Consecutive Marcus Electron and Proton Transfer in Heme Peroxidase Compound II-Catalysed Oxidation Revealed by Arrhenius Plots.

Electron and proton transfer reactions in enzymes are enigmatic and have attracted a great deal of theoretical, experimental, and practical attention. The oxidoreductases provide model systems for testing theoretical predictions, applying experimental techniques to gain insight into catalytic mechanisms, and creating industrially important bio(electro)conversion processes. Most previous and ongoing research on enzymatic electron transfer has exploited a theoretically and practically sound but limited approach that uses a series of structurally similar ("homologous") substrates, measures reaction rate constants and Gibbs free energies of reactions, and analyses trends predicted by electron transfer theory. This approach, proposed half a century ago, is based on a hitherto unproved hypothesis that pre-exponential factors of rate constants are similar for homologous substrates. Here, we propose a novel approach to investigating electron and proton transfer catalysed by oxidoreductases. We demonstrate the validity of this new approach for elucidating the kinetics of oxidation of "non-homologous" substrates catalysed by compound II of Coprinopsis cinerea and Armoracia rusticana peroxidases. This study - using the Marcus theory - demonstrates that reactions are not only limited by electron transfer, but a proton is transferred after the electron transfer event and thus both events control the reaction rate of peroxidase-catalysed oxidation of substrates.

Highly sensitive amperometric biosensor based on alcohol dehydrogenase for determination of glycerol in human urine.

In this paper we report the development of a highly sensitive amperometric glycerol biosensor based on alcohol dehydrogenase from Pseudomonas putida immobilized on graphite electrode modified with carbon nanotubes and a redox mediator tetrathiafulvalene. The designed biosensor demonstrates very high sensitivity towards glycerol (29.2 ±â€¯0.9 µA mM-1 cm-2), low limit of detection (18 µM), linear range from 0.05 to 1.0 mM, high selectivity and satisfactory stability. Biosensor has been successfully used for the determination of glycerol concentration in buffer solutions as well as in the human urine samples. Received results shows a satisfactory agreement with the control measurements carried out using colorimetric commercially available glycerol determination assay kit, thus developed biosensor can be successfully applied for measurements of glycerol concentration in human urine and may be a fast, attractive and non-invasive tool for the determination of glycerol.

Laccase-catalyzed bisphenol A oxidation in the presence of 10-propyl sulfonic acid phenoxazine.

The kinetics of the Coriolopsis byrsina laccase-catalyzed bisphenol A (BisA) oxidation was investigated in the absence and presence of electron-transfer mediator 3-phenoxazin-10-yl-propane-1-sulfonic acid (PPSA) at pH5.5 and 25°C. It was shown that oxidation rate of the hardly degrading compound BisA increased in the presence of the highly reactive substrate PPSA. The increase of reaction rate depends on PPSA and BisA concentrations as well on their ratio, e.g., at 0.2 mmol/L of BisA and 2 µmol/L of PPSA the rate increased 2 times. The kinetic data were analyzed using a scheme of synergistic laccase-catalyzed BisA oxidation. The calculated constant, characterizing reactivity of PPSA with laccase, is almost 1000 times higher than the constant, characterizing reactivity of BisA with laccase. This means that mediator-assisted BisA oxidation rate can be 1000 times higher in comparison to non-mediator reaction if compounds concentration is equal but very low.

Evanescent-field-induced Raman scattering for bio-friendly fingerprinting at sub-cellular dimension.

Evanescent field induced chemical imaging concept has been realized in analytical platform based on the µ-tip-enhanced Raman scattering spectroscopy (µ-TERS). The technique aimed to minimize thermal decomposition of dried biological sample as the result of huge concentration of optical field near the tip by increasing the size of an aperture-less "excitation source". µ-TERS technique is similar to classical biosensor systems based on propagating surface plasmon resonance phenomenon but with sensitive elements a few micrometers in size that can be targeted to the area of interest. The utility of the concept is exemplified by the analysis of dried single cell envelope of genetically modified Saccharomyces cerevisiae yeast cells, which do not have any heat-removing pathways, by water as in the case of the living cell. Practical excitation conditions effective for µ-TERS Raman observation of single layer dried biological samples without photodamage-related spectral distortion have been determined - the allowable limit is above 30s at 13 µW/µm(2). Finally, potential of µ-TERS spectroscopy as new bio-friendly instrumental platform for chemical fingerprinting and analytical characterization of buried nanoscale features is discussed.

Effect of diffusion limitations on multianalyte determination from biased biosensor response.

The optimization-based quantitative determination of multianalyte concentrations from biased biosensor responses is investigated under internal and external diffusion-limited conditions. A computational model of a biocatalytic amperometric biosensor utilizing a mono-enzyme-catalyzed (nonspecific) competitive conversion of two substrates was used to generate pseudo-experimental responses to mixtures of compounds. The influence of possible perturbations of the biosensor signal, due to a white noise- and temperature-induced trend, on the precision of the concentration determination has been investigated for different configurations of the biosensor operation. The optimization method was found to be suitable and accurate enough for the quantitative determination of the concentrations of the compounds from a given biosensor transient response. The computational experiments showed a complex dependence of the precision of the concentration estimation on the relative thickness of the outer diffusion layer, as well as on whether the biosensor operates under diffusion- or kinetics-limited conditions. When the biosensor response is affected by the induced exponential trend, the duration of the biosensor action can be optimized for increasing the accuracy of the quantitative analysis.

Modelling carbon nanotubes-based mediatorless biosensor.

This paper presents a mathematical model of carbon nanotubes-based mediatorless biosensor. The developed model is based on nonlinear non-stationary reaction-diffusion equations. The model involves four layers (compartments): a layer of enzyme solution entrapped on a terylene membrane, a layer of the single walled carbon nanotubes deposited on a perforated membrane, and an outer diffusion layer. The biosensor response and sensitivity are investigated by changing the model parameters with a special emphasis on the mediatorless transfer of the electrons in the layer of the enzyme-loaded carbon nanotubes. The numerical simulation at transient and steady state conditions was carried out using the finite difference technique. The mathematical model and the numerical solution were validated by experimental data. The obtained agreement between the simulation results and the experimental data was admissible at different concentrations of the substrate.

Modelling of amperometric biosensor used for synergistic substrates determination.

In this paper the operation of an amperometric biosensor producing a chemically amplified signal is modelled numerically. The chemical amplification is achieved by using synergistic substrates. The model is based on non-stationary reaction-diffusion equations. The model involves three layers (compartments): a layer of enzyme solution entrapped on the electrode surface, a dialysis membrane covering the enzyme layer and an outer diffusion layer which is modelled by the Nernst approach. The equation system is solved numerically by using the finite difference technique. The biosensor response and sensitivity are investigated by altering the model parameters influencing the enzyme kinetics as well as the mass transport by diffusion. The biosensor action was analyzed with a special emphasis to the effect of the chemical amplification. The simulation results qualitatively explain and confirm the experimentally observed effect of the synergistic substrates conversion on the biosensor response.

Probing reactivity of PQQ-dependent carbohydrate dehydrogenases using artificial electron acceptor.

The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were determined using various electron acceptors. The radical cations of organic compounds and 2,6-dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose. The reactivity of dioxygen with ASDH is low; the bimolecular constant k (ox) = 660 M(-1) s(-1), while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of dehydrogenases carbohydrates specificity. Mono- and disaccharide reactivity with GDH is higher than the reactivity of oligosaccharides. For ASDH, the reactivity increased with the carbohydrate monomer number increase. The specificity of quinoproteins was compared with specificity of flavoprotein Microdochium nivale carbohydrate oxidase due to potential enzymes application for lactose oxidation.

Pyrroloquinoline quinone-dependent carbohydrate dehydrogenase: activity enhancement and the role of artificial electron acceptors.

Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH) offers a variety of opportunities for applications, e.g. in highly sensitive biosensors and electrosynthetic reactions. Here we report on the acceleration (up to 4.9 x 10(4)-fold) of enzymatic ferricyanide reduction by artificial redox mediators (enhancers). The reaction mechanism includes reduction of the PQQ-GDH by glucose followed by oxidation of the reduced PQQ cofactor with either ferricyanide or a redox mediator. A synergistic effect occurs through the oxidation of a reduced mediator by ferricyanide. Using kinetic description of the coupled reaction, the second order rate constant for the reaction of an oxidized mediator with the reduced enzyme cofactor (k(ox)) can be calculated. For different mediators this value is 2.2 x 10(6)-1.6 x 10(8) M(-1)s(-1) at pH 7.2 and 25 degrees C. However, no correlation of the rate constant with the midpoint redox potential of the mediator could be established. For low-potential mediators the synergistic effect is proportional to the ratio of k(ox(med))/k(ox(ferricyanide)), whereas for the high-potential mediators the effect depends on both this ratio and the concentration of the oxidized mediator, which can be calculated from the Nernst equation. The described effect can be applied in various ways, e.g. for substrate reactivity determination, electrosynthetic PQQ cofactor regeneration or building of new highly sensitive biosensors.

Intensification of biocatalytical processes by synergistic substrate conversion. Fungal peroxidase catalyzed N-hydroxy derivative oxidation in presence of 10-propyl sulfonic acid phenoxazine.

Many industrial pollutants, xenobiotics, and industry-important compounds are known to be oxidized by peroxidases. It has been shown that highly efficient peroxidase substrates are able to enhance the oxidation of low reactive substrate by acting as mediators. To explore this effect, the oxidation of two N-hydroxy derivatives, i.e., N-hydroxy-N-phenyl-acetamide (HPA) and N-hydroxy-N-phenyl-carbamic acid methyl ester (HPCM) catalyzed by recombinant Coprinus cinereus (rCiP) peroxidase has been studied in presence of efficient substrate 3-(4a,10a-dihydro- phenoxazin-10-yl)-propane-1-sulfonic acid (PPSA) at pH 8.5. The bimolecular constant of PPSA cation radical reaction with HPA was estimated to be (2.5 +/- 0.2).10(7) M(-1) s(-1) and for HPCM was even higher. The kinetic measurements show that rCiP-catalyzed oxidation of HPA and HPCM can increase up to 33,000 times and 5,500 times in the presence of equivalent concentration of high reactive substrate PPSA. The mathematical model of synergistic rCiP-catalyzed HPA-PPSA and HPCM-PPSA oxidation was proposed. Experimentally obtained rate constants were in good agreement with those calculated from the model confirming the synergistic scheme of the substrate oxidation. In order to explain the different reactivity of substrates, the docking of substrates in the active site of the enzyme was calculated. Molecular dynamic calculations show that the enzyme-substrate complexes are structurally stable. The high reactive PPSA exhibited higher affinity to enzyme active site than HPA and HPCM. Furthermore, the orientation of HPA and HPCM was not favorable for proton transfer to the distal histidine, and different substrate reactivity was explained by these diversities.

Modelling Amperometric Biosensors Based on Chemically Modified Electrodes.

The response of an amperometric biosensor based on a chemically modified electrode was modelled numerically. A mathematical model of the biosensor is based on a system of non-linear reaction-diffusion equations. The modelling biosensor comprises two compartments: an enzyme layer and an outer diffusion layer. In order to define the main governing parameters the corresponding dimensionless mathematical model was derived. The digital simulation was carried out using the finite difference technique. The adequacy of the model was evaluated using analytical solutions known for very specific cases of the model parameters. By changing model parameters the output results were numerically analyzed at transition and steady state conditions. The influence of the substrate and mediator concentrations as well as of the thicknesses of the enzyme and diffusion layers on the biosensor response was investigated. Calculations showed complex kinetics of the biosensor response, especially when the biosensor acts under a mixed limitation of the diffusion and the enzyme interaction with the substrate.

Modelling a Peroxidase-based Optical Biosensor.

The response of a peroxidase-based optical biosensor was modelled digitally.A mathematical model of the optical biosensor is based on a system of non-linear reaction-diffusion equations. The modelling biosensor comprises two compartments, an enzyme layerand an outer diffusion layer. The digital simulation was carried out using finite differencetechnique. The influence of the substrate concentration as well as of the thickness of both theenzyme and diffusion layers on the biosensor response was investigated. Calculations showedcomplex kinetics of the biosensor response, especially at low concentrations of the peroxidaseand of the hydrogen peroxide.

Antioxidants determination with laccase.

The spectrophotometric method of antioxidants determination using recombinant laccase Polyporus pinsitus (rPpL) and Myceliophthora thermophila (rMtL) was developed. The method includes simultaneous oxidation of the antioxidant and high reactive laccase substrate producing chromophoric radical cation. As laccase substrates ABTS and other high reactive phenoxazine derivatives: 2-phenoxazin-10-yl-ethanol (PET), 3-phenoxazin-10-yl-propane-1-sulfonic acid (PPSA) and 3-phenoxazin-10-yl-propionic acid (PPA) were used. The kinetic data were analysed using a scheme of simultaneous oxidation of the antioxidant and the substrate. In a range of (0.9-7.3)x10(-6)M of Trolox the measurings recovered 91 and 99% of the antioxidant if ABTS and both laccases were used. The recovery varied between 82 and 124% if phenoxazine derivatives were used. The antioxidant activity determined in rich with antioxidants food samples, i.e. date-palm, black raisin, golden raisin, skin of red grape, dice of red grape, fitted the literature data.

Laccase-catalysed iodide oxidation in presence of methyl syringate.

The kinetics of potassium triiodide (KI(3)) formation during fungal laccase action was investigated in presence of methyl syringate (MS). The recombinant forms of Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL), and Rhizoctonia solani (rRsL) laccases were used. The triiodide formation rate reached 6.1, 5.5, 6.0, and 2.1 microM/min at saturated rPpL, rCcL, rRsL, and rMtL concentration, respectively, in acetate buffer solution pH 5.5 and in presence of 10 microM of MS and 1 mM of potassium iodide. The triiodide formation rate increased if pH decreased from 6.5 to 4.5. The scheme of laccase-catalysed iodide oxidation includes stadium of MS interaction with oxidized laccase with concomitant production of MS(ox). The reaction of MS(ox) with iodide produced triiodide. The turnover number of MS was 93 and 44 at pH 5.5 for rPpL and rMtL, respectively. The scheme also contained a stadium of reversible reduction of laccase active centre with the mediator explaining the different saturation rate of triiodide production. The fitting kinetic data revealed that the reversibility of the reaction increased for laccases containing lower redox potential of copper type I.

Synergistic substrates determination with biosensors.

High sensitive biosensors for heterocyclic compounds determination were built using oxidases-catalyzed hexacyanoferrate(III) reduction in the presence of these compounds. As oxidases Aspergillus niger glucose oxidase and recombinant Microdochium nivale carbohydrate oxidase were used. The biosensors were build using graphite electrodes and entrapped solution of the oxidases. The sensitivity of the biosensors achieves 5.2-14.5 microA microM-1 cm-2. The detection limit of some heterocyclic compounds was 0.2 microM. The sensitivity of biosensors was 300-10,000 times larger in comparison to hexacyanoferrate(III). To background the scheme of biosensors action kinetics of synergistic substrates oxidation was investigated in homogenous solution. The measurements showed that the rate of the reduction of low reactive substrate (hexacyanoferrate(III)) increased due to synergistic action of high reactive substrates (oxidized heterocyclic compounds). The modeling revealed the limiting step of the process. The increase of hexacyanoferrate(III) reduction rate is determined by the rate of reduced enzymes interaction with oxidized heterocyclic compound. The oxidation of heterocyclic compounds (mediators) with hexacyanoferrate(III) does not limit the process. The analysis of macrokinetics of biosensors action showed that synergistic effect may be realized and high biosensors sensitivity may be achieved if diffusion module of the enzyme reaction with the oxidized mediator and of a cross reaction is larger than 0.5. The calculated relative sensitivity is about three times higher in comparison to experimentally determined that may be caused by the limited stability of oxidized heterocyclic compounds and/or some external diffusion limitation of substrates.

Modelling amperometric enzyme electrode with substrate cyclic conversion.

A mathematical model of amperometric enzyme electrodes in which chemical amplification by cyclic substrate conversion takes place in a single enzyme membrane has been developed. The model is based on non-stationary diffusion equations containing a non-linear term related to Michaelis-Menten kinetic of the enzymatic reaction. The digital simulation was carried out using the finite difference technique. The influence of the substrate concentration, the maximal enzymatic rate as well as the membrane thickness on the biosensor response was investigated. The numerical experiments demonstrate significant (up to dozens of times) gain in biosensor sensitivity at low concentrations of substrate when the biosensor response is under diffusion control.

Amperometric biosensors based on recombinant laccases for phenols determination.

Graphite (GE) or printed graphite electrode (PGE) based biosensors containing recombinant fungal laccase Polyporus pinsitus (rPpL), and Myceliophthora thermophila (rMtL) were developed. The enzymes were immobilized using bovine serum albumin and glutaraldehyde. At pH 5.5 and -0.1 V, the calibration graphs of GE based biosensors were hyperbolic if pyrocatechol was used. The concentration of substrate that results in 50% of steady-state response (EC(50)) was 0.7 mM and sensitivity (S) was 3.8 mA/M. The sensitivity increased up to 4 A/M if larger amount of rPpL was used. The sensitivity of biosensors changed little during 9 days of exploitation, but decreased at longer time. The PGE based biosensors were mounted into the flow-through cell and calibrated under kinetic regime. EC(50) of the biosensors containing rPpL varied from 0.6 to 4.0 mM and sensitivity varied from 0.11 to 1.9 mA/M. The response of biosensor containing thermostable laccase rMtL was less, but response saturated at larger pyrocatechol concentration. The sensitivity changed little during 6 days. Both type of biosensors responded also to 1-naphthol, o-phenylenediamine, guaiacol, o-anizidine, benzidine. The experiments demonstrate recombinant laccases application to biosensor engineering and their use to phenol and related compound determination under steady-state and flow-through regimes.