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n-Alkanethiols (CH3(CH2)nSH, n=3, 5, 11, 13, 15, 17) have been adsorbed at mercury electrodes prom ethanolic solutions. A. c. voltammetry has been carried out of the adsorbed alkanethiol on mercury electrodes in aqueous solutions containing 1M KCl at 25°C. Cyclic d.c. voltammetry of several metal ions also has been performed at these electrodes. The results show that long-chain alka-nethiols n_??_11 can form densely packed monolayers with fully extended alkyl chains in the potential range between 0 and -0.7 volt vs. Ag/AgCl. Metal ions such as Pb2+ are unable to penetrate into the monolayer. In contrast, short-chain alkanethiols with n=3 and 5 form less ordered and loosely packed adsorption layers, which are permeable to the metal ions. A pH dependent a.c. voltammetric peak is observed at a mercury electrode with adsorbed alkanethiol(n=5) in buffer solutions, which is attributable to the redox reaction of the adsorbed thiol: [CH3(CH2)5S]adHg+H++e-_??_[CH3(CH2)SH] adHg.

An enzyme-modified electrode is constructed by immobilizing an oxidoreductase behind a dialysis membrane on a carbon paste electrode containing a redox compound. The redox compound functions as an electron transfer mediator between the electrode and the immobilized enzyme, thus the electrode can oxidize or reduce the substrate electro-enzymatically. Many oxidoreductases, especially dehydrogenases which use artificial electron acceptors are the enzymes suitable for constructing the enzyme-modified electrodes. They can be used as mediated amperometric biosensors which are insensitive to oxygen tension in test solutions. Examples of the mediated enzyme electrodes are given and characteristics of the current response of the electrodes to the substrates are described. Use of the mediated enzyme electrodes with high sensitivity are also described. Enzyme-modified electrodes based on oxidoreductases from bacterial membranes can oxidize the substrates electro-enzymatically in the absence of mediators. Such electrodes can be used as mediatorless enzyme electrodes.

Diaphorase (DI)-catalyzed electrolytic oxidation of NADH to NAD + is studied in the presence of flavins and quinones as electron transfer mediators. The behavior of the catalytic current at the mediate DI-electrode is well explained by theoretical equations based on polarographic catalytic current theory using the reaction layer approximation. The bimolecular reaction rate constants between DI and the mediators are determined by the electrochemical method

Fructose dehydrogenase (EC 1.1.99.11) from bacterial membranes was immobilized on a carbon paste electrode by covering the enzyme layer with a dialysis membrane. The fructose dehydrogenase-modified carbon paste electrode showed a current response to d -fructose without the addition of any external electron transfer mediators. The current response was independent of the oxygen concentration in the solution. Steady-state currents were obtained when measured at fixed electrode potentials. The dependence of the steady-state current on the potential, the pH of the solution and the temperature was studied. On the basis of this investigation, it was shown that the fructose dehydrogenase-modified carbon paste electrode could be used as an unmediated amperometric fructose sensor. d -Fructose in fruits was measured using the present electrode. A method of eliminating the effect of l -ascorbic acid is also described.

Bilirubin oxidase was stored in the solutions containing polyammonium salts for a given time at 30 degrees C, and the activity was assayed. The enzyme catalyzes the reaction: 4[Fe(CN)6]4- + 4H+ + O2 --4[Fe(CN)6]3- + 2H2O, and the activity can be measured by the absorbance at the wavelength for the absorption maxima of [Fe(CN)6)]3-. The results show that polyammonium cations comprising quaternary ammonium in the main chain and hydrophilic groups, such as hydroxyl and amide groups, stabilize the enzyme in solution. These polyammonium cations may act like a protective colloid. The membrane-covered electrode containing the polyammonium cations, the enzyme, and [Fe(CN)6]4-/3- in the internal solution phase was constructed. The electrode gave a well-defined current-potential curve with a steady state limiting current due to the polycation-[Fe(CN)6]4-/3- complex-mediated bioelectrocatalytic current for the reduction of O2. The time-dependent decrease of the limiting current indicates again the stabilizing effect of the polyammonium cations on the enzyme.

The thermal stability of a redox enzyme, bilirubin oxidase (BOD), has been quantitatively evaluated by measuring the inactivation kinetics of BOD at several temperatures. The enzyme activity is directly related to the mediated bioelectrocatalytic current for the BOD-catalyzed reduction of O(2). Thus, the inactivation process is measured by the time-dependent decrease in the bioelectrocatalytic current. The results reveal that the inactivation obeys first-order kinetics, whose rate constants (k) are determined at pH 7.0 and at 50 - 70 degrees C. The half life of BOD activity, calculated from the k value at 50 degrees C is 114 min, which is in harmony with the thermal-stability data given in a catalog by Amano Enzyme Inc. The bioelectrocatalysis method allows in situ measurements of the inactivation kinetics in the period of a few minutes at relatively high temperatures. The rate constants show a large temperature dependence, leading to a large Arrhenius activation energy (E(A)) of 221 kJ mol(-1). The activation Gibbs energy (DeltaG(not equal)), activation enthalpy (DeltaH(not equal)), and activation entropy (DeltaS(not equal)) are also determined.

The voltammetric properties of the coenzyme pyrroloquinoline quinone (PQQox) have been investigated in the pH range from 0 to 8. PQQox gives a quasi-reversible cyclic voltammogram at a platinum electrode, which is ascribed to a two-electron redox reaction of PQQox to pyrroloquinoline quinol (PQQred). The standard redox potential (Eo′) of the PQQox/PQQred couple has been determined using cyclic voltammetry. From the analysis of the Eo′ vs. pH relationship, the four macroscopic acid dissociation constants (Ka) of both PQQox and PQQred have been determined. The pKas have been assigned to the pyridyl nitrogen and three carboxyl groups. The Eo′ of the PQQox/PQQred couple at pH 7.0 has been determined as −0.175 V vs. SCE (a saturated calomel electrode) with a semiquinone (PQQsem) formation constant of 0.02, where PQQox, PQQred, and PQQsem are present as trianions with the three carboxylate anion substituents. The pH dependence of the absorption spectra of PQQox, has been interpreted in terms of the pKa values.

The voltammetric and spectroscopic properties of pyrroloquinoline quinone (PQQ) have been investigated in the pH range from 5 to 13.5. PQQ gives a reversible cyclic voltammogram at the mercury electrode, which is ascribed to a two-step one-electron redox reaction of the o-quinone moiety. Digital simulation analysis of the reversible voltammogram made it possible to evaluate the standard redox potentials of the PQQ/pyrroloquinoline semiquinone (PQQsem) and PQQsem/pyrroloquinoline quinol (PQQred) couples. From the redox potential vs. pH relationships, the acid dissociation constants of PQQ, PQQsem, and PQQred have also been determined. The absorption and electron spin resonance spectroscopic properties are explained by the acid—base and redox equilibria proposed here.

d-Fructose dehydrogenase was immobilized behind a dialysis membrane on a carbon paste electrode containing p-benzoquinone. The electrode showed a current response to d-fructose due to the p-benzoquinone-mediated bioelectrocatalytic oxidation of d-fructose. The dependence of the current response on the potential applied to the electrode, pH of the solution, and temperature was studied. On this basis, measurements of d-fructose were done at 0.5V vs. Ag/AgCl, pH 4.5 and 25°C using the fructose dehydrogenase-modified carbon paste electrode. d-Fructose in fruits was measured by this electrode method, in which the interference from l-ascorbic acid was taken into account. A method of elimination of the interference, which uses ascorbate oxidase, is also described.

A mediated amperometric enzyme electrode, which was constructed by immobilizing oligosacharide dehydrogenase behind a dialysis membrane on the surface of a carbon paste electrode containing p -benzoquinone, showed a current response to d -xylose, d -galactose, d -mannose, lactose, maltose, maltotriose, maltopentaose and maltohexaose. The sensitivity of the current response to these carbohydrates was dependent on the kinetics of the immobilized enzyme reaction and/or the permeation rate of the substrate through the dialysis membrane. Hence the sensitivity could be varied by controlling the amount of the immobilized enzyme and the thickness of the dialysis membrane. The time dependence of the current response ofthe enzyme electrode with a large amount of the immobilized enzyme and a thicker dialysis membrane could be explained by an equation describing diffusion of the substrate in the membrane. The enzyme electrode was used to measure lactose in milk and to assay α-amylase in standard serum.

This paper describes the construction and characterization of a batch-type coulometric system for the detection of D-glucose using a novel FAD-dependent glucose dehydrogenase. In order to overcome the problem of interferents, such as ascorbate and urate, a potential-step method was proposed to separate the electrolysis reactions of interferents and D-glucose by selecting a mediator possessing an appropriate formal potential. The rapid oxidative consumption of the interferents proceeded in the first step, whereas the mediator and glucose remained reduced. In the second step, the mediator was immediately oxidized, and subsequent bioelectrocatalytic oxidation of D-glucose occurred with the aid of aldose 1-epimerase. In this study, potassium octacyanomolybdate (IV) with a formal potential of 0.6 V vs. Ag|AgCl was chosen as a mediator, and the first and second electrolysis potentials were set at 0.4 V and 0.8 V, respectively, by considering the heterogeneous electron-transfer kinetics and the potential window. The background-corrected response in charge corresponded to 99+/-2 % efficiency in terms of the amount of D-glucose.

Bioelectrocatalytic oxidation of acetate was investigated under anaerobic conditions by using Escherichia coli K-12 (IFO 3301) cells cultured on aerobic media containing poly-peptone, glucose or acetate as the sole carbon source. It was found that all E. coli cells cultured on the three media work as good catalysts of the electrochemical oxidation of acetate as well as glucose with Fe(CN)6(3-), 2,3-dimethoxy-5-methyl-1,4-benzo-quinone (Q0), 2,6-dichloro-indophenol, or 2-methyl-1,4-naphthoquinone as artificial electron acceptors (mediators). Acetate-grown E. coli cells exhibited the highest relative activity of the acetate oxidation against the glucose oxidation. On the other hand, all the artificial electron acceptors used work as inhibitors for the catalytic oxidation of acetate at increased concentrations. The inhibition phenomenon can be interpreted in terms of competitive substrate inhibition as a whole. Apparent values of Michaelis constant, catalytic constant, and inhibition constant were evaluated by amperometric methods. Q0 is an effective artificial mediator as evidenced by a large reaction rate constant between the cell and Q0 at least at low concentrations (50 microM). However, Fe(CN)6(3-) is a promising mediator in biosensor applications because the inhibition constant is very large and it works as an electron acceptor even under aerobic conditions.

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.

The 109 kDa quinohemoprotein amine dehydrogenase (QHNDH) from Paracoccus denitrificans contains a novel redox cofactor, cysteine tryptophylquinone (CTQ). This cofactor is derived from a pair of gene-encoded amino acids by post-translational modification and was previously identified and characterized within an 82-residue subunit by chemical methods and crystallographic analysis at 2.05 A resolution. It contains an orthoquinone moiety bound to the indole ring and catalyzes the oxidation of aliphatic and aromatic amines through formation of a Schiff-base intermediate involving one of the quinone O atoms. This paper reports the structural analysis of the complex of QHNDH with the enzyme inhibitor phenylhydrazine determined at 1.70 A resolution. The phenylhydrazone product is attached to the C6 position, identifying the O6 atom of CTQ as the site of Schiff-base formation as postulated by analogy to another amine-oxidizing enzyme, methylamine dehydrogenase. Furthermore, the inner N atom closest to the phenyl ring of phenylhydrazine forms a hydrogen bond to gammaAsp33 in the complex, lending support to the hypothesis that this residue serves as the active-site base for proton abstraction during catalysis.

Paracoccus denitrificans produces two primary enzymes for the amine oxidation, tryptophan-tryptophylquinone (TTQ)-containing methylamine dehydrogenase (MADH) and quinohemoprotein amine dehydrogenase (QH-AmDH). QH-AmDH has a novel cofactor, cysteine tryptophylquinone (CTQ) and two hemes c. In this work, the redox potentials of three redox centers in QH-AmDH were determined by a mediator-assisted continuous-flow column electrolytic spectroelectrochemical technique. Kinetics of the electron transfer from QH-AmDH to three kinds of metalloproteins, amicyanin, cytochrome c550, and horse heart cytochrome c were examined on the basis of the theory of mediated-bioelectrocatalysis. All these metalloproteins work as a good electron acceptor of QH-AmDH and donate the electron to the terminal oxidase of P. denitrificans, which was revealed by reconstitution of the respiratory chain. These properties are in marked contrast with those of MADH, which shows high specificity to amicyanin. These electron transfer kinetics are discussed in terms of thermodynamics and structural property.

Electrochemical studies on the applied aspects of quinoproteins are briefly reviewed. Catalytic reactions of quinoprotein enzymes can be connected to electrochemical reactions directly or by the mediation of molecules functioning as electron acceptors of the enzymes. Such an enzyme-electrochemical reaction is called bioelectrocatalysis. It provides a novel method of kinetic analysis of enzyme catalysis and even whole bacterial cell catalysis. The principle of bioelectrocatalysis is first described, then, the bioelectrocatalysis-based application of quinoproteins in biosensors is mentioned. Characteristics and performance of this type of biosensor is explained by citing our own work. Possible application in bioreactors and biofuel cells is also mentioned.

Bilirubin dehydrogenase, a membrane-bound enzyme that catalyzes the one-step oxidation of ditaurobilirubin and bilirubin to ditaurobiliverdin and biliverdin, respectively, in the presence of an electron acceptor, was found in Aspergillus ochraceus IB-3, and purified from the membrane fraction through solubilization by Triton X-100. Phenazine and quinone derivatives acted as electron acceptors. Accumulation of ditaurobiliverdin and biliverdin by enzyme catalysis increased the absorbance at 660 nm, which is far from the range of wavelengths affected by serum ingredients. The enzyme selectively oxidized ditaurobilirubin at low pH, so changes in the reaction pH enable the enzyme to discriminate between the bilirubin fractions ditaurobilirubin (an example of conjugated bilirubin) and bilirubin (an example of unconjugated bilirubin). Using the enzyme, 2 to 80 microM of ditaurobilirubin were measured accurately by monitoring the changes in absorbance at 660 nm.

Sodium nitrite in acidic solutions produces a voltammetric cathodic wave starting from −0.5 V (vs. Ag/AgCl) in the presence of a small amount of cytochrome c (cyt c), in which cyt c works as a catalyst for the electroreduction of nitric oxide produced by the disproportionation reaction of nitrite. Hemoglobin (Hb) and myoglobin (Mb) are also effective as catalysts for this type of reaction.

Glucose metabolism of bifidobacteria in the presence of 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), a specific growth stimulator for bifidobacteria, and ferricyanide (Fe(CN)(6)(3-)) as an extracellular electron acceptor was examined using resting cells of Bifidobacterium longum and Bifidobacterium breve. NAD(P)H in the cells is oxidized by ACNQ with the aid of diaphorase activity, and reduced ACNQ donates the electron to Fe(CN)(6)(3-). Exogenous oxidation of NADH by the ACNQ/Fe(CN)(6)(3-) system suppresses the endogenous lactate dehydrogenase reaction competitively, which results in the remarkable generation of pyruvate and a decrease in lactate production. In addition, a decrease in acetate generation is also observed in the presence of ACNQ and Fe(CN)(6)(3-). This phenomenon could not be explained in terms of the fructose-6-phosphate phosphoketolase pathway, but suggests rather that glucose is partially metabolized via the hexose monophosphate pathway. This was verified by NADP(+)-induced reduction of Fe(CN)(6)(3-) in cell-free extracts in the presence of ACNQ. Effects of the ACNQ/Fe(CN)(6)(3-) system on anaerobically harvested cells were also examined. Stoichiometric analysis of the metabolites from the pyruvate-formate lyase pathway suggests that exogenous oxidation of NADH is an efficient method to produce ATP in this pathway.

Nonenzymatic reduction of dehydroascorbate into ascorbate by the reduced form (quinol form) of 2-amino-3-carboxy-1,4-naphthoquinone, a strong growth stimulator for bifidobacteria, has been found. The bimolecular reaction rate constant was evaluated as 9 M(-)(1) s(-)(1) at pH 7.0. This reaction has been successfully coupled with enzymatic regeneration of the naphthoquinol by NAD(P)H in cell-free extracts of Bifidobacterium longum 6001. The overall reaction is a regeneration of NAD(P)(+) by dehydroascorbate [or a regeneration of ascorbate by NAD(P)H], in which the naphthoquinone/quinol redox couple functions as an electron transfer mediator. Kinetic study of the reduction of dehydroascorbate with related quinol compounds suggested the significance of the amino substituent of the naphthoquinol. A mechanism of the electron transfer from the quinol to dehydroascorbate is proposed, where the first step of the reaction is a nucleophilic addition of the C(2)-amino substituent of the naphthoquinol to the C(2)-position of dehydroascorbate to form a Schiff base intermediate.

The substrate-oxidizing activity of sake spoilage lactic acid bacteria (hiochi bacteria) was measured using a benzoquinone-mixed carbon paste electrode on which the bacteria had been immobilized. The electrode immersed in a buffer began to produce an anodic current following the addition of D-glucose or gluconate, reflecting the substrate-oxidizing activity of the immobilized bacteria. The time course of the first derivative of the current (dI/dt) exhibited a maximum, the magnitude of which increased with an increase in the number of hiochi bacterial cells immobilized on the electrode. When heat-treated cells were immobilized, the value at the maximum (dI/dt(max)) was lower than that obtained with the electrode on which cells which had not been heat-treated had been immobilized. The decrease in dI/dt(max) increased at higher heating temperatures and longer heating time. The relative decrease in dI/dt(max) due to heat treatment was smaller than the relative decrease in the number of colony formation due to the same treatment, suggesting that cells injured by heat treatment still retain substrate-oxidizing activity.

A new spectroelectrochemical method based on flow column electrolysis has proved to be useful for the determination of the redox potentials of (1) alcohol dehydrogenase formGluconobacter suboxydans(2) soluble glucose dehydrogenase fromAcinetobacter calcoaceticus(3) methylamine dehydrogenase fromParacoccus denitrificansand (4) a new quinohemoprotein amine dehydrogenase fromParacoccus denitrificans.They are (1) 401 (heme 1), 370 (heme 2), 216 (heme 3), 101 (heme 4), and —167 (PQQ) mV vs. NHE at pH 7.0, (2) 33 (PQQox/sem) and —12 (PQQsem/red) mV at pH 7.0, (3) 129 (TTQ) mV at pH 7.5, and (4) 192 (heme), 190(Qox/rem)and 100 (Qsem/red) mV at pH 7.0, Q being a quinone of unknown structure.

2-Amino-3-carboxy-1,4-naphthoquinone (ACNQ) is a novel growth stimulator for bifidobacteria. The role of ACNQ as a mediator of the electron transfer from NAD(P)H to dioxygen (O(2)) and hydrogen peroxide (H(2)O(2)), proposed in our previous paper, was examined using the cell-free extract and whole cells of Bifidobacterium longum. Continuous monitoring of ACNQ, O(2) and H(2)O(2) by several amperometric techniques has revealed that ACNQ works as a good electron acceptor of NAD(P)H diaphorase and that the reduced form of ACNQ is easily autoxidized and also acts as a better electron donor of NAD(P)H peroxidase than NAD(P)H. The generation of H(2)O(2) by B. longum under aerobic conditions is effectively suppressed in the presence of ACNQ. These ACNQ-mediated reactions would play roles as NAD(P)(+)-regeneration processes. The accumulation of ACNQ in the cytosol has been also suggested. These characteristics of ACNQ seem to be responsible for the growth stimulation of bifidobacteria. Vitamin K(3), which has an extremely low growth-stimulating activity and was used as a reference compound, exhibits much lower activity as an electron transfer mediator. The difference in the activity is discussed in terms of the redox potential and partition property of the quinones.

An electrode modified with immobilized whole cells of Desulfovibrio vulgaris (Hildenborough) produces an S-shaped voltammogram with both cathodic- and anodic-catalytic-limiting currents in a methyl viologen-containing buffer saturated with H2. Methyl viologen penetrates into the bacterial cells to serve as an electron carrier in the reversible reaction of hydrogenase in the cells and functions as an electron-transfer mediator between the bacterial cells and the electrode, thus producing the catalytic currents for the evolution and consumption of H2. An equation for the catalytic current that takes into account the reversible hydrogenase reaction explains well the shape of the voltammogram. The potential at null current on the voltammogram agrees with the potential determined by potentiometry with the same electrode, which is equal to the redox potential of the H+/H2 couple in the solution--the standard potential of a hydrogen electrode at the pH of the solution. When D. vulgaris cells are suspended in an argon-saturated buffer containing methyl viologen, the suspension produces a catalytic current at a bare glassy carbon electrode for the evolution of H2. Analysis of the current by a theory for a catalytic current for a unidirectional nonlinear enzyme catalysis allows us to determine the kinetic parameters of the reaction between methyl viologen and hydrogenase in intact D. vulgaris cells. Thus we obtain the apparent Michaelis constant for methyl viologen cation radical, K'MV.+ = 0.16 mM, and the apparent catalytic constant (that is, the turnover number per D. vulgaris cell), zkcat,H+ = 1.2 x 10(7) s-1, for the H2 evolution reaction at pH 5.5 and at 25 degrees C, z being the number of hydrogenases contained in a D. vulgaris cell. The bimolecular reaction rate constant, kcat,H+/K'MV.+, of the reaction between methyl viologen cation radical and oxidized hydrogenase in intact D. vulgaris cells is estimated as 4.2 x 10(7) M-1 s-1. Similarly, the bimolecular reaction rate constant, kcat,H2/K'MV2+, of the reaction between methyl viologen and reduced hydrogenase is estimated to be 1.2 x 10(7) M-1 s-1 at pH 9.5 and 25 degrees C. Both rate constants are large enough for the reactions to be diffusion-limited processes.

Capillary zone electrophoresis (CZE) was applied to the electrophoretic characterization for microorganisms. The electrophoretic peaks detected using light scattering phenomena were characteristic of the microorganisms used. The electrophoretic mobility (mu) evaluated by CZE was in good agreement with that obtained by classical electrophoresis of microorganisms. The migration time was reproducible and depended on the ionic strength (I). Analysis of the mu vs. I relationship provided information regarding the charge density and the hardness of the microbial cell surface. The redox enzymatic activity of microorganisms was also evaluated by CZE using a running buffer containing a corresponding substrate and an appropriate exogenous electron acceptor. A decrease in the concentration of the electron acceptor due to microbial activity can be simultaneously monitored during the electrophoretic process without significant modification of the CZE instrument. Effects of some chemical treatments of microbial cells were also studied using this technique.

PQQ-dependent methanol dehydrogenase (EC 1.1.99.8) was immobilized onto the surface of a carbon paste electrode by the cross-linking method and the electrode was covered with a porous polycarbonate membrane. An electrocatalytic steady-state current for the oxidation of methanol was observed using this electrode in the presence of phenazine methosulphate as an electron transfer mediator and NH4Cl as an activator for enzyme activity. The electrocatalytic current at +0.2 V vs. Ag/AgCl was analyzed. The properties of the electrode were examined for the determination of ammonia. The electrode responded linearly to ammonia in the range of 3 to 60 mM. The assay took 40 to 50 s. The electrode was used repeatedly at room temperature for 15 days and retained 50% of its initial activity.

Diaphorase and 2-amino-3-carboxy-1,4-naphthoquinone as an enzyme and a mediator for NADH oxidation, respectively, were successfully co-immobilized on a glassy carbon electrode with poly-L-lysine and glutaraldehyde as a polymer support and a cross-linking reagent, respectively. The diffusion-controlled oxidation of NADH (10 mM) was achieved around −0.25 V vs Ag|AgCl|KCl(sat.).

An enzyme-modified electrode was prepared producing a diffusion-limited bioelectrocatalytic current for the reduction of O2 to water at neutral pH and at ambient temperature. The electrode used bilirubin oxidase as an enzyme and [Fe(CN)6]3−/4− as a mediator, both of which were immobilized on the surface of a glassy carbon electrode by electrostatic entrapment with poly-L-lysine.

A new redox polymer containing Os (E °′=−0.15 V vs Ag|AgCl) was designed and synthesized as an efficient mediator of diaphorase-catalyzed electrochemical oxidation of NADH and as a support to immobilize enzyme(s) on electrode surfaces. The electrochemical characteristics of the polymer and its application to biosensors and bioanodes of biofuel cells are briefed.

Electrochemical characterization of topa quinone (6-hydroxydopa quinone), the organic cofactor of copper-containing amine oxidases, has been performed with the aid of spectroscopy and ab initio energy minimization technique. Topa quinone exhibits a totally reversible cyclic voltammogram at a mercury electrode, which is ascribed to a two-step one-electron conversion between topa quinone and topa via topa semiquinone intermediate. Digital simulation of the reversible wave has afforded the separated estimation of each one-electron redox potential. The acid-dissociation constants of the phenolic hydroxyl groups of topa quinone, topa semiquinone and topa have been evaluated electrochemically and supported by electronic and electron spin resonance spectra. At pH 7.0, topa quinone is acid-dissociated and has two-electron redox potential of 0.079 V vs. NHE coupled with a three-proton transfer. Redox catalytic activity of topa quinone for the oxidation of amines and NADH was not observed over conventional voltammetric time periods. Energy minimization calculation of acid-dissociated topa quinone anion indicates an intermediate electronic structure between the p - and o -quinone types with three almost equivalent carbonyl groups. The lack of the redox catalytic activity of free topa quinone appears to be attributable to the partial contribution of the p -quinone-type structure.

Nicotinic acid (NA) is efficiently hydroxylated into 6-hydroxynicotinic acid (6HNA) by Pseudomonas fluorescens TN5-immobilized column electrolytic method using K3Fe(CN)6 as an extracellular electron transfer mediator, in which the conversion rate was sufficiently accelerated compared with the aerobic oxidation. The NA conversion system was applied to the continuous-production of 6HNA in 100% yield and the absolute determination of NA in flow-injection analysis.

Direct electron transfer of alcohol dehydrogenase (ADH) from Gluconobacter suboxydans, which contains one pyrroloquinoline quinone (PQQ) and four hemes c, are realized on a carbon-wool column elect...

A carbon electrode modified with immobilized whole cells of Desulfovibrio vulgaris was found to be able to electrochemically drive the reaction: H2↔2H++2e− very effectively in both forward and reverse directions by the use of methyl viologen and vitamin K3 as a mediator, respectively. Similarly, the reduction of dioxygen was electrochemically driven by the use of Thiobacillus ferrooxidans as a biocatalyst and hexacyanoferrate (II) as a mediator.

A carbon electrode modified with immobilized cells of Paracoccus denitrificans has been found to produce catalytic currents for the reduction of nitrate and nitrite in the presence of durohydroquinone. It has been shown that durohydroquinone functions as an electron transfer mediator between the electrode and the bacterial cells to drive electrochemically the denitrifying reaction of Paracoccus denitrificans.

Approximate and more precise empirical equations have been derived for the steady state limiting currents of bioelectrocatalysis based on homogeneous enzyme and mediator at sufficiently high concentrations of the substrate. These equations allow us to evaluate the catalytic constant and the Michaelis constant of the mediator.

FMN(H2) has been found to work as a two-way mediator of diaphorase to reduce NAD+ as well as to oxidize NADH. The FMN/diaphorase/NAD system has been successfully combined with lactate dehydrogenase...

Adriamycin, anthracycline antibiotics, has been found to work as an effective mediator in bioelectrochemical reduction of NAD(P)H catalyzed by ferredoxin-NADP+ reductase or diaphorase. The regeneration system has been satisfactory combined with NAD(P)H-dependent enzymatic reactions.