Salivary biomarkers: novel noninvasive tools to diagnose chronic inflammation.

Several chronic disorders including type 2 diabetes (T2D), obesity, heart disease and cancer are preceded by a state of chronic low-grade inflammation. Biomarkers for the early assessment of chronic disorders encompass acute phase proteins (APP), cytokines and chemokines, pro-inflammatory enzymes, lipids and oxidative stress mediators. These substances enter saliva through the blood flow and, in some cases, there is a close relation between their salivary and serum concentration. Saliva can be easily collected and stored with non-invasive and cost-saving procedures, and it is emerging the concept to use it for the detection of inflammatory biomarkers. To this purpose, the present review aims to discuss the advantages and challenges of using standard and cutting-edge techniques to discover salivary biomarkers which may be used in diagnosis/therapy of several chronic diseases with inflammatory consequences with the pursuit to possibly replace conventional paths with detectable soluble mediators in saliva. Specifically, the review describes the procedures used for saliva collection, the standard approaches for the measurement of salivary biomarkers and the novel methodological strategies such as biosensors to improve the quality of care for chronically affected patients.

Quality-by-Design R&D of a Novel Nanozyme-Based Sensor for Saliva Antioxidant Capacity Evaluation.

Oxidative stress is one of the main causes of cell damage, leading to the onset of several diseases, and antioxidants represent a barrier against the production of reactive species. Saliva is receiving increasing interest as a promising biofluid to study the onset of diseases and assess the overall health status of an individual. The antioxidant capacity of saliva can be a useful indicator of the health status of the oral cavity, and it is nowadays evaluated mainly through spectroscopic methods that rely on benchtop machines and liquid reagents. We developed a low-cost screen-printed sensor based on cerium oxide nanoparticles that can be used to assess the antioxidant capacity of biofluids as an alternative to traditional methods. The sensor development process was investigated via a quality-by-design approach to identify the most critical parameters of the process for further optimization. The sensor was tested in the detection of ascorbic acid, which is used as an equivalent in the assessment of overall antioxidant capacity. The LoDs ranged from 0.1147 to 0.3528 mM, while the recoveries varied from 80% to 121.1%, being therefore comparable with those of the golden standard SAT test, whose recovery value was 96.3%. Therefore, the sensor achieved a satisfactory sensitivity and linearity in the range of clinical interest for saliva and was validated against the state-of-the-art equipment for antioxidant capacity evaluation.

The Potential of Vouacapanes from Pterodon emarginatus Vogel Against COVID-19 Cytokine Storm.

Purpose: The emergence of the COVID-19 pandemic has led to the search for potential therapeutic responses for various aspects of this disease. Fruits of Pterodon emarginatus Vogel (Fabaceae), sucupira, have been used in Brazilian traditional medicine because of their anti-inflammatory properties, which have been proven in vivo, in vitro, and in silico. Therefore, the aim of this work is to evaluate P. emarginatus oleoresin and isolated diterpenes by in vitro anti-inflammatory models. Methods: In this study, the mechanisms underlying the anti-inflammatory activity of P. emarginatus oleoresin and vouacapanes 6α,19ß-diacetoxy-7ß,14ß-dihydroxyvouacapan (V1), 6α-acetoxy-7ß,14ß-dihydroxyvouacapan (V2), and methyl 6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate (V3) were investigated in HaCaT cells. Results: Oleoresin, V2, and V3 inhibited phospholipase A2 (30.78%, 24.96%, and 77.64%, respectively). Both vouacapanes also inhibited the expression of COX-2 (28.3% and 33.17%, respectively). The production of interleukin 6 (IL-6) was inhibited by oleoresin by 35.47%. However, oleoresin did not interfere with Nrf-2 expression or IL-8 production. Conclusion: The results support the ethnomedicinal use of P. emarginatus oleoresin as an anti-inflammatory herbal medicine, and also highlight P. emarginatus oleoresin and isolated vouacapanes as an attractive therapeutic approach for COVID-19 through the reduction or chronological control of the inflammatory mediators IL-6, cyclooxygenase-2 (COX-2), phospholipase A2, and INF-y (indirectly) during the SARS-CoV-2 infection process.

LQFM212, a piperazine derivative, exhibits potential antioxidant effect as well as ameliorates LPS-induced behavioral, inflammatory and oxidative changes.

AIMS: Oxidative stress, impaired antioxidant defense and neuroinflammation are often associated with the onset and progression of neuropsychiatric diseases. Conversely, several piperazine compounds presents beneficial neuropharmacological effects as well as antioxidant activity, and some derivatives combine both activities. LQFM212 (2,6-di-tert-butyl-4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)phenol) was synthesized to produce effects on CNS and to have an additional antioxidant effect. Previous preclinical tests have been shown anxiolytic- and antidepressant-like effects of LQFM212 in mice. Herein, the main objective was to verify the possible antioxidant potential and the effects of LQFM212 against behavioral changes, inflammatory and oxidative markers induced by lipopolysaccharide (LPS). MAIN METHODS: Initially, antioxidant potential of LQFM212 was evaluated by electrochemical assays. Afterwards, the effects of oral treatment with LQFM212 were evaluated in mice using LPS-induced models of systemic or local inflammation. KEY FINDINGS: In LPS-induced neuroinflammation, LQFM212 treatment reverted changes caused by LPS, demonstrated by attenuated anxiogenic- and depressive-like behaviors, reduced pro-inflammatory cytokines (TNF-α and IL-1ß) and increased anti-inflammatory cytokines (IL-4 and IL-10) on serum, and also improved oxidative stress-related changes (levels of nitrite, malondialdehyde, glutathione and carbonylated protein, and superoxide dismutase, catalase, myeloperoxidase and cholinesterase activities) on brain cortex and hippocampus. However, LQFM212 treatment did not attenuate the inflammatory changes in LPS-induced pleurisy model. SIGNIFICANCE: LQFM212 presents antioxidant activity and ameliorates behavioral, inflammatory and oxidative changes after LPS-induced neuroinflammation model. These effects do not seem to be secondary to a peripheral anti-inflammatory action of LQFM212, since this compound failed to attenuate the inflammatory changes in LPS-induced pleurisy model.

Comparative homology of Pleurotus ostreatus laccase enzyme: Swiss model or Modeller?

Laccases stand out in the industrial context due to their versatile biotechnological applications. Although these enzymes are frequently investigated, currently, Pleurotus ostreatus laccase structural model is unknown. Therefore, this research aims to predict and validate a P. ostreatus laccase theoretical model by means of comparative homology. The laccase target's primary structure (AOM73725.1) was obtained from the NCBI database, the model was predicted from homologous structures obtained from the PDB (PDB-ID: 5A7E, 2HRG, 4JHU, 1GYC) using the Swiss-Model and Modeller, and was refined in GalaxyRefine. The models were validated using PROCHECK, VERIFY 3D, ERRAT, PROVE and QMEAN Z-score servers. Moreover, molecular docking between the laccase model (Lacc4MN) and ABTS was performed on AutoDock Vina. The models that were generated by the Modeller showed superior stereochemical and structural characteristics to those predicted by the Swiss Model. The refinement made it difficult to stabilize the copper atoms which are typical of laccases. The Lacc4MN model showed the interactions between the amino acids in the active site of the laccase and the copper atoms, thereby hinting the stabilization of the metal through electrostatic interactions with histidine and cysteine. The molecular docking between Lacc4MN and ABTS showed negative free energy and the formation of two hydrogen bonds involving the amino acids ASP 208 and GLY 268, and a Pi-sulfur bond between residue HIS 458 and ABTS, which demonstrates the typical catalytic functionality of laccases. Furthermore, the theoretical model Lacc4MN presented stereochemical and structural characteristics that allow its use in silico tests.Communicated by Ramaswamy H. Sarma.

Poloxamer-Based Hydrogel as Drug Delivery System: How Polymeric Excipients Influence the Chemical-Physical Properties.

Thermogelling amphiphilic block copolymers have been widely investigated in the development of pharmaceutical drug carriers. In particular, thermosensitive gels based on poloxamer 407 (P407) have great potential for periodontal disease treatment, thanks to their ability to be liquid at room temperature and become viscous gels at body temperature. However, some problems, related to short in situ residence time, reduce their feasible clinical use. Thus, in order to improve the effective applicability of these materials, we studied how P407 thermogels are affected by the pH and by the inclusion of different hydrophilic polymers, used as excipients for increasing the gel stiffness. For this scope, a complete chemical-physical characterization of the synthesized gels is provided, in terms of determination of sol-gel transition temperature, viscosity and erosion degree. The data are correlated according to a statistical multivariate approach based on Principal Component Analysis and their mucoadhesion properties are also tested by Tapping mode-Atomic Force Microscopy (TM-AFM) imaging. Finally, we studied how the different P407 formulations are able to influence the release pathway of two antibacterial drugs (i.e., chlorhexidine digluconate and doxycycline hyclate) largely used in oral diseases.

An insight into polyscopoletin electrosynthesis by a quality-by-design approach.

Scopoletin (SP) as a functional monomer for electropolymerization has recently been investigated in the context of molecularly imprinted polymers for biosensing applications. Herein we describe an in-depth analysis of the mechanisms involved in the electropolymerization of SP toward the optimization of the experimental conditions for applications in sensor studies. PolySP films have been in situ synthesized on a standard glassy carbon electrodes by varying three independent experimental parameters, and the output of the analysis has been evaluated in terms of the resulting electroactive area and surface coverage. A quality-by-design approach including design-of-experiments principles and response surface methodology produced unbiased observations on the most relevant parameters to be controlled during the electropolymerization of SP. By combining the output of electroactive area and surface overage, we highlighted a strong dependence on the monomer concentration and scan rate. Thus, an appropriate selection of these two parameters should be sought to have an optimal electropolymerization process, leading to uniform films and homogeneous surface behavior. This study shows that the application of multi-factorial analysis in a comprehensive design of experiments allows the systematic study of polymer electrosynthesis. Therefore, this research is expected to guide further efforts in the electropolymerization of several functional monomers. Supplementary Information: The online version contains supplementary material available at 10.1007/s10853-022-07349-8.

Redox Profiling of Selected Apulian Red Wines in a Single Minute.

Wine is a complex bioproduct whose chemical composition is highly variable across production regions. In order to shed light on affordable ways to promote the characterization of wines and explore the physicochemical basis of their antioxidant capacity, this work reported on the quick and easy redox profiling of selected red wines from Apulia, Italy. Therefore, an affordable and quickly performed semiempirical quantum chemistry approach, i.e., the extended Hückel method, was used to compute the bandgaps of the main phytochemical markers attributed to red wines. The findings of these calculations were then compared to an electroanalytical investigation in the form of cyclic and square-wave voltammetry, and the electric current of the redox profiles was used as the input dataset for principal component analysis. Results showcased that the semiempirical quantum chemistry calculations allowed the correlation of the bandgaps to the observed faradaic signals upon voltammetry; thereby, also providing insights on their antioxidant appeal by highlighting the feasibility of charge-transfer processes at low electric potentials. Furthermore, the principal component analysis showed that the electric current dataset gathered in a time span of 55 s allowed the appropriate separation of the samples, which hints at the possible use of quick voltammetric assays as fingerprinting tools.

Anxiolytic- and antidepressant-like effects of new phenylpiperazine derivative LQFM005 and its hydroxylated metabolite in mice.

The current treatments available for anxiety and depression are only palliative. Full remission has remained elusive, characterizing unmet medical needs. In the scope of an academic drug discovery program, we describe here the design, synthesis, in vitro metabolism prediction and pharmacological characterization of a new piperazine compound, 1-(4-methoxyphenyl)-4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazine (LQFM005), and of its main putative metabolite, 4-(4-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)- 1H-pyrazol-1-yl)phenol (LQFM235). The production of the metabolite was initially performed by in vitro biotransformation of LQFM005 using Aspergillus candidus and then by chemical synthesis. Oral administration of either 12 or 24 µmol/kg LQFM005 to mice did not affect spontaneous locomotor activity but increased the time spent in the center of the open field. Both LQFM005 and LQFM235 (24 µmol/kg) increased the time spent by the mice in the open arms of the elevated plus maze (EPM), a good indication of anxiolytic-like effect, and decreased the immobility time in the forced swimming test (FST), suggesting an antidepressant-like effect. The previous administration of WAY-100635 (a 5-HT1A antagonist) abolished the effects of LQFM005 in both EPM and FST. Binding experiments showed that LQFM005 and its metabolite bind to the 5-HT1A receptor with a moderate affinity (Ki around 5-9 µM). The two compounds are relatively safe, as indicated by cytotoxic assessment using the 3T3 fibroblast cell line and estimated LD50 around 600 mg/kg. In conclusion, oral administration of the newly synthesized phenylpiperazines produced anxiolytic- and antidepressant-like effects in behavioral tests, putatively in part through the activation of 5-HT1A receptors.

Application of Deconica castanella ligninolytic enzymatic system in the degradation of hexachlorobenzene in soil.

Hexachlorobenzene (HCB) is a pollutant still found in the environment despite being widely banned. Considering that basidiomycetes are useful to degrade a variety of organochlorinated pollutants, we therefore report the influence of HCB on the ligninolytic enzymatic system of Deconica castanella. The inoculum was prepared with sugarcane bagasse and soybean flour and was added in soil with and without HCB (2000 mg kg soil-1 ), 5% emulsion containing soybean oil and Tween 20 at proportion 9:1, v:v; with 70% moisture at 25°C. Fungal biomass was quantified by widely acknowledged growth biomarker ergosterol. The extraction of the enzymatic complex was performed and laccase, Mn-dependent peroxidase (MnP), and lignin peroxidase (LiP) activities were determined. Furthermore, HCB and its metabolites were quantified by gas chromatography and chlorides by potentiometric titration. Results evidenced that HCB did not interfere in fungal growth, though the only detected enzymatic activity was laccase. MnP and Lip were not detected during D. castanella growth in soil. The peak of laccase enzymatic activity occurred in the presence of HCB. In addition, the laccase exhibited thermostability. Therefore, we hereby shed light on the role of laccase in the degradation of HCB by an efficient low-cost and environmentally safe detoxification mechanism.

Development and Optimization of Solanum Lycocarpum Polyphenol Oxidase-Based Biosensor and Application towards Paracetamol Detection.

Purpose: The development biosensing technologies capable of delivering fast and reliable analysis is a growing trend in drug quality control. Considering the emerging use of plant-based polyphenol oxidases (PPO) as biological component of electrochemical biosensors, this work reports the first Solanum lycocarpum PPO biosensor and its use in the pharmaceutical analysis of paracetamol in tablet formulations. Methods: The biosensor was optimized regarding fruit maturation (immature and mature-ripe), vegetal extract volume to be used in biosensor construction as well as optimal pH of electrochemical cell fluid. Results: Results evidenced that the extract which rendered the biosensor with best analytical performance was from immature fruits, and the biosensor produced using 100 µL of crude plant extract promoted better faradaic signal gathering. Moreover, when neutral pH media was used in the electrochemical cell, the biosensor showcased best faradaic signal output from the used redox probe (catechol), suggesting thence that the method presents high sensibility for phenolic compounds detection. Furthermore, the biosensor was able to quantify paracetamol in a linear range from 50 to 300 µM, showcasing LoD and LoQ of 3 µM and 10 µM, respectively. Conclusion: after careful evaluation, this biosensor might be a low-cost alternative for conventional pharmaceutical quality control methods.

Quality by Design-driven investigation of the mechanical properties of mucoadhesive films for needleless anesthetics administration

Objectives: To systematically evaluate the effects of hydroxypropyl methyl cellulose (HPMC) type (E5LV, E15LV, and K100LV); plasticizer type (glycerol and mannitol), plasticizer loading (0.12 and 0.24% w/w); and loading of prilocaine and lidocaine hydrochlorides combined at 1:1 ratio (0 and 47 mg/cm2) in the mechanical properties of buccal films. Methods: A quality by design (QbD) approach based on a full factorial design (3 x 23) and complementarily multivariate statistical tools i.e., principal component analysis (PCA), response surface methodology (RSM), and correlation matrix were used in this pursuit. The thickness, elongation at break, tensile strength, force at break, and Young`s modulus of the anesthetic buccal films obtained by solvent casting were assessed. Results: The QbD, PCA and RSM altogether demonstrated that all studied formulation variables, mainly the drug loading, affect the mechanical properties of the films at different significance levels. The multivariate analysis yielded the modelling of elongation at break, tensile strength, and force at break, which significantly correlated with each other. The drugs exerted a synergic plasticizing effect on the films, and the use of HPMC K100 LV (with greater hydroxypropyl substitution degree and viscosity) and mannitol favored their elasticity and resistance. Furthermore, the majority of the films fulfilled the requirements for buccal administration due to their softness and mechanical resistance. Conclusion: Mannitol is suitable plasticizer for manufacturing HPMC anesthetic buccal films with improved mechanical properties. These results are a step forward in the rational development of formulations for the replacement of needles in dentistry

Physicochemical Investigation of Psoralen Binding to Double Stranded DNA through Electroanalytical and Cheminformatic Approaches.

This work showcased the first physicochemical investigation of psoralen (PSO) binding to double stranded DNA (dsDNA) through electroanalytical methods. Results evidenced that PSO presents one non-reversible anodic peak at electric potential (Epa) ≈ 1.42 V, which is associated with its oxidation and the formation of an epoxide derivative. Moreover, PSO analytical signal (i.e., faradaic current) decreases linearly with the addition of dsDNA, while the electric potential associated to PSO oxidation shifts towards more positive values, indicating thence that dsDNA addition hinders PSO oxidation. These findings were corroborated by the chemoinformatic study, which evidenced that PSO intercalated noncovalently at first between base-pairs of the DNA duplex, and then irreversibly formed adducts with both DNA strands, leading up to the formation of a cross-link which bridges the DNA helix, which explains the linear dependence between the faradaic current generated by PSO oxidation and the concentration of DNA in the test-solution, as well as the dependence between Ep and the addition of dsDNA solution. Therefore, the findings herein reported evidence of the applicability of electroanalytical approaches, such as voltammetry in the study of DNA intercalating agents.

Piroxicam voltammetric determination by ultra low cost pencil graphite electrode

Piroxicam (PRX) was determined in pharmaceutical capsules with differential pulse voltammetry (DPV) in a three electrode system consisting of a pencil graphite electrode (PGE) as working electrode, a Pt wire and a reference electrode of Ag/AgCl/KCl 3 M. An irreversible oxidation peak was observed in Epa c.a. 0.6 V, which correlates to the oxidation of PRX. The coefficient of linear correlation obtained was 0.9946, with limit of detection of 2.1 µM and limit of quantification of 4.7 µM. PGE assays showed good analytical performance compared to high performance liquid chromatography and spectrophotometry, showing the potential to be further developed and employed in quick and simple analyses.

Determination of Methyldopa and Paracetamol in Pharmaceutical Samples by a Low Cost Genipa americana L. Polyphenol Oxidase Based Biosensor.

Purpose: Jenipapo fruit (Genipa americana L) is a natural source of polyphenol oxidases (PPOs) whose potential in pharmaceutical analysis is noteworthy. Henceforth, this work reports the electrochemical study of a low-cost PPO-based biosensor produced from the crude extract of Jenipapo fruits and accounts a practical approach to employ this biosensor in the determination of methyldopa and paracetamol in pharmaceutical samples. Methods: In order to investigate the electrochemical properties of the biosensor, theoretical and practical approaches were employed, and both samples and the biosensor were analyzed through electrochemical impedance spectroscopy (EIS) and voltammetric techniques, namely: differential pulse voltammetry (DPV) and cyclic voltammetry (CV). Results: showcased that the biosensor presented good analytical features, as well as low detection limits (8 µmol L-1 for methyldopa and 5 µmol L-1 for paracetamol). The relative standard deviation was less than 5% mid-assay. Conclusion: The use of this biosensor is a reliable, low cost and useful alternative in the pharmaceutic determination of phenolic drugs (e.g. methyldopa and paracetamol).

Electrochemical Characterization of Central Action Tricyclic Drugs by Voltammetric Techniques and Density Functional Theory Calculations.

This work details the study of the redox behavior of the drugs cyclobenzaprine (CBP), amitriptyline (AMP) and nortriptyline (NOR) through voltammetric methods and computational chemistry. Results obtained in this study show that the amine moiety of each compound is more likely to undergo oxidation at 1a at Ep1a ≈ 0.69, 0.79, 0.93 V (vs. Ag/AgCl/KClsat) for CBP, AMP and NOR, respectively. Moreover, CBP presented a second peak, 2a at Ep2a ≈ 0.98 V (vs. Ag/AgCl/KClsat) at pH 7.0. Furthermore, the electronic structure calculation results corroborate the electrochemical assays regarding the HOMO energies of the lowest energy conformers of each molecule. The mechanism for each anodic process is proposed according to electroanalytical and computational chemistry findings, which show evidence that the methods herein employed may be a valuable alternative to study the redox behavior of structurally similar drugs.

Characterization of a thermostable Deconica castanella Laccase and application toward pentachlorophenol degradation.

Pentachlorophenol (PCP) is an organochlorine pesticide whose toxicity led it to be banned in several countries. In Brazil however, this compound is widely accessible, and its indiscriminate use leads to extensive soil contamination, which requires henceforth, the development of new approaches to manage PCP presence in environment. Considering that PCP is susceptible to undergo enzymatic degradation, this investigation is thence, aimed at the purification, and characterization, of a thermostable fungal enzyme (i.e. Laccase of Deconica castanella (Dc-Lac), and assess its role in PCP in vitro biodegradation. Results evidenced that, molecular mass of the partially purified Dc-Lac was estimated to be of 64 kDa, presenting apparent Km of 0.47 µmol, and Vmax of 11.56 U mg-1. The optimum temperature and pH were 55 °C and 2.5, respectively. The T½ verified at 55, 60, and 80 °C were 19 and 17 hr, and 47 min, respectively. The highest PCP biodegradation was of 23% at pollutant concentration of 100 µg L-1, which evidenced that Dc-Lac is a thermostable enzyme that acted directly in PCP degradation and may be a useful asset to remediate this pollutant.

Voltammetric Evaluation of Diclofenac Tablets Samples through Carbon Black-Based Electrodes.

Diclofenac (DIC) is a non-steroidal anti-inflammatory drug of wide use around the world. Electroanalytical methods display a high analytical potential for application in pharmaceutical samples but the drawbacks concerning electrode fouling and reproducibility are of major concern. Henceforth, the aim of this work was to propose the use of alternative low-cost carbon black (CB) and ionic liquid (IL) matrix to modify the surface of pencil graphite electrodes (PGE) in order to quantify DIC in raw materials, intermediates, and final products, as well as in stability assays of tablets. The proposed method using CB+IL/PGE displayed good recovery (99.4%) as well as limits of detection (LOD) of 0.08 µmol L-1 and limits of quantification (LOQ) of 0.28 µmol L-1. CB+IL/PGE response was five times greater than the unmodified PGE. CB+IL-PGE stands as an interesting alternative for DIC assessment in different pharmaceutical samples.

Optimization of laccase-alginate-chitosan-based matrix toward 17 α-ethinylestradiol removal.

Laccase extract (LE) from Pycnoporus sanguineus was immobilized on calcium and copper alginate-chitosan beads and applied for the removal of 17α-ethinylestradiol (EE2). Effects of immobilization conditions such as: sodium alginate (SA) concentration; LE/SA ratio and chitosan/ion (Ca+2 or Cu+2) ratio on the immobilization yield were investigated. Immobilized LE on Ca-beads and Cu-beads was then used to degrade an EE2 solution. The optimal conditions for LE immobilization on Ca-beads were: 1.5% (w/v) SA, 1:5 (v/v) LE/SA and 3:7 (v/v) chitosan/ion (Ca+2). The optimal conditions for immobilization on Cu-beads were 2.0% (w/v) SA, 0.5:5 (v/v) LE/SA and 3:7 (v/v) chitosan/ion (Cu+2). The best result was obtained for immobilized LE on Ca-beads in buffer-absent medium. Furthermore, the immobilized enzyme was reused in five cycles for EE2 removal. The formation of EE2 dimers by LE treatment has been demonstrated by electrospray ionization coupled to time of flight mass spectrometer (ESI-TOF-MS). The results evidenced that immobilized LE in alginate-chitosan-divalent cation bead is an effective alternative for EE2 removal.

Stability of immobilized laccase on Luffa Cylindrica fibers and assessment of synthetic hormone degradation.

In this work were studied the pH, thermal, and storage stability of free and immobilized laccases. Enzymes were produced by Pleurotus ostreatus on potato dextrose (PD) broth and potato dextrose modified (PDM) broth, and immobilized using Luffa cylindrica fibers as support. Both free and immobilized enzymes were assessed on their respective enzymatic activities and for 17α-ethinylestradiol (EE2) degradation. The optimum pH conditions concerning laccase activity ranged from 3.6 to 4.6, while temperature ranged between 30 °C and 50 °C for both free and immobilized enzyme. Laccase produced using PD broth presented greater storage stability and thermal stability than that of PDM. Best EE2 removals were of 79.22% and 75.00% for the free and immobilized enzymes, respectively. Removal rates were assessed during 8 h at pH 5. The removal of 17α-ethinylestradiol was stabilized in the fourth cycle of use. Results imply that immobilization promoted stability towards pH and temperature variations, although media played a decisive role in the enzymatic activity. Both free and immobilized laccases of P. ostreatus were able to degrade EE2, whereas immobilized laccase in PDM medium presented possible reuse applicability, albeit removal was not optimal when compared to other reports.