS and up to four cycles in the degradation of BPA. The results suggest that the laccase from T. versicolor may be useful in biological strategies aiming at degrading endocrine disruptors, such as BPA. Full article

Emerging pollutants such as organophosphate flame retardants (OPFRs) pose a critical threat to environmental and human health, while conventional wastewater treatments often fail to remove them. This study addresses this issue by evaluating the bioremediation potential of white-rot fungi for the removal of two OPFRs: tris(2-chloroethyl) phosphate (TCEP) and tributyl phosphate (TBP). Three fungal species—Ganoderma lucidum, Trametes versicolor, and Phanerochaete velutina—were screened for their degradation capabilities. Among these, G. lucidum and T. versicolor demonstrated removal efficiencies exceeding 99% for TBP, while removal rates for TCEP were significantly lower, with a maximum of 30%. The exploration of the enzyme role showed that cytochrome P450 is involved in the degradation while the extracellular laccase is not involved. Continuous batch experiments were performed using a trickle-bed reactor (TBR) operating under non-sterile conditions, a setting that closely resembles real-world wastewater treatment environments. G. lucidum was immobilized on oak wood chips, and the removal efficiencies were measured to be 85.3% and 54.8% for TBP and TCEP, respectively, over 10 cycles. Microbial community analysis showed that G. lucidum remained the dominant species in the reactor. These findings demonstrate the efficacy of fungal-based trickle-bed bioreactors, offering a sustainable and efficient alternative for addressing environmental pollution caused by highly recalcitrant pollutants. Full article

Lipase from Pseudomonas fluorescens (PFL) and laccase from Trametes versicolor were co-immobilized onto nanocellulose (NC), using a layer-by-layer approach. Initially, PFL was adsorbed onto NC through ionic and hydrophobic interactions. To achieve higher PFL immobilization yield and activity, NC was functionalized with aldehyde groups through periodate oxidation (NCox) or glutaraldehyde activation (NC-GA). FTIR analysis confirmed these chemical modifications. Among the functionalized NCs, NCox showed the best capacity to retain higher amounts of PFL (maximum load: 20 mg/g), and this support was selected to proceed with the co-immobilization experiments. In this process, NCox-250-PFL (NCox activated with 250 µmol/g of aldehyde groups) was covered with polyethyleneimine (PEI), laccase was co-immobilized, and a crosslinking step using glutaraldehyde was used to covalently attach the enzymes to the support, producing the biocatalyst NCox-250-PFL-PEI-Lac-GA. Co-immobilized enzymes presented higher thermal stability (50 °C) than soluble enzymes; co-immobilized laccase retained 61.1% of its activity after 24 h, and PFL retained about 90% after 48 h of deactivation at 50 °C. In operational stability assays, the heterogeneous biocatalysts maintained more than 45% of their activity after five cycles of pNPB hydrolysis and ABTS oxidation. This co-immobilized biocatalyst, with its high stability and activity retention, is a promising multi-active heterogeneous biocatalyst for use in cascade reactions of industrial interest. Full article

The direct discharge of cationic surfactants into environmental matrices has exponentially increased due to their wide application in many products. These compounds and their degraded products disrupt microbial dynamics, hinder plant survival, and affect human health. Therefore, there is an urgent need to develop electroanalytical assessment techniques for their identification, determination, and monitoring. In our study, ZnO-PANI nanocomposites were electrodeposited on a glassy carbon electrode (GCE), followed by the immobilization of laccase enzymes and the electrodeposition of polypyrrole (PPy), to form a biosensor that was used for the detection of CTAB. A UV-Vis analysis showed bands corresponding to the π-π* transition of benzenoid and quinoid rings, π-polaron band transition and n-π*polaronic transitions associated with the extended coil chain conformation of PANI, and the presence and interaction of ZnO with PANI and type 3 copper in the laccase enzymes. The FTIR analysis exhibited peaks corresponding to N-H and C-N stretches and bends for amine, C=C stretches for conjugated alkenes, and a C-H bend for aromatic compounds. A high-resolution scanning electron microscopy (HRSEM) analysis proved that PANI and ZnO-PANI were deposited as fibres with hairy topography resulting from covalent bonding with the laccase enzymes. The modified electrode (PPy-6/GCE) was used as a platform for the detection of CTAB with three linear ranges of 0.5–100 µM, 200–500 µM, and 700–1900 µM. The sensor displayed a high sensitivity of 0.935 μA μM⁻¹ cm⁻², a detection limit of 0.0116 µM, and acceptable recoveries of 95.02% and 87.84% for tap water and wastewater, respectively. Full article

Methylene blue is a cationic organic dye commonly found in wastewater, groundwater, and surface water due to industrial discharge into the environment. This emerging pollutant is notably persistent and can pose risks to both human health and the environment. In this study, we developed a Surface Plasmon Resonance Biosensor employing a BK7 prism coated with 3 nm chromium and 50 nm of gold in the Kretschmann configuration, specifically for the detection of methylene blue. For the first time, laccases immobilized on a gold surface were utilized as bio-receptors for this organic dye. The enzyme was immobilized using carbodiimide bonds with EDC/NHS crosslinkers, allowing for the analysis of samples with minimal preparation. The method demonstrated validation with a limit of detection (LOD) of 4.61 mg L⁻¹ and a limit of quantification (LOQ) of 15.37 mg L⁻¹, a working range of 0–100 mg L⁻¹, and an R² value of 0.9614 during real-time analysis. A rainwater sample spiked with methylene blue yielded a recovery rate of 122.46 ± 4.41%. The biosensor maintained a stable signal over 17 cycles and remained effective for 30 days at room temperature. Full article