Enzyme Glucose Dehydrogenase Research Articles

Enantioselective cascade biocatalysis for deracemization of 2-hydroxy acids using a three-enzyme system.

BackgroundEnantiopure 2-hydroxy acids are key intermediates for the synthesis of pharmaceuticals and fine chemicals. We present an enantioselective cascade biocatalysis using recombinant microbial cells for deracemization of racemic 2-hydroxy acids that allows for efficient production of enantiopure 2-hydroxy acids.ResultsThe method was realized by a single recombinant Escherichia coli strain coexpressing three enzymes: (S)-2-hydroxy acid dehydrogenase, (R)-2-keto acid reductase and glucose dehydrogenase. One enantiomer [(S)-2-hydroxy acid] is firstly oxidized to the keto acid with (S)-2-hydroxy acid dehydrogenase, while the other enantiomer [(R)-2-hydroxy acid] remains unchanged. Then, the keto acid obtained reduced to the opposite enantiomer with (R)-2-keto acid reductase plus cofactor regeneration enzyme glucose dehydrogenase subsequently. The recombinant E. coli strain coexpressing the three enzymes was proven to be a promising biocatalyst for the cascade bioconversion of a structurally diverse range of racemic 2-hydroxy acids, giving the corresponding (R)-2-hydroxy acids in up to 98.5 % conversion and >99 % enantiomeric excess.ConclusionsIn summary, a cascade biocatalysis was successfully developed to prepare valuable (R)-2-hydroxy acids with an efficient three-enzyme system. The developed elegant cascade biocatalysis possesses high atom efficiency and represents a promising strategy for production of highly valued (R)-2-hydroxy acids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0560-1) contains supplementary material, which is available to authorized users.

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea.

Regulation of Pyrroloquinoline Quinone-Dependent Glucose Dehydrogenase Activity in the Model Rhizosphere-Dwelling Bacterium Pseudomonas putida KT2440.

Soil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme. While GDH-dependent phosphate solubilization has been observed in numerous bacteria, little is known concerning the mechanism by which this process is regulated. Here we use the model rhizosphere-dwelling bacterium Pseudomonas putida KT2440 to explore GDH activity and PQQ synthesis, as well as gene expression of the GDH-encoding gene (gcd) and PQQ biosynthesis genes (pqq operon) while under different growth conditions. We also use reverse transcription-PCR to identify transcripts from the pqq operon to more accurately map the operon structure. GDH specific activity and PQQ levels vary according to growth condition, with the highest levels of both occurring when glucose is used as the sole carbon source and under conditions of low soluble phosphate. Under these conditions, however, PQQ levels limit in vitro phosphate solubilization. GDH specific activity data correlate well with gcd gene expression data, and the levels of expression of the pqqF and pqqB genes mirror the levels of PQQ synthesized, suggesting that one or both of these genes may serve to modulate PQQ levels according to the growth conditions. The pqq gene cluster (pqqFABCDEG) encodes at least two independent transcripts, and expression of the pqqF gene appears to be under the control of an independent promoter and terminator. Plant growth promotion can be enhanced by soil- and rhizosphere-dwelling bacteria by a number of different methods. One method is by promoting nutrient acquisition from soil. Phosphorus is an essential nutrient that plants obtain through soil, but in many cases it is locked up in forms that are not available for plant uptake. Bacteria such as the model bacterium Pseudomonas putida KT2440 can solubilize insoluble soil phosphates by secreting gluconic acid. This chemical is produced from glucose by the activity of the bacterial enzyme glucose dehydrogenase, which requires a coenzyme called PQQ. Here we have studied how the glucose dehydrogenase enzyme and the PQQ coenzyme are regulated according to differences in bacterial growth conditions. We determined that glucose dehydrogenase activity and PQQ production are optimal under conditions when the bacterium is grown with glucose as the sole carbon source and under conditions of low soluble phosphate.

Dual Enzymatic Detection by Bulk Electrogenerated Chemiluminescence.

The combination of enzymes, as recognition elements for specific analytes, and of electrogenerated chemiluminescence (ECL) as a readout method has proven to be a valuable strategy for sensitive and specific analytical detection. However, ECL is intrinsically a 2D process which could potentially limit the analysis of inhomogeneous samples. Here, we show how a bulk ECL signal, generated by thousands of carbon microbeads remotely addressed via bipolar electrochemistry, are implemented as a powerful tool for the concomitant ECL sensing and imaging of two enzymatic substrates. We selected two enzymes (glucose dehydrogenase and choline oxidase) that react with their respective model substrates and produce in situ chemical species (β-nicotinamide adenine dinucleotide (NADH) and H2O2) acting as coreactants for the ECL emission of different luminophores ([Ru(bpy)3](2+) at λ = 620 nm and luminol at λ = 425 nm, respectively). Both enzymes are spatially separated in the same capillary. We demonstrate thus the simultaneous quantitative determination of both glucose and choline over a wide concentration range. The originality of this remote approach is to provide a global chemical view through one single ECL image of inhomogeneous samples such as a biochemical concentration gradient in a capillary configuration. Finally, we report the first proof-of-concept of dual biosensing based on this bulk ECL method for the simultaneous imaging of both enzymatic analytes at distinct wavelengths.

High performance glucose/O2 compartment-less biofuel cell using DNA/CNTs as platform for immobilizing bilirubin oxidase as novel biocathode and integrated NH2-CNTs/dendrimer/glucose dehydrogenase/nile blue as bioanode

Herein, deoxyribonucleic acid (DNA)/multi-walled carbon nanotube (MWCNTs) with enhanced negative charged density was used as a novel electrochemical platform for oriented immobilization of bilirubin oxidase. The proposed support improved the direct electron transfer kinetics of BOD and its catalytic activity toward oxygen reduction reaction (ORR). In comparison to BOD enzyme which immobilized directly onto MWCNTs the current density increased three folds and reached to 270μAcm−2 at 0.405V with an onset potential of 0.57V (vs. Ag/AgCl). The ability of this modified electrode as a biocathode is investigated after assembling with bioanode. The bioanode prepared with covalent attachment of glucose dehydrogenase enzyme (GDH) and nile blue (NB) as an efficient mediator for coenzyme regeneration onto glassy carbon electrode modified with amino-carbon nanotubes(MWCNTs-NH2) and carboxyl terminated polyamidoamin dendrimer (PAMAM-Den) as a multifunctional linker. Finally, the performance of one-compartment glucose/O2 biofuel cell without separators is also investigated. The open circuit voltage of the cell and maximum current density are obtained 660mV and 172μAcm−2, respectively, while the maximum power density of 45μWcm−2 is achieved at 428mV of the cell voltage in buffer solution saturated with O2 and containing 50mM of glucose. The stability of the constructed EBFC is investigated under continuous operation at maximum power. It is observed that the biofuel cell can retain more than 85% of its power and potential performance after 24h.

Paper-Based Device for Rapid Visualization of NADH Based on Dissolution of Gold Nanoparticles.

We describe a paper-based device that enables rapid and sensitive room-temperature detection of dihydronicotinamide adenine dinucleotide (NADH) via a colorimetric readout and demonstrate its value for monitoring NAD+-driven enzymatic reactions. Our system is based on NADH-mediated inhibition of gold nanoparticle (AuNPs) dissolution in a Au3+-cetyltrimethylammonium bromide (CTAB) solution. We fabricated a device consisting of a mixed cellulose ester paper featuring a wax-encircled, AuNP-coated film atop a cotton absorbent layer sandwiched between two plastic cover layers. In the absence of NADH, the Au3+-CTAB complex dissolves the AuNP layer completely, generating a white color in the test zone. In the presence of NADH, Au3+ is rapidly reduced to Au+, greatly decreasing the dissolution of AuNPs and yielding a red color that becomes stronger at increasing concentrations of NADH. This device exploits capillary force-assisted vertical diffusion, allowing us to apply a 25 μL sample to a surface-confined test zone to achieve a detection limit of 12.5 μM NADH. We used the enzyme glucose dehydrogenase as a model to demonstrate that our paper-based device can monitor NAD+-driven biochemical processes with and without selective dehydrogenase inhibitors by naked-eye observation within 4 min at room temperature in a small sample volume. We believe that our paper-based device could offer a valuable and low-cost analytical tool for monitoring NAD+-associated enzymatic reactions and screening for dehydrogenase inhibitors in a variety of testing contexts.

Coupling of an enzymatic biofuel cell to an electrochemical cell for self-powered glucose sensing with optical readout

A miniaturized biofuel cell (BFC) is powering an electrolyser invoking a glucose concentration dependent formation of a dye which can be determined spectrophotometrically. This strategy enables instrument free analyte detection using the analyte-dependent BFC current for triggering an optical read-out system. A screen-printed electrode (SPE) was used for the immobilization of the enzymes glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) for the biocatalytic oxidation of glucose and reduction of molecular oxygen, respectively. The miniaturized BFC was switched-on using small sample volumes (ca. 60μL) leading to an open-circuit voltage of 567mV and a maximal power density of (6.8±0.6) μWcm−2. The BFC power was proportional to the glucose concentration in a range from 0.1 to 1.0mM (R2=0.991). In order to verify the potential instrument-free analyte detection the BFC was directly connected to an electrochemical cell comprised of an optically-transparent SPE modified with methylene green (MG). The reduction of the electrochromic reporter compound invoked by the voltage and current flow applied by the BFC let to MG discoloration, thus allowing the detection of glucose.

Nickel-phendione complex covalently attached onto carbon nanotube/cross linked glucose dehydrogenase as bioanode for glucose/oxygen compartment-less biofuel cell

Here, [Ni(phendion) (phen)]Cl2 complex, (phendion and phen are 1,10-phenanthroline-5,6-dione and 5-amino-1, 10-phenanthrolin) covalently attached onto carboxyl functionalized multi walls carbon nanotube modified glassy carbon electrode (GCE/MWCNTs-COOH) using solid phase interactions and combinatorial approaches.The attached [Ni(phendion) (phen)]Cl2 complex displays a surface controlled electrode process and it acts as an effective redox mediator for electrocatalytic oxidation of dihydronicotinamide adenine dinucleotide (NADH) at reduced overpotentials. With co-immobilization of glucose dehydrogenase enzyme (GDH) by crosslinking an effective biocatalyst for glucose oxidation designed. The onset potential and current density are −0.1 V versus Ag/AgCl electrode and 0.550 mA cm−2, which indicate the applicability of the proposed system as an efficient bioanode for biofuel cell (BFC) design. A GCE/MWCNTs modified with electrodeposited gold nanoparticles (AuNPs) as a platform for immobilization of bilirubin oxidase (BOD) and the prepared GCE/MWCNTs/AuNPs/BOD biocathode exhibits an onset potential of 0.56 V versus Ag/AgCl. The performance of the fabricated bioanode and biocathode in a membraneless enzyme based glucose/O2 biofuel cell is evaluated. The open circuit voltage of the cell and maximum current density are 520 mV and 0.233 mA cm−2, respectively, while maximum power density of 40 μWcm−2 achieves at voltage of 280 mV with stable output power after 24 h continues operation.

Kinetic model for a threshold filter in an enzymatic system for bioanalytical and biocomputing applications.

A recently experimentally observed biochemical "threshold filtering" mechanism by processes catalyzed by the enzyme malate dehydrogenase is explained in terms of a model that incorporates an unusual mechanism of inhibition of this enzyme that has a reversible mechanism of action. Experimental data for a system in which the output signal is produced by biocatalytic processes of the enzyme glucose dehydrogenase are analyzed to verify the model's validity. We also establish that fast reversible conversion of the output product to another compound, without the additional inhibition, cannot on its own result in filtering.

Implications of FAD Electrode Reaction Kinetics for Electrocatalysis of NADH Oxidation and Development of NAD‐Dependent Enzyme Electrodes

AbstractFAD entrapped into the polyethyleneimine (PEI) matrix underwent 2e−/2H+ redox transformation, in contrast to the 1e−/1H+ reaction observed with non‐stabilized FAD‐modified electrodes. Under conditions of the 2e−/2H+ reaction, FAD/PEI‐modified electrodes catalytically oxidized NADH starting from −100 mV vs. Ag/AgCl. These electrodes may be used for construction of NAD‐dependent dehydrogenase electrodes for clinically important analytes such as glucose when coupled to the model enzyme glucose dehydrogenase. Latter allows detection of glucose starting from −100 mV within 0.05–5 mM concentration range. These results suggest the FAD stabilization by different matrices as a simple and promising approach to achieve its 2e−/2H+ redox chemistry resulting in catalysis of NADH oxidation.

Paper-Based, Multi-Fueled Enzymatic Fuel Cell with Passive Microfluidic Flow

Enzymatic fuel cell (EFC) technology offers several advantages over the conventional electrochemical power sources: higher energy density, low-cost, environmentally-friendly and renewable biocatalysts, room temperature and pH neutral operating environment, and fuel flexibility via a variety of renewable fuels (e.g. sugars and alcohols). The development of a flexible, paper-based system can significantly increase the utility of the device.The developed system was based on microfluidic passive evaporative pump action provided by the paper and ensured continuous delivery of fuel to the electrodes. The multi-enzymatic anode, consisting of several oxidative enzymes, was capable of simultaneously or separately converting glucose and/or ethanol fuels to electrical energy. Coupled with an air-breathing enzymatic cathode the complete cell produced 800 µW /cm2 at 0.3V.Employing our previously developed carbon nanotube (CNT) based ink we developed a series of enzymatic anodes: with immobilized glucose dehydrogenase (GDH); with immobilized alcohol dehydrogenase (ADH); and with co-immobilized GDH and ADH enzymes. Additionally, in order to investigate multi-oxidation of a single fuel (ethanol) anodes were developed with co-immobilized ADH and aldehyde dehydrogenase (AlDH). Half-cell experiments were performed on the various enzymatic anodes to determine limits of performance as well as stability and compatibility with non-complementary fuels. Results are depicted in Figure 1.Bilirubin oxidase (BOx) air-breathing cathode was also developed, characterized and optimized for best performance. The composite cathode consisted of three layers: Toray paper (TP) current collector layer; Teflon-treated carbon black (XC-35) gas diffusional layer, and high conductivity CNT paper catalytic layer. All three layers were fused together in a hydraulic press at 500 psi. BOx was tethered to the surface of the catalytic layer through the use of bi-functional chemical linker (1-Pyrenebutanoic acid, succinimidyl ester, PBSE), which forms peptide bonds with the enzyme through the succinimidyl moiety and π-π stacks on CNTs through the pyrene moiety. Such approach resulted in increased stability of the enzyme at the electrode surface when compared to physical adsorption deposition method.Once the individual components of the fuel cell were developed and optimized, the complete EFC was constructed and tested. The GDH-based and ADH/AlDH-based systems were analyzed in respective fuels. The experimental power curve for the GDH EFC is illustrated in Figure 2. Finally, in order to demonstrate actual application, three paper-based EFCs were connecter in series and immersed in Gatorade® (fuel). This system was then employed to power a digital clock continuously for several days (Figure 3).

Photocurrent Measurements at Quantum Dot Electrodes for Bioanalytical Applications

Quantum dots (QDs) allow the generation of electron-hole pairs upon illumination. When these particles are fixed to an electrode a photocurrent can be generated. This allows their use as a light-switchable layer on the surface. The QDs can not only exchange electrons with the electrode, but can also show reactions with donor or acceptor compounds in solution. This provides access to the construction of signal chains starting from an analyte molecule to be detected.The direction and magnitude of the photocurrent depend on several factors such as electrode polarization, solution pH and composition. These defined dependencies have been evaluated with respect to the combination of QD-electrodes with enzyme reactions for sensorial purpose [1,2].Thus, it has been found that the cofactor of many dehydrogenase-based reactions NADH can be oxidized at the illuminated QDs without applying high potentials. This provides the basis for a coupling strategy with the enzyme glucose dehydrogenase for the analysis of glucose concentrations in solution by photocurrent measurements.CdSe/ZnS-QD-modified electrodes can also be used to follow enzymatic reactions in solution based on their capability to reduce molecular oxygen while being excited [3]. In order to develop a photoelectrochemical biosensor, glucose oxidase (GOD) is immobilized on the QD-electrode. One immobilization strategy applies the layer-by-layer-technique of GOD and a polyelectrolyte (polyallylamine hydrochloride). Photocurrent measurements of such a sensor show a clear concentration dependent behavior for millimolar glucose concentrations. Sensitivity can be influenced by the enzyme concentration and the number of layers deposited. The principle of combining QD electrodes with a layered protein architecture and light-triggered read-out can also be transferred to other enzymes such sarcosine oxidase and sarcosine detection. The sensing properties of quantum dot electrodes can be significantly influenced by additional nanoparticles giving access to the detection of other substances, but also by immobilizing multiple layers of the QDs.In another direction of research it can be demonstrated that direct electron transfer from excited quantum dots can be achieved with the redox protein cytochrome c provided the QD surface is properly modified. This allows the detection of the protein in different concentrations and redox states, but also of interaction partners which show a defined reaction with the protein. Examples are the cyt c reduction by superoxide radicals resulting in an anodic photocurrent and the cyt c oxidation by nitrate reductase in the presence of nitrate resulting in a cathodic photocurrent .[1] Ch. Stoll, S. Kudera, W.J. Parak, F. Lisdat, SMALL 2 (6) (2006) 741-743).[2] K. Schubert, W. Khalid, Z. Yue, W. Parak, F. Lisdat, Langmuir 26 (2) (2010) 1395-1400[3] J. Tanne, D. Schäfer, W. Khalid, W. J. Parak, F. Lisdat, Analytical Chemistry 83 (20) (2011) 7778–7785

Purification of Xylitol Dehydrogenase and Improved Production of Xylitol by Increasing XDH Activity and NADH Supply in Gluconobacter oxydans

Gluconobacter oxydans is known to be a suitable candidate for producing xylitol from d-arabitol. In this study, the enzyme responsible for reducing d-xylulose to xylitol was purified from G. oxydans NH-10 and characterized as xylitol dehydrogenase. It has been reported that XDH depends exclusively on NAD(+)/NADH as cofactors with a relatively low activity, which was proposed to be the direct reason for its limiting the overall conversion process. To better produce xylitol, an engineered G. oxydans PXPG was constructed to coexpress the XDH gene and a cofactor regeneration enzyme (glucose dehydrogenase) gene from Bacillus subtilis. Activities for both enzymes were more than twofold higher in the G. oxydans PXPG than in the wild strain. Approximately 12.23 g/L xylitol was obtained from 30 g/L d-arabitol by resting cells of the engineered strain with a conversion yield of 40.8%, whereas only 7.56 g/L xylitol was produced by the wild strain with a yield of 25.2%. These results demonstrated that increasing the XDH activity and the cofactor NADH supply could improve the xylitol productivity notably.

Biofabricated film with enzymatic and redox-capacitor functionalities to harvest and store electrons

Exciting opportunities in bioelectronics will be facilitated by materials that can bridge the chemical logic of biology and the digital logic of electronics. Here we report the fabrication of a dual functional hydrogel film that can harvest electrons from its chemical environment and store these electrons by switching the film's redox-state. The hydrogel scaffold was formed by the anodic deposition of the aminopolysaccharide chitosan. Electron-harvesting function was conferred by co-depositing the enzyme glucose dehydrogenase (GDH) with chitosan. GDH catalyzes the transfer of electrons from glucose to the soluble redox-shuttle NADP+. Electron-storage function was conferred by the redox-active food phenolic chlorogenic acid (CA) that was enzymatically grafted to the chitosan scaffold using tyrosinase. The grafted CA undergoes redox-cycling reactions with NADPH resulting in the net transfer of electrons to the film where they are stored in the reduced state of CA. The individual and dual functionalities of these films were demonstrated experimentally. There are three general conclusions from this proof-of-concept study. First, enzymatically-grafted catecholic moieties confer redox-capacitor function to the chitosan scaffold. Second, biological materials (i.e. chitosan and CA) and mechanisms (i.e. tyrosinase-mediated grafting) allow the reagentless fabrication of functional films that should be environmentally-friendly, safe and potentially even edible. Finally, the film's ability to mediate the transfer of electrons from a biological metabolite to an electrode suggests an approach to bridge the chemical logic of biology with the digital logic of electronics.

Electrochemistry and Current Control in Surface Films Based on Silica-Azure Redox Nanoparticles, Carbon Nanotubes, Enzymes, and Polyelectrolytes

The redox active nanoparticles were developed by covalently attaching redox dye Azure C (AZU) to commercial silica nanoparticles (SN) via the silylated amine and glutaric dialdehyde links. The SN-AZU nanoparticles were studied as redox mediators for the oxidation of reduced β-nicotinamide adenine dinucleotide (NADH) in two polymeric films. The first film (F1) was composed of SN-AZU, carbon nanotubes, and cationic polyelectrolyte chitosan. The second film (F2) contained also added enzyme glucose dehydrogenase and its cofactor β-nicotinamide adenine dinucleotide (NAD(+)). The films F1 and F2 were cast on the glassy carbon electrodes, covered with an anionic polyelectrolyte Nafion, and their electrochemical properties were probed with NADH and glucose, respectively, using voltammetry, amperometry, and potentiometry. The Nafion overcoat reduced the sensitivity of F1/Nafion film electrodes to NADH by >98%. In contrast, depending on the concentration of Nafion, the sensitivity of the F2/Nafion film electrodes (reagentless biosensors) to glucose increased by up to 340%. The amplification of glucose signal was ascribed to the Donnan exclusion and ensuing Nafion-gated ionic fluxes, which enhanced enzyme activity in films F2. The proposed model predicts that such signal amplification should be also feasible in the case of other enzyme-based biosensors.

Repression of oxalic acid-mediated mineral phosphate solubilization in rhizospheric isolates of Klebsiella pneumoniae by succinate

Two strains of Klebsiella (SM6 and SM11) were isolated from rhizospheric soil that solubilized mineral phosphate by secretion of oxalic acid from glucose. Activities of enzymes for periplasmic glucose oxidation (glucose dehydrogenase) and glyoxylate shunt (isocitrate lyase and glyoxylate oxidase) responsible for oxalic acid production were estimated. In presence of succinate, phosphate solubilization was completely inhibited, and the enzymes glucose dehydrogenase and glyoxylate oxidase were repressed. Significant activity of isocitrate lyase, the key enzyme for carbon flux through glyoxylate shunt and oxalic acid production during growth on glucose suggested that it could be inducible in nature, and its inhibition by succinate appeared to be similar to catabolite repression.

Amperometric biosensor based on multilayer containing carbon nanotube, plasma-polymerized film, electron transfer mediator phenothiazine, and glucose dehydrogenase

We report on a novel fabrication approach of amperometric biosensor based on multilayer films containing carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), electron transfer mediator phenothiazine (PT), and enzyme glucose dehydrogenase (GDH). The configuration of the electrochemical electrode is sequentially composed of sputtered gold, acetonitrile PPF, PT, GDH, and acetonitrile PPF (denoted as PPF/GDH/PT/CNT/PPF/Au). First PPF deposited on Au acts as a permselective membrane and as a scaffold for CNT layer formation. Second PPF directly deposited on GDH acts as a matrix for enzyme immobilization. To facilitate the electrochemical communication between the CNT layer and GDH, CNT was treated with nitrogen plasma. The electron transfer mediator PT plays a role as the mediator in which the electron caused by enzymatic reaction transports to the electrode. The synergy between the mediator and CNT provides benefits in terms of lowering the operational potential and enhancing the sensitivity (current). The optimized glucose biosensor revealed a sensitivity of 5.1 ± 0.9 μA mM − 1 cm − 2 at + 0.2 V vs. Ag/AgCl, linear dynamic range of 4.9–19 mM, and a response time of 5 ± 1 s. Unlike conventional wet-chemical processes that are incompatible with mass production techniques, this dry-chemistry procedure has great potential for enabling high-throughput production of bioelectronic devices. Furthermore, those devices can be applied and expands for the cell biological functional field as a useful, helpful, or indispensable tool.

Pyrroloquinoline Quinone Biosynthesis GenepqqC, a Novel Molecular Marker for Studying the Phylogeny and Diversity of Phosphate-Solubilizing Pseudomonads

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.

Repression of mineral phosphate solubilizing phenotype in the presence of weak organic acids in plant growth promoting fluorescent pseudomonads

Two phosphate solubilizing bacteria (PSB), M3 and SP1, were obtained from the rhizosphere of mungbean and sweet potato, respectively and identified as strains of Pseudomonas aeruginosa. Their rock phosphate (RP) solubilizing abilities were found to be due to secretion high amount of gluconic acid. In the presence of malate and succinate, individually and as mixture, the P solubilizing ability of both the strains was considerably reduced. This was correlated with a nearly 80% decrease in the activity of the glucose dehydrogenase (GDH) but not gluconate dehydrogenase (GAD) in both the isolates. Thus, GDH enzyme, catalyzing the periplasmic production of gluconic acid, is under reverse catabolite repression control by organic acids in P. aeruginosa M3 and SP1. This is of relevance in rhizospheric conditions and is a new explanation for the lack of field efficacy of such PSB.

An autonomous drug release system based on chemo-mechanical energy conversion “Organic Engine” for feedback control of blood glucose

A novel autonomous drug release system was fabricated and tested. The system consists of two integrated units: decompression unit and drug release unit. The decompression unit was fabricated by separating a cylindrical cell into a top cell (gas phase) and a bottom cell (liquid phase) by glucose oxidase (GOD) enzyme immobilized membrane. The enzyme membrane recognizes glucose and converts chemical energy found in glucose to mechanical energy. The linear correlation between glucose concentration and de-pressure slope of the top cell was revealed as applying glucose solution to the bottom cell. Afterward, the drug release unit which utilizes the energy of the decompression unit as a power source was fabricated and evaluated by recording its release actions. The drug release unit was made to release at a constant quantity of drug in the liquid phase. The system was then fabricated by combining the decompression unit and the drug release unit. And it was evaluated in an open loop and in a closed loop by applying a mixture of glucose solution (100 mmol/l) and NADH + using glucose dehydrogenase enzyme (GDH) as a glucose reducer. Glucose concentration decreased gradually in the closed loop and, as a consequence, interval time of the GDH release became longer. In other words, an inverse correlation between actuation interval of the system and glucose concentration was shown. As a result, the possibility of feedback control of glucose concentration by the drug release system without external energy was confirmed.