Diaphorase Research Articles

Photoswitchable Enzymatic Biofuel Cell Based on Fusion Protein with Natural Photoreceptor Vivid.

Enzymatic biofuel cells (EBFCs) have increasingly been the subject of research, but the control of the EBFC output remains difficult. In this study, we fuse glucose 6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) with the natural photoreceptor Vivid named "Mag". The output current and power density of EBFCs with the fusion protein exhibit a sensitive and efficient response to blue light. Following optimizations, the power density increases nearly 4-fold from 1.32 to 6.26 μW cm-2, whereas the current rises from 5.9 to 10.8 μA after 20 min of illumination, dropping back within 30 min under dark conditions.

Biphasic Bioelectrocatalytic Synthesis of Chiral β-Hydroxy Nitriles

Two obstacles limit the application of oxidoreductase-based asymmetric synthesis. One is the consumption of high stoichiometric amounts of reduced equivalent. The other is the low solubility of organic substrates, intermediates, and products in the aqueous phase. In order to address the two obstacles of oxidoreductase-based asymmetric synthesis, a biphasic bioelectrocatalytic system was constructed and applied.. In this study, the preparation of chiral β-hydroxy nitriles catalyzed by alcohol dehydrogenase (AdhS) and halohydrin dehalogenase (HHDH) was investigated as a high-value organic product. Diaphorase (DH) was immobilized by cobaltocene-modified poly(allylamine) redox polymer on the electrode surface (DH/Cc-PAA bioelectrode) to realize the effective bioelectrocatalytic NADH regeneration. AdhS is a NAD-dependent dehydrogenase, so the diaphorase modified biocathode was used to regenerate NADH to support the conversion from ethyl 4-chloroacetoacetate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) catalyzed by AdhS. The addition of methyl tert-butyl ether (MTBE) as organic phase not only increased the uploading of COBE, but also prevented the spontaneous hydrolysis of COBE and extended the lifetime of DH/Cc-PAA bioelectrode, and finally increased the Faradaic efficiency and the concentration of generated (R)-ethyl-4-cyano-3-hydroxybutyrate ((R)-CHCN). After 10 hours of reaction, the highest concentration of (R)-CHCN in biphasic bioelectrocatalytic system achieve 25.5 mM with 81.2% enantiomeric excess (ee p). The conversion ratio of COBE achieved 85%, which was 8.8 times higher than that of single phase system. Besides COBE, the other two substrates with aromatic ring structures were also used in this biphasic bioelectrocatalytic system to prepare corresponding chiral β-hydroxy nitriles. The results indicate that the biphasic bioelectrocatalytic system has the potential to produce a variety of β-hydroxy nitriles with different structures.

Electrochemical detection of interleukin-8 in human saliva using a polyenzyme label based on diaphorase and neutravidin

Polyenzymes have been commonly employed as catalytic labels to obtain high signal amplification in immunoassays, but they are mainly constructed using horseradish peroxidase that requires the use of H2O2, whose storage is difficult. Here, we present a new polyenzyme label based on diaphorase (DI), a dehydrogenase redox enzyme with high catalytic activity and long-term stability. The polyenzyme label is constructed using a complex of a biotinylated DI and an avidin analog. Among the five commercially available DIs, the one that shows the highest signal amplification using electrochemical-enzymatic redox cycling involving an electron mediator, a DI, and NADH is chosen. Among the three avidin analogs (avidin, neutravidin, and streptavidin), neutravidin provides the lowest nonspecific binding of the polyenzyme label. The polyenzyme label shows higher signal amplification and lower nonspecific binding than a single enzyme label of DI. Comparative studies reveal that the detection limit for the detection of interleukin-8 (IL-8) in saliva using the polyenzyme label (∼1 pg/mL) is one order of magnitude lower than that using a single enzyme label (∼10 pg/mL). IL-8 concentrations measured in clinical saliva samples agree well with those measured using a conventional microplate immunoassay. Thus, the polyenzyme label can be applied for the noninvasive detection of other clinically relevant proteins in saliva.

Glucose Sensor Using NAD-Dependent Glucose Dehydrogenase, Diaphorase, and Osmium Complex

It is still challenging to detect glucose selectively in serum and blood, and quickly using nicotinamide adenine dinucleotide (NAD-GDH). In this paper, we report a selective and rapid glucose sensor, based on electrochemical-enzymatic-enzymatic (ENN) redox cycling involving bis(2,2-bipyridyl)dichloroosmium(II)[Os(bpy)2Cl2], diaphorase (DI), NAD+, NAD-GDH, and glucose. DI and Os(bpy)2Cl2 are used to obtain fast mediated oxidation of NADH that is generated as a result of glucose oxidation by NAD-GDH. DI and NAD-GDH are co-immobilized via affinity binding on an avidin-modified indium tin oxide electrode to obtain fast and stable ENN redox cycling. Two enzymes (DI and NAD-GDH) and two electron mediators [Os(bpy)2Cl2 and NAD+] are efficient. The reason is that they are insensitive to oxygen. The applied potential (0.0 V vs Ag/AgCl) is low enough to minimize interfering electrochemical reactions, and the redox reactions of Os(bpy)2Cl2 with interfering species are slow. NAD-GDH is much less reactive to problematic monosaccharides such as xylose, fructose, galactose, and mannose than glucose. Artificial serum containing 5% (w/v) human serum albumin shows a similar electrochemical back-ground level in serum. All results enable us to obtain selective and reproducible glucose detection. The fast ENN redox cycling allows sensitive glucose detection with a wide range of concentrations in artificial serum with a short measuring time (5 s) without an incubation period.Keywords: Glucose sensor, Glucose dehydrogenase, Diaphorase, Osmium complex

Biphasic Bioelectrocatalytic Synthesis of Chiral β-Hydroxy Nitriles.

Two obstacles limit the application of oxidoreductase-based asymmetric synthesis. One is the consumption of high stoichiometric amounts of reduced cofactor. The other is the low solubility of organic substrates, intermediates, and products in the aqueous phase. In order to address these two obstacles to oxidoreductase-based asymmetric synthesis, a biphasic bioelectrocatalytic system was constructed and applied. In this study, the preparation of chiral β-hydroxy nitriles catalyzed by alcohol dehydrogenase (AdhS) and halohydrin dehalogenase (HHDH) was investigated as a model bioelectrosynthesis, since they are high-value intermediates in statin synthesis. Diaphorase (DH) was immobilized by a cobaltocene-modified poly(allylamine) redox polymer on the electrode surface (DH/Cc-PAA bioelectrode) to achieve effective bioelectrocatalytic NADH regeneration. Since AdhS is a NAD-dependent dehydrogenase, the diaphorase-modified biocathode was used to regenerate NADH to support the conversion from ethyl 4-chloroacetoacetate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) catalyzed by AdhS. The addition of methyl tert-butyl ether (MTBE) as an organic phase not only increased the uploading of COBE but also prevented the spontaneous hydrolysis of COBE, extended the lifetime of DH/Cc-PAA bioelectrode, and increased the Faradaic efficiency and the concentration of generated (R)-ethyl-4-cyano-3-hydroxybutyrate ((R)-CHCN). After 10 h of reaction, the highest concentration of (R)-CHCN in the biphasic bioelectrocatalytic system was 25.5 mM with 81.2% enantiomeric excess (eep). The conversion ratio of COBE achieved 85%, which was 8.8 times higher than that achieved with the single-phase system. Besides COBE, two other substrates with aromatic ring structures were also used in this biphasic bioelectrocatalytic system to prepare the corresponding chiral β-hydroxy nitriles. The results indicate that the biphasic bioelectrocatalytic system has the potential to produce a variety of β-hydroxy nitriles with different structures.

Amelioration of cadmium stress in Withania somnifera by ROS management: active participation of primary and secondary metabolism

Cadmium (Cd) is considered as a non-essential heavy metal with substantial toxicity on environment. Withania somnifera, a reputed therapeutic herb exhibits vast pharmacological activities due to the presence of steroidal lactones-withanolides. The present study deals with reactive oxygen species (ROS) management through primary and secondary metabolism as adaptive response, on exogenous Cd exposure of W. somnifera. Increased invertase enzyme activity resulted in higher reducing sugars in plant under Cd stress to provide additional carbon source and NADH or NADPH. Higher activities of proline metabolic pathway enzymes such as ornithine aminotransferase (OAT) and pyrroline-5-corboxylate synthase (P5CS) resulted in elevated proline accumulation. The considerable participation of phenylpropanoid metabolism also found dominantly upregulated. Increased enzyme activities of phenylalanine ammonia lyase (PAL), shikimate dehydrogenase (SKDH), glucose-6-phosphate dehydrogenase (G6PDH), and cinnamyl alcohol dehydrogenase (CADH) resulted in phenolics and flavonoids accumulation under higher Cd stress. Upregulation of glutathione-S-transferase (GST) activity conferred its role in Cd chelation. In addition, glutamate oxaloacetate transaminase (GOT), amyl esterase (EST) and diaphorase (DIA) established their participation in Cd tolerance mechanism. Thus, present study elucidated major role of proline and phenylpropanoid metabolism in providing the osmoticum and antioxidants to the plants under Cd stress. The information may be helpful in developing stress resistant plants by targeting these pathways using conventional and molecular approaches.

Specific and Rapid Glucose Detection Using NAD‐dependent Glucose Dehydrogenase, Diaphorase, and Osmium Complex

AbstractSelective glucose measurement in serum and blood and rapid glucose measurement using nicotinamide adenine dinucleotide (NAD)‐dependent glucose dehydrogenase (NAD‐GDH) are still very challenging. Here, we report a selective and rapid glucose sensor, based on electrochemical‐enzymatic‐enzymatic (ENN) redox cycling involving bis(2,2‐bipyridyl)dichloroosmium(II) [Os(bpy)2Cl2], diaphorase (DI), NAD+, NAD‐GDH, and glucose. DI and Os(bpy)2Cl2 are used to obtain fast mediated oxidation of NADH that is generated as a result of glucose oxidation by NAD‐GDH. DI and NAD‐GDH are co‐immobilized via affinity binding on an avidin‐modified indium tin oxide electrode to obtain fast and stable ENN redox cycling. Two enzymes (DI and NAD‐GDH) and two electron mediators [Os(bpy)2Cl2 and NAD+] are insensitive to oxygen. The applied potential (0.0 V vs Ag/AgCl) is low enough to minimize interfering electrochemical reactions, and the redox reactions of Os(bpy)2Cl2 with interfering species are slow. NAD‐GDH is much less reactive to problematic monosaccharides such as xylose, fructose, galactose, and mannose than glucose. Artificial serum containing 5 % (w/v) human serum albumin shows a similar electrochemical background level in serum. All results enable us to obtain selective and reproducible glucose detection. The fast ENN redox cycling allows sensitive glucose detection with a wide range of concentrations in artificial serum with a short measuring time (5 s) without an incubation period.

A Recombinant 12-His Tagged Pyrococcus furiosus Soluble [NiFe]-Hydrogenase I Overexpressed in Thermococcus kodakarensis KOD1 Facilitates Hydrogen-Powered in vitro NADH Regeneration.

Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H2 ). Here, the authors expressed the four αβγδ subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin. Size-exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half-life of 70 h at 80 °C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H2 gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN-containing diaphorase (DI) is used to match NADP-preferred SHI and NAD-preferred lactate dehydrogenase (LDH). This study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production.

An enzyme-modified capillary as a platform for simultaneous fluorometric detection of d-glucose and l- lactate

The preparation of a glass capillary pattered with lipid layers on which lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH) were regionally adsorbed and its application for simultaneous detection of d-glucose and l-lactate in human serum is described. A lipid layer was formed on the surface of BSA-unabsorbed octadecyltrichlorosilane (OTS) inner wall of a glass capillary. The electrostatic charge of the lipid layer was a key factor for adsorbing the enzymes on the lipid layer. The fluorescence intensities were observed at each enzyme site in the presence of diaphorase (DIA), β-nicotinamide-adenine dinucleotide oxidized (NAD), resazurin, d-glucose and l-lactate. The fluorescence intensities at each enzyme site increased with an increase in the concentration of d-glucose and l-lactate=with the detection limits of 32 μM and 4.9 μM, respectively.

Washing-Free Displacement Immunosensor for Cortisol in Human Serum Containing Numerous Interfering Species.

Simple and sensitive competitive immunosensors for small molecules are difficult to obtain, especially in serum containing numerous interfering species (ISs) with different concentrations. Herein, we report a washing-free and sensitive (competitive) displacement immunosensor for cortisol in human serum, based on electron mediation of Os(bpy)2Cl2 between an electrode and a redox label [oxygen-insensitive diaphorase (DI)] (i.e., electrochemical-enzymatic redox cycling). The anticortisol IgG-DI conjugate bound to a cortisol-immobilized electrode is displaced by competitive binding of cortisol in serum and diffuses away from the electrode during incubation; therefore, the concentration of the displaced conjugate near the electrode becomes very low, even without washing. Electrochemically interfering ascorbic acid is converted to a redox-inactive species by ascorbate oxidase during incubation. The remaining bound conjugate mainly contributes to electrochemical currents. Compared with ferrocene methanol, Fe(CN)64-, and Ru(NH3)63+, the electrochemical and redox cycling behaviors of Os(bpy)2Cl2 are influenced significantly less by ISs in serum. Comparative studies reveal that washing-free displacement assay shows better cortisol-induced signal change than three other assays. The surface concentration of cortisol immobilized on the electrode is optimized, because the electrochemical signal is highly dependent on the surface concentration. When the washing-free displacement immunosensor is applied for the detection of cortisol in artificial serum, cortisol is measured with a detection limit of ∼30 pM within 12 min. The cortisol concentrations measured in clinical serum samples agree well with those obtained using a commercial instrument. The new immunosensor is highly promising for the simple, sensitive, and rapid point-of-care detection of small molecules.

STUDI KERAGAMAN GENETIK Diospyros rumpii Bakh DI SULAWESI UTARA BERDASARKAN PENANDA ISOENZIM

Diospyros rumphii Bakh. is one of tropical commerciall wood species grouped in ebony. Ebony is the name of some species of commercial timber from genus of Diospyros (Ebenaceae). Seven species of them are commercial wood, which in the trade is known as ebony namely; D.celebica, D.rumphii, D.pilosanthera, D.lolin, D.ebenum, D.ferrea and D.macrophylla. Two of seven species ebony have the highest economic values namely; D.celebica and D.rumphii. Nowadays, the population D. rumphii in the nature are decreases due to over harvesting in the past. This study aims to determine genetic diversity of D. rumphii within populations, between populations and its relationship. The samples of D. rumphii in this study from four populations in North Sulawesi i.e Bitung, Talise, Talaud and Bolaang Mongondow. Genetic diversity was detected by isoenzym markers with seven enzyme i.e Glutamate oxaloacetate transmirase (GOT), Shikimate dehydrogenase (SHD), Esterase (EST), Acid phoshatase (ACP), Diaphorase (DIA), 6-Phosphogluconate dehydrogenase (6PG) and Glutamate dehydrogenase (GDH). The results showed that there are three enzym was detected polymorphisme i.e EST, GOT and GDH. Genetic diversity generated by three enzyme systems who are controlled by 5 loci and 15 alleles. The average percentage of polymorphic loci was100%. The number of total genetic diversity is 0.64,which are distributed within population 0.59 (91.24%) and between population 0.05(8.76%). Cluster analysis using Unweighted Pair Group Method with Aritmatic Means (UPGMA) based on Nei standard genetic distance showed three clusters:Bitung and Bolaang Mongondow, Talise, and Talaud.

Robust l-malate bienzymatic biosensor to enable the on-site monitoring of malolactic fermentation of red wines

Monitoring the malolactic fermentation process is strictly required to guarantee the sensorial quality and freshness of red wines. This could be achieved by in-field and real-time continuous measurements of l-malate concentration in the fermentation tanks. The potential of a miniaturized amperometric bienzymatic biosensor as an analytical tool to be applied in such scenario is described in this paper. The biosensor comprises a thin-film gold electrode as transducer, malate dehydrogenase (MDH) and diaphorase (DP) enzymes together with nicotinamide adenine dinucleotide (NAD+) cofactor as the selective receptor and an adequate redox mediator to record the corresponding amperometric signal. Three different biosensor architectures are studied, whose main differences lie in the immobilization of the different chemical components onto the electrode surface. In all cases a fast-electrosynthethized polypyrrole (PPy) membrane is generated for this purpose. The experimental conditions are optimized and the best architecture shows a sensitivity of 1365 ± 110 mA M−1 cm−2 and a detection limit of 6.3 × 10−8 M in a concentration range of 1 × 10−7 M – 1 × 10−6 M. The biosensor presents an excellent working stability as it retains above 90% of its sensitivity after 37 days, thus enabling the monitoring of the malolactic fermentation of three red wines. The obtained results show excellent agreement with the standard colorimetric method.

Cholesterol as a Promising Alternative Energy Source: Bioelectrocatalytic Oxidation Using NAD-Dependent Cholesterol Dehydrogenase in Human Serum

Cholesterol is an essential structural component of mammalian cells, although elevated concentrations of cholesterol in the human body are linked to atherosclerotic disease. In this context, monitoring physiological cholesterol concentrations is of great interest in medical fields and the concept of producing electrical energy from cholesterol is interesting. In this work, we report the bioelectrocatalytic oxidation of cholesterol with the use of nicotinamide adenine dinucleotide-(NAD) dependent cholesterol dehydrogenase (ChDH) as an alternative enzyme to the commonly-used cholesterol oxidase (ChOx) in bioelectronic applications. To achieve bioelectrocatalytic cholesterol oxidation, ChDH was combined with diaphorase (DH) in a bilayer fashion, whereby DH acts as an intermediary enzyme to facilitate NADH oxidation at a ferrocene redox polymer. Characterization of the resulting bioanode identifies a linear cholesterol response range from 5 μM to 200 μM, with an associated sensitivity of 60.12 mA cm−2 M−1. Furthermore, an O2-reducing biocathode incorporating bilirubin oxidase (BOx) was combined with the cholesterol bioanode into a complete cholesterol/O2 membrane-less enzymatic fuel cell (EFC).

Harvesting Energy using Biocatalysts

<i>Harvesting Energy using Biocatalysts</i>

KERAGAMAN GENETIK CENDANA PADA TEGAKAN PENGHASIL BENIH DAN TEGAKAN REHABILITASI DI NUSA TENGGARA TIMUR BERDASARKAN PENANDA ISOZIM

This study aimed to determine the genetic diversity of sandalwood on seed production stand and rehabilitation stand in East Nusa Tenggara using isozyme genetic marker. The study was conducted using samples collected from three stands, i.e. Seed Production Area (APB) representing seed production stand, KHDTK rehabilitation stand and CSR rehabilitation stand representing rehabilitation stands. Samples of sandalwood juvenile leaves are taken randomly from each location. Sample materials taken were juvenile leaves collected randomly from each stand. There were 57 samples taken from APB, while each 25 samples were collected from rehabilitation stands of KHDTK and CSR. Isozymes analyses were carried out in the laboratory using three kinds of enzyme systems i.e Esterase (EST), Diaphorase (DIA) and Shikimate Dehydrogenase (SHD). The results showed that for genetic diversity within stands, the mean of polymorphic loci was 88.89%, with a mean number of alleles per locus 2.1667 and a mean of effective alleles 1.2103. The expected heterozygosity within stands (HS) was 0.1558, with the observed heterozygosity (HO) of 0.1402, while the mean index of fixation (FIS) was 0.1118. On genetic diversity among stands, DST, and GST values were 0.0090 and 0.0545 respectively, while total expected heterozygosity of the three stands (HT) was 0.1648. To anticipate sandalwood genetic diversity decline, it is essential to identify and record the remaining sandalwood populations, then conserve rare alleles either through in-situ or ex-situ conservation programs.

Automatic bionalyzer using an integrated amperometric biosensor for the determination of L-malic acid in wines

A new automatic bioanalyzer for L-malic acid using an integrated amperometric biosensor as detector is reported for the first time in this work. The biosensor is constructed by gold film sputtering deposition on a stainless steel disk electrode and co-immobilization of the enzymes malate dehydrogenase (MDH) and diaphorase (DP) together with the redox mediator tetrathiafulvalene (TTF) by means of dialysis membrane. The analytical performance of the biosensor was evaluated when it was used as amperometric detector in three different analytical methodologies: stirred solutions, semiautomatic FIA system and automatic bioanalyzer. The bienzyme biosensor exhibited great analytical performance in terms of sensitivity, selectivity and reproducibility of the measurements and its usefulness was demonstrated by analyzing wine reference materials with certified content of L-malic acid. The attractive analytical and operational characteristics demonstrated by the automatic bioanalyzer make it a promising simple, rapid and field-based tool for routine wine and fruit control.

A Hidden Transhydrogen Activity of a FMN-Bound Diaphorase under Anaerobic Conditions.

BackgroundRedox cofactors of NADH/NADPH participate in many cellular metabolic pathways for facilitating the electron transfer from one molecule to another in redox reactions. Transhydrogenase plays an important role in linking catabolism and anabolism, regulating the ratio of NADH/NADPH in cells. The cytoplasmic transhydrogenases could be useful to engineer synthetic biochemical pathways for the production of high-value chemicals and biofuels.Methodology/Principal FindingsA transhydrogenase activity was discovered for a FMN-bound diaphorase (DI) from Geobacillus stearothermophilus under anaerobic conditions. The DI-catalyzed hydride exchange were monitored and characterized between a NAD(P)H and a thio-modified NAD+ analogue. This new function of DI was demonstrated to transfer a hydride from NADPH to NAD+ that was consumed by NAD-specific lactate dehydrogenase and malic dehydrogenase.Conclusions/SignificanceWe discover a novel transhydrogenase activity of a FMN-DI by stabilizing the reduced state of FMNH2 under anaerobic conditions. FMN-DI was demonstrated to catalyze the hydride transfer between NADPH and NAD+. In the future, it may be possible to incorporate this FMN-DI into synthetic enzymatic pathways for balancing NADH generation and NADPH consumption for anaerobic production of biofuels and biochemicals.

Implementation of a new integrated d-lactic acid biosensor in a semiautomatic FIA system for the simultaneous determination of lactic acid enantiomers. Application to the analysis of beer samples

An integrated amperometric d-lactic acid biosensor involving a gold film deposited by sputtering on a stainless steel disk electrode where the enzymes d-lactic acid dehydrogenase (DLDH) and diaphorase (DP) as well as the redox mediator tetrathiafulvalene (TTF) are coimmobilized by using a dialysis membrane, is reported in this work. Amperometry in stirred solutions at a detection potential of +0.15V (vs Ag/AgCl reference electrode) provided a linear calibration plot for d-lactic acid over the 1.0×10−4 to 3.8×10−3g L−1 concentration range, with a limit of detection of 3.1×10−5g L−1. The usefulness of the biosensor was demonstrated by determining d-lactic acid in beer samples with good results. Additionally, the biosensor was implemented together with a commercial l−lactic amperometric biosensor in a semiautomatic flow-injection analysis (FIA) system able to perform a rapid and simple stereo-specific determination of d- and d-lactic without a previous separation step. The operational characteristics of the biosensors under flow conditions were evaluated and its applicability was demonstrated through the simultaneous determination of both enantiomers in beer samples.

Diaphorase-Viologen Conjugates as Bioelectrocatalysts for NADH Regeneration

Redox enzymes are widely used as powerful biocatalysts owing to their high selectivity and sustainability. Since most of the enzymatic reactions require the expensive coenzyme (the reduced form of nicotinamide adenine dinucleotide, NADH) in a stoichiometric amount, efficient NADH regeneration is essential for various biocatalytic syntheses. Even though an electroenzymatic recycling method has many advantages, it requires low-molecular-weight redox mediators to transfer electrons from electrodes to an enzymes' active site, which are generally toxic and make it difficult to purify products after the reaction. In the present study, we have conjugated a mediator to enzyme catalyst to solve the problem of the electroenzymatic NADH regeneration. Mediators bound to enzymes can be separated from the reaction medium and then reused readily. Moreover, the conjugation can promote a faster electron transfer between mediators and enzymes. For the conjugation, a viologen with a carboxyl group, ethyl carboxyethyl viologen (ECV) was synthesized and covalently linked to diaphorase (DI) using a cross-linker. Cyclic voltammetry showed that the ECV conjugated to DI displayed electrochemical behavior similar to free ECV. In marked contrast to native DI, the DI-ECV conjugate reduced NAD+ to NADH without any mediator, and this bioelectrocatalyst regenerated NADH successfully for enzymatic lactate synthesis.

Optimization of an Enzyme-based Multi-parameter Biosensor for Monitoring Biogas Processes

An enzyme-based multi-parameter sensor chip for the simultaneous measurement of formate, D- and L-lactate is presented. Thereby, the combination of a diaphorase (DIA) from Clostridium kluyveri with different NAD+-dependent dehydrogenases enables the specific recognition of each compound. The amperometric detection is performed via monitoring of the oxidation current of enzymatically produced hexacyanoferrate(II) at +300mV vs. Ag/AgCl. Chemical cross-linking with glutaraldehyde is used for enzyme immobilization. Thereby, the sensor signal is investigated at different concentrations of the cross-linker in the enzyme membrane. The obtained results indicate that by optimization of the immobilization matrix, the sensor performance can be improved.