Oxidation Of ABTS Research Articles

Selection of organic solvents as reaction media for laccase from Trametes versicolor

The ability of laccases to work effectively in properly selected aqueous-organic solutions significantly expands the scope of their application, making them commercially attractive and more competitive. However, it must be considered that molecules of organic solvent are very aggressive towards enzymes and destroy weak non-covalent interactions that maintain the spatial structure of the protein. In this work, we quantitatively described the influence of the nature of water-miscible organic solvents and their concentration on the activity and stability of Trametes versicolor laccase during the oxidation of ABTS (2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) through the maximum rates and the Michaelis constants KM. The obtained kinetic parameters make it possible to compare the effect of various organic solvents on laccase and select the most optimal one, which minimizes the decrease in enzyme activity, maintains its stability and allows repeated use. Among the solvents studied, polyhydroxy compounds have the least negative effect on laccase activity; however, in all cases, a high concentration of organic solvent (≥30% (v/v)) leads to suppression of enzymatic activity. It has been shown that the longer the alkyl chain (hydrophobic fragment of the structure), the stronger the negative effect of alcohols on enzymes. The most toxic solvent for Trametes versicolor laccase is isopropanol. In addition, numerous examples of various uses of Trametes versicolor in aquatic-organic environments are discussed here.

Flexible microfluidic colorimetric detection chip integrated with ABTS·+ and Co@MnO2 nanozyme catalyzed TMB reaction systems for bio-enzyme free detection of sweat uric acid

BackgroundThe development of wearable detection devices that can achieve noninvasive, on-site and real-time monitoring of sweat metabolites is of great demand and practical significance for point-of-care testing and healthcare monitoring. Monitoring uric acid (UA) content in sweat provides a simple and promising way to reduce the risk of gout and hyperuricemia. Traditional bioenzyme based UA assays suffer from high cost, poor stability, inconvenience for storage and easy deactivation of bioenzymes. Wearable microfluidic colorimetric detection device for sweat UA detection has not been reported. The development of novel wearable microfluidic colorimetric detection chip with no requirement of bioenzymes for sweat UA detection is of great importance for health care monitoring. ResultsFirstly, Co@MnO2 nanozyme with high oxidase-like activity was synthesized and characterized. Co@MnO2 can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) directly to generate blue-green colored ox-TMB. Green colored 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical (ABTS·+) was produced by the oxidation of ABTS by potassium persulfate. UA exhibits distinct quenching effect on Co@MnO2 catalyzed TMB colorimetric reaction system and ABTS·+ based colorimetric system, leading to obvious color fading of the two colorimetric systems. Then, a flexible microfluidic colorimetric detection chip for UA detection was fabricated by assembling Co@MnO2/TMB modified paper chips and ABTS·+ modified paper chips into a polydimethylsiloxane (PDMS) microfluidic chip. The fabricated microfluidic colorimetric detection chip exhibits good linear relationship for sweat UA detection. The linear range is from 20 to 200 μmol/L with detection limit as low as 6.6 μmol/L. Good results were obtained for the detection of UA in actual sweat from three volunteers. SignificanceThis work provides two bio-enzyme free colorimetric detection systems for UA detection. Furthermore, a simple, low-cost and selective flexible wearable microfluidic colorimetric detection chip was fabricated for noninvasive and on-site detection of sweat UA, which holds great application potential for personal health monitoring and point-of-care testing.

Production and characterization of novel thermostable CotA-laccase from Bacillus altitudinis SL7 and its application for lignin degradation

Laccases are multi-copper oxidases and found in ligninolytic bacteria catalyzing the oxidation of both phenolic and non-phenolic compounds, however its application in lignin degradation suffers due to low oxidation rate, which have intensified the search for new laccases. In the present study, spore coat A protein (CotA) encoding gene having laccase like activity from Bacillus altitudinis SL7 (CotA-SL7) was cloned and expressed in Escherichia coli. The purified CotA-SL7 was active at wide range of temperature and pH with optimum activity at 55 °C and pH 5.0. The kinetic parameters of CotA-SL7 was determined with Km, Vmax, and kcat values 0.4 mM, 2777 μmol/min/mg, and 5194 s−1, respectively. Molecular docking revealed the presence of Pro, Phe, Asp, Asn, His, and Ile residues at the active site taking part in the oxidation of ABTS. The purified CotA-SL7 reduced lignin contents by 31 % and changes in lignin structure were analyzed through fourier transformed infrared spectroscopy (FTIR), scanning electron microsscopy (SEM) and gas chromatography mass-spectrometry (GC-MS). The appearance of low molecular size compounds clearly indicates the cleavage of lignin polymer and opening of the benzene ring by purified CotA-SL7. Thus, high catalytic efficiency of CotA-SL7 makes it a suitable bio-catalyst for remediation of lignin contaminated wastewater from pulp and paper industries with clear insights into lignin degradation at molecular level.

Oxidation of anti-thyroid drugs and their selenium analogs by ABTS radical cation

Lactoperoxidase was previously used as a model enzyme to test the inhibitory activity of selenium analogs of anti-thyroid drugs with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. Peroxidases oxidize ABTS to a metastable radical ABTS•+, which is readily reduced by many antioxidants, including thiol-containing compounds, and it has been used for decades to measure antioxidant activity in biological samples. We showed that anti-thyroid drugs 6-n-propyl-2-thiouracil, methimazole, and selenium analogs of methimazole also reduced it rapidly. This reaction may explain the anti-thyroid action of many other compounds, particularly natural antioxidants, which may reduce the oxidized form of iodine and/or tyrosyl radicals generated by thyroid peroxidase thus decreasing the production of thyroid hormones. However, influence of selenium analogs of methimazole on the rate of hydrogen peroxide consumption during oxidation of ABTS by lactoperoxidase was moderate. Direct hydrogen peroxide reduction, proposed before as their mechanism of action, cannot therefore account for the observed inhibitory effects. 1-Methylimidazole-2-selone and its diselenide were oxidized by ABTS•+ to relatively stable seleninic acid, which decomposed slowly to selenite and 1-methylimidazole. In contrast, oxidation of 1,3-dimethylimidazole-2-selone gave selenite and 1,3-dimethylimidazolium cation. Accumulation of the corresponding seleninic acid was not observed.

Fungal Ligninolytic Enzymes and Their Application in Biomass Lignin Pretreatment.

Lignocellulosic biomass is a significant source of sustainable fuel and high-value chemical production. However, due to the complex cross-linked three-dimensional network structure, lignin is highly rigid to degradation. In natural environments, the degradation is performed by wood-rotting fungi. The process is slow, and thus, the use of lignin degradation by fungi has not been regarded as a feasible technology in the industrial lignocellulose treatment. Fungi produce a wide variety of ligninolytic enzymes that can be directly introduced in industrial processing of lignocellulose. Within this study, screening of ligninolytic enzyme production using decolorization of ABTS and Azure B dyes was performed for 10 fungal strains with potentially high enzyme production abilities. In addition to standard screening methods, media containing lignin and hay biomass as carbon sources were used to determine the change in enzyme production depending on the substrate. All selected fungi demonstrated the ability to adapt to a carbon source limitation; however, four strains indicated the ability to secrete ligninolytic enzymes in all experimental conditions-Irpex lacteus, Pleurotus dryinus, Bjerkandera adusta, and Trametes versicolor-respectively displayed a 100%, 82.7%, 82.7%, and 55% oxidation of ABTS on lignin-containing media and 100%, 87.9%, 78%, and 70% oxidation of ABTS on hay-containing media after 168 h of incubation. As a result, the most potent strains of fungi were selected to produce lignocellulose-degrading enzymes and to demonstrate their potential application in biological lignocellulose pretreatment.

Nanostructured binuclear Fe(III) and Mn(III) porphyrin materials: Tuning the mimics of catalase and peroxidase activity

Tuning of the structures and electronic effects of nanocrystals is of great relevance to the preparation of specialized materials such as artificial enzymes.In this study, binary porphyrin materials have been prepared by ionic self-assembly of oppositely charged Fe(III) and Mn(III) porphyrins. The materials have been characterized by SEM, XPS and UV–vis spectroscopy and tested as biomimetic models of catalase. The heterometallic material [Mn/Fe], prepared by association of MnTHPyP and FeTSPP, showed the highest activity for H2O2 dismutation reaction, 89–95 U·mg−1, over a pH range of 4–7. An enhancement in activity of one to two orders of magnitude can be observed relatively to the individual Fe(III) or Mn(III) porphyrins and to the mono-metallic materials that were prepared by association of a metalloporphyrin (MP) and a metal free porphyrin (H2P). These results support the occurrence of cooperative mechanisms between two metal ion centers within the structure. The materials with the highest catalase-like activity were also tested as peroxidase mimetics in ABTS oxidation. Best activity was observed for the [Mn/Fe] material at pH 4, while at pH 6 the activity decreases by 97%. EPR studies at pH 6 demonstrate the absence of free hydroxyl or superoxide radicals in the mechanism and thus support the activity of the [Mn/Fe] material as a catalytic antioxidant under these conditions.

Antibodies-Abzymes with Antioxidant Activities in Two Th and 2D2 Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology.

The exact mechanisms of multiple sclerosis development are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis) in Th and 2D2 mice is associated with the infringement of the differentiation profiles of bone marrow hematopoietic stem cells which are bound with the production of compounds that are harmful for human autoantibodies-abzymes that hydrolyze myelin oligodendrocyte glycoprotein, myelin basic protein, and DNA. It showed that autoimmune patients' antioxidant IgG antibodies oxidise some compounds due to their peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent) activities more effectively than those in healthy humans can. It was interesting to identify whether the redox activities of the antibodies change during the development of autoimmune diseases. Here, we analyzed the change in these redox activities of the IgGs from the blood of Th and 2D2 mice, which corresponded to different stages of the EAE development. The peroxidase activity in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in the Th (4-fold) and 2D2 (2-fold) mice IgGs, on average, is higher than the oxidoreductase activity is. The peroxidase activity of the Th (1.9-fold) and 2D2 (3.5-fold) mice IgGs remarkably increased during the 40 days of the spontaneous development of EAE. Forty days after the immunization of the MOG peroxidase activity, the IgGs of the Th and 2D2 mice increased 5.6-6.0 times when they were compared with those that presented no increase (3 months of age). The mice IgGs were oxidized with 3,3'-diaminobenzidine (2.4-4.3-fold) and o-phenylenediamine (139-143-fold) less efficiently than they were with ABTS. However, the temper of the change in the IgG activity in the oxidation of these substrates during the spontaneous and MOG-induced development of EAE was close to that which occurred for ABTS. All of the data show that the IgG peroxidase and oxidoreductase activities of EAE mice can play an important role in their protection from toxic compounds and oxidative stress.

Efficient Biocatalytic System for Biosensing by Combining Metal-Organic Framework (MOF)-Based Nanozymes and G-Quadruplex (G4)-DNAzymes.

A high catalytic efficiency associated with a robust chemical structure are among the ultimate goals when developing new biocatalytic systems for biosensing applications. To get ever closer to these goals, we report here on a combination of metal-organic framework (MOF)-based nanozymes and a G-quadruplex (G4)-based catalytic system known as G4-DNAzyme. This approach aims at combining the advantages of both partners (chiefly, the robustness of the former and the modularity of the latter). To this end, we used MIL-53(Fe) MOF and linked it covalently to a G4-forming sequence (F3TC), itself covalently linked to its cofactor hemin. The resulting complex (referred to as MIL-53(Fe)/G4-hemin) exhibited exquisite peroxidase-mimicking oxidation activity and an excellent robustness (being stored in water for weeks). These properties were exploited to devise a new biosensing system based on a cascade of reactions catalyzed by the nanozyme (ABTS oxidation) and an enzyme, the alkaline phosphatase (or ALP, ascorbic acid 2-phosphate dephosphorylation). The product of the latter poisoning the former, we thus designed a biosensor for ALP (a marker of bone diseases and cancers), with a very low limit of detection (LOD, 0.02 U L-1), which is operative in human plasma samples.

Cloning, expression and biochemical characterization of lignin-degrading DyP-type peroxidase from Bacillus sp. Strain BL5

Lignin is a major byproduct of pulp and paper industries, which is resistant to depolymerization due to its heterogeneous structure. The enzymes peroxidases can be utilized as potent bio-catalysts to degrade lignin. In the current study, an Efeb gene of 1251bp encoding DyP-type peroxidase from Bacillus sp. strain BL5 (DyPBL5) was amplified, cloned into a pET-28a (+) vector and expressed in Escherichia coli BL21 (DE3) cells. A 46 kDa protein of DyPBL5 was purified through ion-exchange chromatography. Purified DyPBL5 was active at wide temperature (25−50 °C) and pH (3.0–8.0) range with optimum activity at 35 °C and pH 5.0. Effects of different chemicals on DyPBL5 were determined. The enzyme activity was strongly inhibited by SDS, DDT and β-mercaptoethanol, whereas stimulated in the presence of organic solvents such as methanol and ethanol. The kinetic parameters were determined and Km, Vmax and Kcat values were 1.06 mM, 519.75 μmol/min/mg and 395 S̶ 1, respectively. Docking of DyPBL5 with ABTS revealed that, Asn 244, Arg 339, Asp 383 and Thr 389 are putative amino acids, taking part in the oxidation of ABTS. The recombinant DyPBL5 resulted in the reduction of lignin contents up to 26.04 %. The SEM and FT-IR analysis of test samples gave some indications about degradation of lignin by DyPBL5. Various low molecular weight lignin degradation products were detected by analyzing the samples through gas chromatography mass spectrometry. High catalytic efficiency and lignin degradation rate make DyPBL5 an ideal bio-catalyst for remediation of lignin-contaminated sites.

ABTS as an electron shuttle to accelerate the degradation of diclofenac with horseradish peroxidase-catalyzed hydrogen peroxide oxidation

Horseradish peroxidase (HRP)-catalyzed hydrogen peroxide (H2O2) oxidation could degrade a variety of organic pollutants, but the intrinsic drawback of slow degradation rate limited its widespread application. In this study, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was introduced into HRP/H2O2 system as an electron shuttle to enhance diclofenac degradation under neutral pH conditions. The green-colored ABTS radical (ABTS•+), generated by the oxidation of ABTS with HRP-catalyzed H2O2 oxidation, was proved to be the main reactive species for the rapid degradation of diclofenac in HRP/H2O2/ABTS system. There was no destruction of ABTS/ABTS•+ in HRP/H2O2/ABTS system, and ABTS was verified as an ideal electron shuttle. The reaction conditions including solution pH (4.5–10.5), HRP concentration (0–8 units mL−1) and H2O2 concentration (0–500 μM) would impact the formation of ABTS•+, and affect the degradation of diclofenac in HRP/H2O2/ABTS system. Moreover, compared with Fenton and hydroxylamine/Fenton systems, HRP/H2O2/ABTS system had better diclofenac degradation efficiency, higher H2O2 utilization efficiency and stronger anti-interference capacity in actual waters. Overall, the present study provided a meaningful and promising way to enhance the degradation of organic pollutants in water with HRP-catalyzed H2O2 oxidation.

Designing biopolymer-based artificial peroxidase for oxidative removal of dibenzothiophene from a model diesel fuel

Naturally occurring peroxidases are important for living organisms and have manifold utility in industries. However, lack of stability in harsh reaction conditions hinders wide applicability of such enzymes. Thus, suitable alternative is vital which can endure severe reaction conditions. As a substitute of natural peroxidase, herein, biopolymer-based polyelectrolyte complexes (PECs) coordinated with Fen+ is proposed as macromolecular peroxidase mimicking systems. Three PECs were engineered via complexation of protonated chitosan and alginate with Fe2+ (Fe2+-PEC), Fe3+ (Fe3+-PEC), and Fe3O4 (Fe3O4-PEC), respectively. Computational study showed the Fe3+-PEC was highly stable with abundant electrostatic and intramolecular hydrogen bonding interactions. The versatility of the Fe-PECs as artificial peroxidase biocatalysts was probed by two types of peroxidase assays – ABTS oxidation in buffer systems (pH 4.0 and 7.0) and pyrogallol oxidation in organic solvents (acetonitrile, ethyl acetate and toluene). Overall, Fe3+-PEC showed remarkably high peroxidase activity both in aqueous buffers and in organic solvents, whereas, Fe3O4-PEC showed least catalytic activity. Finally, as a proof of concept, the ability of the biocatalyst to carry out deep oxidative desulphurization was demonstrated envisaging removal of dibenzothiophene from model fossil fuel in a sustainable way.

Increase in Autoantibodies-Abzymes with Peroxidase and Oxidoreductase Activities in Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology.

The exact mechanisms of multiple sclerosis (MS) development are still unknown, but the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice is associated with the violation of bone marrow hematopoietic stem cells (HSCs) differentiation profiles associated with the production of harmful for human’s autoantibodies hydrolyzing myelin basic protein, myelin oligodendrocyte glycoprotein (MOG35–55), and DNA. It was shown that IgGs from the sera of healthy humans and autoimmune patients oxidize many different compounds due to their H2O2-dependent peroxidase and oxidoreductase activity in the absence of H2O2. Here we first analyzed the change in the relative redox activities of IgGs antibodies from the blood of C57BL/6 mice over time at different stages of the EAE development. It was shown that the peroxidase activity of mice IgGs in the oxidation of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is on average 6.9-fold higher than the oxidoreductase activity. The peroxidase activity of IgGs increased during the spontaneous development of EAE during 40 days, 1.4-fold. After EAE development acceleration due to mice immunization with MOG35–55 (5.3-fold), complexes of bovine DNA with methylated bovine serum albumin (DNA-metBSA; 3.5-fold), or with histones (2.6-fold), the activity was increased much faster. The increase in peroxidase activity after mice immunization with MOG35–55 and DNA-metBSA up to 40 days of experiments was relatively gradual, while for DNA-histones complex was observed its sharp increase at the acute phase of EAE (14–20 days). All data show that IgGs’ redox activities can play an important role in the protection of mice from toxic compounds and oxidative stress.

On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy.

The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surface-exposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. So far in B-type DyPs, there has been no evidence for protein radical formation after activation by hydrogen peroxide. Interestingly, B-type Klebsiella pneumoniae dye-decolorizing peroxidase (KpDyP) displays a persistent organic radical in the resting state composed of two species that can be distinguished by W-band electron spin echo electron paramagnetic resonance (EPR) spectroscopy. Here, on the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of KpDyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in KpDyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. This suggests the existence of long-range electron path(s) in B-type DyPs. The mechanistic and physiological relevance of the reaction mechanism of B-type DyPs is discussed.

Determination of Peracetic Acid in the Presence of Hydrogen Peroxide Based on the Catalytic Oxidation of ABTS

Determination of Peracetic Acid in the Presence of Hydrogen Peroxide Based on the Catalytic Oxidation of ABTS

Microalgae: A new source of healthy ingredients. Antioxidant and antimicrobial properties and incorporation as a functional ingredient for cheese making

AbstractAntimicrobial and antioxidant activities of nine species of microalgae have been determined to study its potential as healthy ingredients for functional foods. In order to determine the antioxidant capacity, two different techniques have been used. Firstly, a colorimetric test was carried out in order to detect the presence of lipophilic and hydrophilic antioxidant compounds, this method is based on the detection of the radical ABTS + , which is produced by the oxidation of ABTS in the presence of potassium persulfate, detection of the radical ABTS + decreases in the presence of antioxidant compounds. Also the ORAC test was also applied for this purpose. Results with both methods showed very promising results for Rhodomonas lens. Thus this species was chosen to develop a functional cheese and study the healthy properties that the microalgae confer on it.To determine the antimicrobial activity five food food-borne pathogenic strains were used: Escherichia coli CECT 516, Enterococcus faecalis CECT 481, Listeria monocytogenes CECT 935, Salmonella enterica CECT 4594 and S. aureus CECT 435. First, extracts of different polarity were obtained from microalgae. From each species, 4 extracts were obtained using different solvents of higher to less polarity (MilliQ Water, ethanol, ethyl acetate and hexane). The use of these solvents is common in the preparation of plant extracts, since they ensure the solubility of most of the compounds of interest and do not show toxicity after evaporation to determine the antimicrobial activity. The agar diffusion technique was used and the plates were kept refrigerated at 4 ° C for 1 h to allow the extracts to diffuse through the agar and subsequently incubated at 37 ° C for 24 h to allow microbial growth. After that time the presence of inhibition halos was observed around the perforations of the plates. Results showed that the ethanolic extract presented more antimicrobial activity than the extracts using MilliQ Water, ethyl acetate or hexane. Salmonella was not inhibited by any of the extracts studied.

Kinetic characterization of laccase from Bacillus atrophaeus, and its potential in juice clarification in free and immobilized forms

In the present study, a laccase gene (BaLc) from a lignin degrading bacterium, Bacillus atrophaeus, has been cloned and expressed in Escherichia coli. The optimal catalytic activity of the protein was achieved at 5.5 pH and 35°C temperature, measured by oxidation of ABTS. The Km and Vmax values were determined as 1.42 mM and 4.16 µmole/min, respectively. To achieve the enzyme recovery, the biocatalyst (BaLc) was covalently attached onto the functionalized iron magnetic-nanoparticles. The nanoparticles were characterized by zeta-potential and FTIR analyses. The immobilized BaLc enzyme was physico-kinetically characterized, exhibiting retention of 60% of the residual activity after ten reaction cycles of ABTS oxidation. The immobilized biocatalyst system was tested for its biotechnological exploitability in plant juice processing, achieving 41-58% of phenol reduction, 41-58% decolorization, 50-59% turbidity reduction in the extracts of banana pseudo-stem and sweet sorghum stalk, and apple fruit juice. This is the first study to demonstrate the use of nanoparticle-laccase conjugate in juice clarification. The findings suggest that B. atrophaus laccase is a potential catalytic tool for plant juice bioprocessing activities.

Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching

Carbonate radicals (CO3-) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO3- formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared to previously reported methods using electron paramagnetic resonance spin trapping with DMPO, and the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid). The kinetics of PGR consumption elicited by CO3- was followed by visible spectrophotometry. Solutions containing PGR (5–200 μM), SOD-1 (0.3–3 μM), H2O2 (2 mM) in bicarbonate buffer (200 mM, pH 7.4) showed a rapid loss of the PGR absorption band centered at 540 nm. The initial consumption rate (Ri) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3- release of 24.6 ± 4.3 μM min−1 for 3 μM bSOD-1. Both bSOD-1 and hSOD-1 showed a similar peroxidase activity, with enzymatic inactivation occurring over a period of 20 min. The single Trp residue (Trp32) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 ± 0.1 μM min−1) with this occurring more rapidly than hSOD-1 inactivation, suggesting that these processes are not directly related. Added free Trp was rapidly oxidized in competition with PGR. These data indicate that PGR reacts rapidly and efficiently with CO3- resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3- release from bSOD-1 and hSOD-1 peroxidase activity.

Signal peptide replacement resulted in recombinant homologous expression of laccase Lcc8 in Coprinopsis cinerea

Although the model agaricomycete Coprinopsis cinerea possess 17 different laccase genes, up to now only four C. cinerea laccases have been purified and characterized to some degree. By exchanging the nucleotide sequence of the deduced signal peptide of Lcc8 it was possible to homologously express lcc8 in C. cinerea under control of the Agaricus bisporus gdpII promoter and the C. cinerea lcc1 terminator. The purified Lcc8 showed two bands in the SDS-PAGE with a molecular weight of 64 kDa and 77 kDa, respectively. The IEF determined pI values of 3.3 and 3.4 for both bands. The optimal pH for oxidation of the substrates ABTS, 2,6-dimethoxyphenol, guaiacol and syringaldazine was pH 4.0, pH 5.0, pH 4.5 and pH 5.0, respectively. Best pH for enzyme storage was pH 8.0. The optimal temperature for oxidation of ABTS was 63 °C, while Lcc8 showed activity of at least 50% over 300 min at 50 °C. The comparable high stability of Lcc8 at alkaline pH and higher temperatures can be of interest for biotechnical applications.

Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects.

Dye-decolorizing peroxidases (DyPs) are a family of H2O2-dependent heme peroxidases, which have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been uncovered for the B-class ElDyP from Enterobacter lignolyticus. Its structure revealed that a water molecule acts as the sixth axial ligand with two channels at diameters of ~3.0 and 8.0 Å leading to the heme center. A conformational change of ERS* to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs' bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type ElDyP with D2O2 at pH 3.5 suggested that cmpd 0 deprotonation by the distal aspartate is rate-limiting in the formation of cmpd I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with cmpd I instead of the free enzyme. The significant inverse sKIEs of kcat/KM and kERS* suggested that the aquo release in DyPs is mechanistically important and may explain the enzyme's adoption of two-electron reduction for cmpd I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining DyPs' acidic pH optimum. The kinetic mechanism of D143H-ElDyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs' lignolytic activity.

Protein adsorption and activity on carbon xerogels with narrow pore size distributions covering a wide mesoporous range

Four carbon xerogels (CXs) were used to study the effect of the average pore size (APS) on the adsorption and activity of cytochrome c (cyt c). Pore size distributions of the CXs were relatively narrow with APSs covering the whole mesoporosity range (5, 15, 30 and 55 nm), as determined by mercury porosimetry. Selected techniques verified that all carbons were identical in terms of composition and surface chemistry. The best APS for cyt c adsorption (in terms of capacity) was 30 nm, with loadings of 180 mg of protein (g of support)−1. The CX with APS of 15 nm also hosted high amounts of cyt c, followed by the material with 55 nm. The CX with 5 nm APS did not adsorb cyt c. The pH of adsorption had little effect on the final amount adsorbed, thus stressing the hydrophobic nature of the protein/carbon surface interaction. The activity of the resulting materials towards the ABTS oxidation was similar regardless the amount of cyt c adsorbed on their surface/pores. Their performance in successive cycles of re-use was different depending on the carbon support; xerogels bearing APSs ≥15 nm presented an increasing activity with increasing the number of cycles.