Practical applications of copper-based enzymes: synthesis of sulfonated polyaniline through laccase-catalyzed oxidation.

Known for its tunable conductivity and stability, Polyaniline (PANI) is a valuable polymer for electronics and sensing devices. Challenges in solubility have been addressed by creating sulfonated PANI (SPANI), enhancing its practical use. Synthesizing SPANI from sulfonated aniline is intricate, but laccase biocatalysis offers an eco-conscious solution, effective even against high redox potential obstacles. This research monitored the Trametes versicolor laccase-induced oxidation of 3-ABSa via UV-vis spectroscopy, with a notable peak at 565 nm signifying SPANI synthesis, effective even at suboptimal pH. Mediators further boost this process. Moreover, NMR and spectroelectrochemistry confirm the green synthesis of SPANI by laccase, hinting that pH fine-tuning could improve yields, alongside the concurrent creation of azobenzene derivatives.

Ingestion of chitosan-starch blends: Effect on the survival of supralittoral amphipods.

Sandy beach ecosystems are particularly affected by plastic pollution. Supralittoral amphipods are important components of the food web in sandy beaches and their ability to ingest microplastics and bioplastics has been assessed. Chitosan, a polysaccharide obtained by deacetylation of chitin, the second most abundant polymer in the world, represents an interesting component to produce novel bioplastics in combination with other biopolymers like starch. Here, the possibility of ingesting chitosan-starch blends and the possible effects on the amphipod Talitrus saltator were investigated. Groups of adult individuals were fed with sheets containing mixtures of chitosan and starch in different percentages for 7 and 14 days. The results showed that chitosan ingestion is dependent on the percentage of starch present in the mixture. Moreover, FTIR analyses of both sheets and faecal pellets after consumption show that chitosan is not digested. Furthermore, the survival rate of amphipods fed with a mixture of chitosan and starch decreases after one week compared to the control groups (100 % starch and paper), and drops drastically to 0 % after two weeks the experiment began. In addition, consumption of 100 % chitosan is negligible. Therefore, the results of the experimental observations evidenced that chitosan is avoided as food resource and its consumption significantly affects the survival capacity of T. saltator. It is emphasized that the release of mixtures of chitosan and starch into the marine environment appears to be dangerous for littoral amphipods.

A Novel Bio-Adhesive Based on Chitosan-Polydopamine-Xanthan Gum for Glass, Cardboard and Textile Commodities.

The escalating environmental concerns associated with petroleum-based adhesives have spurred an urgent need for sustainable alternatives. Chitosan, a natural polysaccharide, is a promising candidate; however, its limited water resistance hinders broader application. The aim of this study is to develop a new chitosan-based adhesive with improved properties. The polydopamine association with chitosan presents a significant increase in adhesiveness compared to pure chitosan. Polydopamine is synthesized by the enzymatic action of laccase from Trametes versicolor at pH = 4.5, in the absence or presence of chitosan. This pH facilitates chitosan's solubility and the occurrence of catechol in its reduced form (pH < 5.5), thereby increasing the final adhesive properties. To further enhance the adhesive properties, various crosslinking agents were tested. A multi-technique approach was used for the characterization of formulations. The formulation based on 3% chitosan, 50% polydopamine, and 3% xanthan gum showed a spectacular increase in adhesive properties when tested on glass, cardboard and textile. This formulation increased water resistance, maintaining the adhesion of a sample soaked in water for up to 10 h. For cardboard and textile, material rapture occurred, in mechanical tests, prior to adhesive bond failure. Furthermore, all the samples showed antiflame properties, expanding the benefits of their use. Comparison with commercial glues confirms the remarkable adhesive properties of the new formulation.

Special Issue on Biotechnological Applications of Oxidoreductases.

This Special Issue was launched in conjunction with the 10th edition of the OxiZymes meeting in Siena (Italy) in 2022 [...].

Nanoparticles of Lignins and Saccharides from Fishery Wastes as Sustainable UV-Shielding, Antioxidant, and Antimicrobial Biofillers.

Lignin nanoparticles containing saccharides from fishery wastes were prepared as sustainable biofillers for advanced materials. Organosolv lignin and Kraft lignin were used as polyphenol components in association with chitosan and chitooligosaccharides. The chemophysical and biological activities of lignin/saccharide nanoparticles, such as UV-shielding, antioxidant, and antimicrobial activities, were found to be dependent on both molecular weight and deacetylation degree of saccharides, with the best performance being obtained in the presence of low-molecular-weight and highly deacetylated chitooligosaccharides. In addition, chitooligosaccharides showed a synergistic antioxidant effect with lignins, associated with antimicrobial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive).

Quantification and Improvement of the Dynamics of Human Serum Albumin and Glycated Human Serum Albumin with Astaxanthin/Astaxanthin-Metal Ion Complexes: Physico-Chemical and Computational Approaches.

Glycated human serum albumin (gHSA) undergoes conformational changes and unfolding events caused by free radicals. The glycation process results in a reduced ability of albumin to act as an endogenous scavenger and transporter protein in diabetes mellitus type 2 (T2DM) patients. Astaxanthin (ASX) in native form and complexed with metal ions (Cu2+ and Zn2+) has been shown to prevent gHSA from experiencing unfolding events. Furthermore, it improves protein stability of gHSA and human serum albumin (HSA) as it is shown through molecular dynamics studies. In this study, the ASX/ASX-metal ion complexes were reacted with both HSA/gHSA and analyzed with electronic paramagnetic resonance (EPR) spectroscopy, rheology and zeta sizer (particle size and zeta potential) analysis, circular dichroism (CD) spectroscopy and UV-Vis spectrophotometer measurements, as well as molecular electrostatic potential (MEP) and molecular docking calculations. The addition of metal ions to ASX improves its ability to act as an antioxidant and both ASX or ASX-metal ion complexes maintain HSA and gHSA stability while performing their functions.

Stereoselective Access to Antimelanoma Agents by Hybridization and Dimerization of Dihydroartemisinin and Artesunic acid.

A library of five hybrids and six dimers of dihydroartemisinin and artesunic acid has been synthetized in a stereo-controlled manner and evaluated for the anticancer activity against metastatic melanoma cell line (RPMI7951). Among novel derivatives, three artesunic acid dimers showed antimelanoma activity and cancer selectivity, being not toxic on normal human fibroblast (C3PV) cell line. Among the three dimers, the one bearing 4-hydroxybenzyl alcohol as a spacer showed no cytotoxic effect (CC50 >300 µM) and high antimelanoma activity (IC50 =0.05 µM), which was two orders of magnitude higher than that of parent artesunic acid, and of the same order of commercial drug paclitaxel. In addition, this dimer showed cancer-type selectivity towards melanoma compared to prostate (PC3) and breast (MDA-MB-231) tumors. The occurrence of a radical mechanism was hypothesized by DFO and EPR analyses. Qualitative structure activity relationships highlighted the role of artesunic acid scaffold in the control of toxicity and antimelanoma activity.

Homogentisic Acid and Gentisic Acid Biosynthesized Pyomelanin Mimics: Structural Characterization and Antioxidant Activity.

Pyomelanin mimics from homogentisic acid (HGA) and gentisic acid (GA) were biosynthesized by the oxidative enzyme T. versicolor laccase at physiological pH to obtain water soluble melanins. The pigments show brown-black color, broad band visible light absorption, a persistent paramagnetism and high antioxidant activity. The EPR approach shows that at least two different radical species are present in both cases, contributing to the paramagnetism of the samples. This achievement can also shed light on the composition of the ochronotic pigment in the Alkaptonuria disease. On the other hand, these soluble pyomelanin mimics, sharing physico-chemical properties with eumelanin, can represent a suitable alternative to replace the insoluble melanin pigment in biotechnological applications.

Laccase-Catalyzed 1,4-Dioxane-Mediated Synthesis of Belladine N-Oxides with Anti-Influenza A Virus Activity.

Belladine N-oxides active against influenza A virus have been synthetized by a novel laccase-catalyzed 1,4-dioxane-mediated oxidation of aromatic and side-chain modified belladine derivatives. Electron paramagnetic resonance (EPR) analysis confirmed the role of 1,4-dioxane as a co-oxidant. The reaction was chemo-selective, showing a high functional-group compatibility. The novel belladine N-oxides were active against influenza A virus, involving the early stage of the virus replication life cycle.

Production and characterization of chitooligosaccharides by the fungal chitinase Chit42 immobilized on magnetic nanoparticles and chitosan beads: selectivity, specificity and improved operational utility.

Chitin-active enzymes are of great biotechnological interest due to the wide industrial application of chitinolytic materials. Non-stability and high cost are among limitations that hinder industrial application of soluble enzymes. Here we report the production and characterization of chitooligosaccharides (COS) using the fungal exo-chitinase Chit42 immobilized on magnetic nanoparticles and food-grade chitosan beads with an immobilization yield of about 60% using glutaraldehyde and genipin linkers. The immobilized enzyme gained operational stability with increasing temperature and acidic pH values, especially when using chitosan beads-genipin that retained more than 80% activity at pH 3. Biocatalysts generated COS from colloidal chitin and different chitosan types. The immobilized enzyme showed higher hydrolytic activity than free enzyme on chitosan, and produced COS mixtures with higher variability of size and acetylation degree. In addition, biocatalysts were reusable, easy to handle and to separate from the reaction mixture.

Structure and Bioactive Properties of Novel Textile Dyes Synthesised by Fungal Laccase.

Novel sustainable processes involving oxidative enzymatic catalysts are considered as an alternative for classical organic chemistry. The unique physicochemical and bioactive properties of novel bio-products can be obtained using fungal laccase as catalyst. Among them are textile biodyes synthesised during oxidation of substrates belonging to the amine and methoxy organic derivatives. The process of synthesis occurs in mild conditions of pH, temperature, and pressure, and without using harmful oxidants. The effect of fungal laccase activity on the substrates mixture transformation efficiency was analysed in terms of antimicrobial dye synthesis on a large scale. Three new phenazine dyes, obtained in the presence of laccase from Cerrena unicolor, were studied for their structure and properties. The phenazine core structure of the products was a result of tri-molecular transformation of aminomethoxybenzoic acid and aminonaphthalene sulfonic acid isomers. One of the compounds from the synthesised dye, namely 10-((2-carboxy-6-methoxyphenyl)amino)-11-methoxybenzo[a]phenazine-8-carboxylic acid, was able to inhibit the growth of Staphylococcus aureus. The high concentration of substrates (5 g/L) was efficiently transformed during 72 h in the mild conditions of pH 4 with the use of laccase with an activity of 200 U per g of the substrates mixture. The new bioactive dye exhibited excellent dyeing properties with concomitant antibacterial and antioxidative activity. The proposed enzyme-mediated synthesis represents an alternative eco-friendly route for the synthesis of novel antimicrobial compounds with high importance for the medical textile industry.

Paramagnetism and Relaxation Dynamics in Melanin Biomaterials.

Spectroscopical characterization of melanins is a prior requirement for the efficient tailoring of their radical scavenging, ultraviolet-visible radiation absorption, metal chelation, and natural pigment properties. Electron paramagnetic resonance (EPR), exploiting the common persistent paramagnetism of melanins, represents the elective standard for the structural and dynamical characterization of their constituting radical species. Although melanins are mainly investigated using X-band (9.5 GHz) continuous wave (CW)-EPR, an integration with the application of Q-band (34 GHz) in CW and pulse EPR for the discrimination of melanin pigments of different compositions is presented here. The longitudinal relaxation times measured highlight faster relaxation rates for cysteinyldopa melanin, compared to those of the most common dopa melanin pigment, suggesting pulse EPR spin-lattice relaxation time measurements as a complementary tool for characterization of pigments of interest for biomimetic materials engineering.

Synthesis and Evaluation of Artemisinin-Based Hybrid and Dimer Derivatives as Antimelanoma Agents.

A library of hybrid and dimer compounds based on the natural scaffold of artemisinin was synthesized. These derivatives were obtained by coupling of artemisinin derivatives, artesunate, and dihydroartemisinin with a panel of phytochemical compounds. The novel artemisinin-based hybrids and dimers were evaluated for their anticancer activity on a cervical cancer cell line (HeLa) and on three complementary metastatic melanoma cancer cell lines (SK-MEL3, SK-MEL24, and RPMI-7951). Two hybrid compounds obtained by coupling of artesunate with eugenol and tyrosol, and one of the dimer compounds containing curcumin, emerged as the most active and cancer-selective derivatives.

Biomimetic synthesis of galantamine via laccase/TEMPO mediated oxidative coupling.

Laccase-mediated intramolecular oxidative radical coupling of N-formyl-2-bromo-O-methylnorbelladine afforded a novel and isolable spirocyclohexadienonic intermediate of galantamine. High yield and conversion of substrate were obtained in the presence of the redox mediator 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). This laccase procedure, with an overall yield of 34%, represents a scalable and environmentally friendly alternative to previously reported syntheses of galantamine based on the use of potassium ferricyanide as an unspecific radical coupling reagent.

Novel textile dye obtained through transformation of 2-amino-3-methoxybenzoic acid by free and immobilised laccase from a Pleurotus ostreatus strain.

Transformation of 2-amino-3-methoxybenzoic acid into novel and eco-friendly orange dye (N15) was performed using native and immobilised laccase (LAC) from Pleurotus ostreatus strain. A several parameters affecting laccase-mediated transformation efficiency included the selection of type and pH value of buffer, reaction temperature, substrate and laccase concentration as well as the type of carrier and LAC storage conditions were evaluated. The optimal conditions for N15 dye synthesis were 40 mM sodium-tartrate buffer pH 5.5 containing 3 mM of the substrate, efficiently transformed by 2 U of free laccase per 1 mmol of the substrate. Laccase was immobilised on porous Purolite® carriers, which had never been tested as a support for oxidoreductases. Immobilised laccase, characterised by a high immobilisation yield, was obtained by adsorption of laccase on a porous acrylic carrier with octadecyl groups (C18) incubated in optimum conditions of 40 mM phosphate buffer pH 7.0 containing 1 mg of laccase per 1 g of the carrier (wet mass). The immobilised LAC showed the highest storage stability for 21 days and higher thermostability at 40 ℃ and 60 ℃ in comparison to its native form. The N15 dye showed good dyeing properties towards natural fibres, and the dyed fibre demonstrated resistance to different physicochemical factors during use, which was confirmed by commercial quality tests. The N15 dye is a phenazine, i.e. a heterogenic compound containing amino-, methoxy-, and three carboxyl functional groups with the molecular weight of approximately 449.37 U.

Spectroscopic Characterisation of the Naphthalene Dioxygenase from Rhodococcus sp. Strain NCIMB12038.

Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O2-saturated naphthalene increased the intensity of the mononuclear contribution. A study of the "peroxide shunt", an alternative mechanism for the oxidation of substrate in the presence of H2O2, showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes.

Bridged bicyclic 2,3-dioxabicyclo[3.3.1]nonanes as antiplasmodial agents: Synthesis, structure-activity relationships and studies on their biomimetic reaction with Fe(II).

Despite recent advancements in its control, malaria is still a deadly parasitic disease killing millions of people each year. Progresses in combating the infection have been made by using the so-called artemisinin combination therapies (ACTs). Natural and synthetic peroxides are an important class of antimalarials. Here we describe a new series of peroxides synthesized through a new elaboration of the scaffold of bicyclic-fused/bridged synthetic endoperoxides previously developed by us. These peroxides are produced by a straightforward synthetic protocol and are characterized by submicromolar potency when tested against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. To investigate their mode of action, the biomimetic reaction of the representative compound 6w with Fe(II) was studied by EPR and the reaction products were characterized by NMR. Rationalization of the observed structure-activity relationship studies was performed by molecular docking. Taken together, our data robustly support the hypothesized mode of activation of peroxides 6a-cc and led to the definition of the key structural requirements responsible for the antiplasmodial potency. These data will pave the way in future to the rational design of novel optimized antimalarials suitable for in vivo investigation.

Effect of Electrode Shape and Flow Conditions on the Electrochemical Detection with Band Microelectrodes.

In this work, we report the analysis of the electrochemical detection of electroactive species with band microelectrodes that operate under controlled convection. The study focuses on the determination of the collection efficiency of the analyte as a function of inlet flow velocity and microband geometry (inlaid, bumped and recessed), also providing a straightforward method for the theoretical determination of the lower detection limit. The analysis has been carried out by simulating the dimensionless mass transport with the finite element method, delivering the stationary limiting current density. Simulations have been performed on systems consisting of single and double band electrodes to investigate the trail effect on the electrochemical detection. We show that the obtained dimensionless results can be easily turned into dimensional data, providing a tool for the design of devices. The proposed method is general and can easily be extended to systems with different geometry.

Spectroscopic Characterization of Natural Melanin from a Streptomyces cyaneofuscatus Strain and Comparison with Melanin Enzymatically Synthesized by Tyrosinase and Laccase.

An actinobacteria strain was isolated from Algerian Sahara soil and assigned to Streptomyces cyaneofuscatus Pridham et al. 1958 species. This strain was selected for its ability to produce melanin exopigments in liquid and solid media. Melanin synthesis was associated with tyrosinase activity and the enzyme from this strain was isolated and biochemically characterized. Synthetic melanin was then enzymatically produced using the S. cyaneofuscatus Pridham et al. 1958 tyrosinase. As this enzyme showed a higher diphenolase activity, a synthetic melanin from the enzymic oxidation of 3,4-dihydroxyphenylalanine (dopa) was obtained by the use of a Trametes versicolor (L.) Lloyd laccase for comparison. The natural and synthetic pigments were physico-chemically characterized by the use of ultraviolet (UV)-Visible, and Fourier transform infrared (FT-IR) and multifrequency electron paramagnetic resonance (EPR) spectroscopies. All the melanin samples displayed a stable free radical when analyzed by X-band EPR spectroscopy. Once the samples were recorded at Q-band EPR, a copolymer derived from a mixture of different constituents was evident in the natural melanin. All radical species were analyzed and discussed. The use of water-soluble melanin naturally produced by S. cyaneofuscatus Pridham et al. 1958 represents a new biotechnological alternative to commercial insoluble pigments.

Isolation and characterization of a novel tyrosinase produced by Sahara soil actinobacteria and immobilization on nylon nanofiber membranes.

In the present study different actinomycete strains were collected and isolated from Algerian Sahara soil with the aim to select novel enzymes with promising features for biotechnological applications. The Ms1 strain was selected, amongst the others, for its capability to produce melanin in different solid media. Ms1 chromosomal DNA was sequenced and the strain assigned to Streptomyces cyaneofuscatus sp. A tyrosinase (MW∼30kD) encoding sequence was identified and the corresponding enzyme was isolated and biochemically characterized. The tyrosinase showed the highest activity and stability at neutral and alkaline pH and it was able to oxidize l-DOPA at T=55°C and pH 7. The enzyme showed variable stability in presence of various water-miscible organic solvents, while it was inactivated by reducing agents. The tyrosinase activity was unaffected by NaCl and enhanced by different cations. Furthermore, the enzyme showed a higher specificity for diphenols than monophenols showing a higher diphenolase than monophenolase activity. Finally, tyrosinase was stabilized by immobilization on nylon nanofiber membranes with a payload of 82% when 1% glutaraldeyde was used. Taken all together, these results show that the enzyme displays interesting properties for biotechnological purposes.