PQQ-dependent Glucose Dehydrogenase Research Articles

Operation of a carbon nanotube-based glucose/oxygen biofuel cell in human body liquids—Performance factors and characteristics

The performance of an enzymatic biofuel cell in physiological media depends not only on the electrode architecture but is also influenced by substances in these media which can be directly oxidized or reduced at the anode and/or cathode or have an impact on the biocatalytic processes.For the anode construction carbon nanotubes are modified with a polyaniline film onto which pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) from Acinetobacter calcoaceticus is covalently coupled. The cathode is based on bilirubin oxidase (BOD) from Myrothecium verrucaria which is coupled to a PQQ-modified carbon nanotube electrode. The resulting biofuel cell achieves power maxima of more than 100μW/cm2.Galvanodynamic performance measurements in urine and saliva show a significant loss of the maximum power density compared to an EBFC in 5mM glucose containing buffer. The EBFC achieves 12% in urine and 18% in saliva. The initial open cell potential of about 710mV for the EBFC is reduced to 400mV in urine and to 665mV in saliva.To elucidate the effect of potential interfering substances in the real media the individual electrodes are investigated by cyclic voltammetry in human urine and saliva and also in the presence of urea, uric acid and ascorbic acid.Reasons for the low power output in the both body liquids are the very low glucose concentrations. In urine the cell potential is significantly decreased because of the strong influence of an oxidation process at the cathode. The low anode performance in saliva can be attributed to the diminished biochemical catalysis.

DNA Release from a Bioelectronic Interface Stimulated by a DNA Signal – Amplification of DNA Signals

AbstractA new bioelectronic system activated by a DNA‐signal and releasing another DNA has been designed. The system was composed of two modified electrodes: one electrode functionalized with enzymes was activated with a DNA‐signal, while another electrode coated with an alginate film released pre‐loaded DNA species, when the first electrode was activated. The sensing electrode was functionalized with pyrroloquinoline quinone‐dependent glucose dehydrogenase (PQQ‐GDH) producing negative potential in the presence of glucose. This bioelectrocatalytic process was inhibited by the presence of glucose oxidase (GOx) at the external surface of the biomolecular system organized on the electrode surface through a DNA‐linker. GOx consumed incoming glucose and did not allow its penetration to the internal layer functionalized with PQQ‐GDH, thus leaving the electrode mute. The DNA‐signal resulted in the detachment of GOx and activated the process biocatalyzed by PQQ‐GDH due to penetration of glucose to the internal layer at the electrode surface. Finally, formation of a negative potential on the sensing electrode resulted in the activation of the second electrode and DNA release from the alginate matrix. The Sense‐and‐Act system allowed amplification of the DNA‐signal by releasing much higher amount of DNA comparing with the applied DNA‐signal. The DNA‐signal amplification will find applications in various biosensor and biomolecular computing systems.

Computational Approaches for Rational Design of Electrodes for Biofuel Cells and Biosensors Applications

Redox enzymes have been extensively exploited as electrocatalysts in biofuel cells and biosensors due to the advantages they have over commonly used inorganic catalysts. For example, they often have higher activity and selectivity than the inorganic catalysts and can operate at biologically benign conditions. However, additional improvements have to be made for the development of enzymatic electrodes with high activity, stability, and ease of production that can lead to broader applications of enzyme-based systems. Immobilization and optimal interaction of the proteins with the support material is one of the key components in the design of enzymatic electrodes. Therefore, rational design of the electrode surface provides one of the most promising routes for creating biofuel cells and biosensors with enhanced performances. Such a design, however, demands deeper understanding of the bio-nano interface interactions as well as efficient enzyme orientation, which can be achieved by the use of various computational approaches. Density Functional Theory (DFT) and docking simulations were used to gain a fundamental understanding of the role of different molecules explored as orienting and/or mediating agents in the design and operation of several enzymatic electrodes. Different computational approaches were first used to explain the role of 1,2-benzoquinone, 1,4-benzoquinone, and ubiquinone on the operation and mechanism of electron transfer in PQQ-dependent soluble glucose dehydrogenase anodes (1, 2). The same approach was also used to understand the role of bilirubin in the bilirubin-modified oxygen reducing bio-cathodes based on bilirubin oxidase. We further studied the interaction of unsubstituted phenothiazine with glucose, lactate and cholesterol oxidases. Namely, it has been shown that the hydrophobic phenothiazine can be utilized for efficient electron transfer in various oxidases. In addition, docking simulations were used to discuss the nature of the interactions between flavins and outer membrane cytochromes of Shewanella spp., which has practical implication in the development of microbial fuel cells. 1. Babanova S, Matanovic I, Atanassov P. Quinone-modified surfaces for enhanced enzyme-electrode interactions in PQQ-dependent glucose dehydrogenase anodes. Chem Electro Chem. 2014;1(11):2017–28. 2. Babanova S, Matanovic I, Chavez MS, Atanassov P. The role of quinones in electron transfer of PQQ-glucose de-hydrogenase anodes – mediation or orientation effect. J. Am. Chem. Soc. 2015;137(24):7754-7762. Figure 1

Power Harvesting from Human Serum in Buckypaper-Based Enzymatic Biofuel Cell

The requirement for a miniature, high density, long life, rechargeable power source is common to a vast majority of microsystems, including the implantable devices for medical applications. A model biofuel cell system operating in human serum has been studied for future applications of biomedical and implantable medical devices. Anodic and cathodic electrodes were made of carbon nanotube –buckypaper modified with PQQ-dependent glucose dehydrogenase and laccase, respectively. Modified electrodes were characterized electrochemically and assembled in a biofuel cell set-up. Power density of 16.12 μW/cm2 was achieved in human serum for lower than physiological glucose concentrations. Increasing the glucose concentration and biofuel cell temperature caused an increase on power output leading up to 49.16 μW/cm2.

Towards a novel bioelectrocatalytic platform based on "wiring" of pyrroloquinoline quinone-dependent glucose dehydrogenase with an electrospun conductive polymeric fiber architecture.

Electrospinning is known as a fabrication technique for electrode architectures that serve as immobilization matrices for biomolecules. The current work demonstrates a novel approach to construct a conductive polymeric platform, capable not only of immobilization, but also of electrical connection of the biomolecule with the electrode. It is produced upon electrospinning from mixtures of three different highly conductive sulfonated polyanilines and polyacrylonitrile on ITO electrodes. The resulting fiber mats are with a well-retained conductivity. After coupling the enzyme pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) to polymeric structures and addition of the substrate glucose an efficient bioelectrocatalysis is demonstrated. Depending on the choice of the sulfonated polyanilline mediatorless bioelectrocatalysis starts at low potentials; no large overpotential is needed to drive the reaction. Thus, the electrospun conductive immobilization matrix acts here as a transducing element, representing a promising strategy to use 3D polymeric scaffolds as wiring agents for active enzymes. In addition, the mild and well reproducible fabrication process and the active role of the polymer film in withdrawing electrons from the reduced PQQ-GDH lead to a system with high stability. This could provide access to a larger group of enzymes for bioelectrochemical applications including biosensors and biofuel cells.

Biofuel Cell Based on Carbon Fiber Electrodes Functionalized with Graphene Nanosheets

Carbon fiber electrodes were electrochemically modified with graphene nanosheets in situ deposited in the 3D-structure, characterized by field emission scanning electron microscopy (SEM), X-ray diffraction (XRD) and Raman scattering methods. The graphene-modified electrodes were further functionalized with PQQ-dependent glucose dehydrogenase and laccase and used for assembling a biofuel cell operating in the presence of oxygen and glucose “fuel”. The paper represents novel approach to the production of graphene-functionalized electrodes and their use in biofuel cells, potentially applicable in implantable bioelectronics devices.

Application of Modified Carbon Nanotube Materials for Enzymatic Biofuels Cells Based on Direct Enzyme-Electrode Contacts

Carbon nanotubes (CNTs) arranged in 3 dimensional structures represent an interesting material for the development of biocatalytic electrodes. Due to their architecture they can provide docking places for enzymes and after modification of the surface properties often a direct electrochemistry can be observed. The direct electron transfer where the catalytic starts near the E0 of the enzymes redox center can avoid a loss of in cell potential and allows the development of efficient enzymatic biofuel cells (EBFC). Also the membrane-less construction of the EBFCs is advantageous for high power output. With this respect the pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ) GDH) is an interesting enzyme since it is insensitive towards oxygen which is the terminal electron acceptor at the cathode. Here two types of carbon nanotubes materials - bucky paper (BP) and vertically aligned carbon nanotubes (vaCNTs) - are used for the development of glucose/oxygen biofuel cells. For the anode development these materials are modified with poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic) acid (PABMSA) for covalent coupling of the glucose oxidizing (PQQ) GDH. The cathode is based on the oxygen reducing Bilirubin oxidase (BOD) which is covalently coupled to PQQ modified BP and vaCNTs electrodes. For the electrochemical characterisation of the individual electrodes voltammetric measurements are performed. The voltammograms for the different anode preparations show that the modification of both carbon nanotube materials with an aniline-based polymer film (PABMSA) and covalent enzyme coupling result in an direct enzyme-electrode contact. The influence of the polymer concentration during the electrode preparation and the impact of the buffer composition on the current density are investigated. Both electrode materials show the highest current density in 100 mM citrate phosphate (CiP) buffer containing 10 mM glucose and applying 5 mg/ml PABMSA for CNTs modification. For the BP-based anode current densities up to 0.75 mA/cm2 can be detected while electrodes made of vaCNTs reveal a maximum current density of 1.3 mA/cm2 at +0.1 V vs. Ag/AgCl. The cathode construction with PQQ as interlayer and a covalent attachment of the BOD to the carboxylic groups shows the highest electrocatalytic activity under air saturated conditions – for bucky paper about 1 mA/cm2 at 0.1 V vs. Ag/AgCl. Applying vaCNTs for the BOD-cathode development a local maximum current of 1.3 mA/cm2 and a steady-state catalytic current of 0.55 mA/cm2 at +0.1 V vs. Ag/AgCl can be obtained. A combination of the BP-based (PQQ)GDH/PABMSA and BOD/PQQ electrodes in a biofuel cell application achieves a power output of 107 µW/cm2 at a cell potential of 490 mV. The same modification procedures and enzymes applied in a vaCNTs fuel cell lead to a power density of 122 µW/cm2 at cell potential of 540 mV. The separate evaluation of both carbon nanotubes based materials reveal a better stability of the vaCNTs-based enzyme electrodes.

Combined Experimental and Computational Approach for Rational Design of Bio-Nano Interfaces

Various approaches have been developed in the last 5 years regarding targeted enzyme orientation on the electrode surface but no explanation of enzyme orientation on electron transfer properties have been provided. The same can be said about the utilization of mediators for enhancement of the interfacial electron transfer. Facing this fundamental challenge we started using computational approaches along with experimental efforts to gain knowledge of the underlying processes. In our computational and experimental benchmark studies soluble PQQ-dependent glucose dehydrogenase (PQQ-sGDH) have been explored since this enzyme is known as capable of direct electron transfer (DET). Various techniques for PQQ-sGDH immobilization and/or enhanced electron transfer have been developed over the years and a key question regarding the “proper” orientation of PQQ-sGDH has been raised. Should the substrate-binding pocket and thus PQQ be placed facing the electrode surface or it should be fully exposed to the electrolyte? To resolve this question and to enhance the electron transfer at the enzyme-electrode interface, quinones were explored in solution and immobilized on carbon support material. In both cases the current densities generated form the glucose oxidation through PQQ-sGDH were increased from 1.5 to 5 times depending on the properties of the explored quinones. In addition to the electrochemical characterization of the designed enzymatic anodes, molecular computational calculations, such as Density Functional Theory (DFT) and Molecular Docking Simulations, were used to gain insight into interactions between the quinones and PQQ-sGDH from one hand and the quinones and the support material, from another (Fig. 1). The role of the two types of benzoquinones, 1,2- and 1,4-benzoquinone, and ubiquinone in the operation and mechanism of the electron transfer in PQQ-sGDH anodes has been addressed1. It was established that the higher electrochemical performance of PQQ-sGDH anodes in the presence of free 1,2- and 1,4-benzoquinones is due to the smaller distance between these molecules and the enzyme cofactor (PQQ). It was proposed that when 1,4-benzoquinone is adsorbed, it plays the role of a mediator as a result of its high electron transfer rate ability and smaller PQQ-quinone distance. At the same time when 1,2-benzoquinone is adsorbed on the electrode surface, the enzyme will transfer the electrons directly to the support and the modifier will mostly play the role of an orienting agent. Similar conclusion can be made for the PQQ-sGDH anode modified with physically adsorbed ubiquinone for which purely orientation role was proposed. 1.Babanova, S., Matanovic, I. & Atanassov, P. Quinone-modified surfaces for enhanced enzyme-electrode interactions in PQQ-dependent glucose dehydrogenase anodes. Chem. Electro. Chem. 1, 2017–2028 (2014). Figure 1

Bioelectrocatalysis of Fructose Dehydrogenase at Polyanilline-Modified Electrodes

Polymers can provide a suitable chemical environment for the immobilization of biomolecules in an active form on surfaces. Furthermore they can be used to wire redox proteins and enzymes with electrodes. For this purpose mainly redox polymers with well-defined redox centres and conductive polymers with intrinsic electron conductivity are applied. Polyanillines are belonging the latter class of polymers. Their properties can be easily tuned by the substitution pattern on the monomeric units. Recently we have shown that sulfonated polyanillines are suitable partners in the interaction with the enzyme PQQ-dependent glucose dehydrogenase [1]. On the basis of efficient electron exchange different enzyme electrodes can be constructed [2-4]. In this study we have investigated whether it is possible to connect the multi-domain enzyme fructose dehydrogenase FDH with electrodes using the same group of polymers. The interaction of a sulfonated and methoxy-substituted polyanilline with FDH is studied in solution using UV/Vis spectroscopy. It can be demonstrated that electron transfer from the substrate reduced enzyme to the polymer is feasible, but also that this interaction is enhanced by the presence of calcium ions. Subsequently the reaction has been investigated by electrochemical means with all partners in solution verifying the spectroscopic results. In a next step of development the enzyme has been fixed on planar ITO electrodes by means of a polymer layer. Such an enzyme electrode exhibits effective, substrate-induced bioelectrocatalysis starting at rather low potential (~0mV vs Ag/AgCl). The efficiency can be significantly enhanced when macroporous ITO electrodes are used. They have pore size diameters of ~ 300nm and allow incorporation of higher amounts of polymer and enzyme. A current enhancement of ~ 35 is found compared to the flat ITO. The study demonstrates that sulfonated polyanillines can be considered as valuable matrices in connecting redox enzymes with electrodes. [1] D. Sarauli; C.G. Xu; B. Dietzel; B. Schulz; F. Lisdat, Acta Biomater. 2013, 9, 8290 [2] I. Schubart; G. Göbel; F. Lisdat, Electrochimica Acta 2012, 82, 224 [3] D. Sarauli, C.G. Xu, B. Dietzel, B. Schulz, F. Lisdat, Journal of Materials Chemistry B 2 (21) 2014 3151-3404 [4] D. Sarauli, K. Peters, Xu, B. Schulz, D. Fattakhova-Rohlfing, F. Lisdat, ACS Appl. Mater. Interfaces 2014, 6, 17887

Role of Quinones in Electron Transfer of PQQ-Glucose Dehydrogenase Anodes—Mediation or Orientation Effect.

In this study, the influence of two quinones (1,2- and 1,4-benzoquinone) on the operation and mechanism of electron transfer in PQQ-dependent glucose dehydrogenase (PQQ-sGDH) anodes has been determined. Benzoquinones were experimentally explored as mediators present in the electrolyte. The electrochemical performance of the PQQ-sGDH anodes with and without the mediators was examined and for the first time molecular docking simulations were used to gain a fundamental understanding to explain the role of the mediator molecules in the design and operation of the enzymatic electrodes. It was proposed that the higher performance of the PQQ-sGDH anodes in the presence of 1,2- and 1,4-benzoquinones introduced in the solution is due to the shorter distance between these molecules and PQQ in the enzymatic molecule. It was also hypothesized that when 1,4-benzoquinone is adsorbed on a carbon support, it would play the dual role of a mediator and an orienting agent. At the same time, when 1,2-benzoquinone and ubiquinone are adsorbed on the electrode surface, the enzyme would transfer the electrons directly to the support, and these molecules would primarily play the role of an orienting agent.

Surface modifications for enhanced enzyme immobilization and improved electron transfer of PQQ-dependent glucose dehydrogenase anodes

Pyrroloquinoline quinone dependent soluble glucose dehydrogenase (PQQ-sGDH) enzymatic MWCNT electrodes were p roduced using 1-pyrenecarboxylic acid (PCA) activated through carbodiimide functionalization and 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) as tethering agents. At 600mV potential, the current density generated by the activated-PCA tethered PQQ-sGDH anode was significantly greater than the current density generated by the untethered PQQ-sGDH and PBSE tethered anodes, and performance was nearly identical to the performance of a covalently bound PQQ-sGDH anode. A technique for covalently bonding heme-b (hemin), a natural quinohemoprotein porphyrin redox cofactor, to carbon nanotubes modified with arylamine groups is reported. The resulting performance of the covalently bound hemin PQQ-sGDH anode is considerably higher than that of any other PQQ-sGDH anodes tested.

Simplifying Enzymatic Biofuel Cells: Immobilized Naphthoquinone as a Biocathodic Orientational Moiety and Bioanodic Electron Mediator

An elegant method to perform bioelectocatalysis with different oxidoreductases at the cathode and at the anode of an enzymatic biofuel cell is presented. Noncovalent functionalization of multiwalled carbon nanotubes (MWCNTs) was accomplished via π–π interactions of pyrene derivatives. 1-[Bis(2-naphthoquinonyl)aminomethyl]pyrene was synthesized and successfully immobilized on MWCNTs. The incorporation of the quinone-modified MWCNTs within enzymatic bioelectrocatalytic applications was evaluated. The hydrophobic nature of the naphthoquinone aided orientation of laccase and bilirubin oxidase toward the electrode, which enhanced their ability to undergo the direct bioelectrocatalysis of oxygen. In contrast, the electrochemical properties of the quinone were used at the bioanode to mediate electrons from the bioelectrocatalytic oxidation of glucose by pyrroloquinoline quinone-dependent glucose dehydrogenase. This method demonstrates how the smart modification of MWCNTs can develop materials, which can be used ...

Controlling the charge of pH-responsive redox hydrogels by means of redox-silent biocatalytic processes. A biocatalytic off/on switch

Coupling of redox-silent biocatalytic processes for analyte detection with enzyme-catalyzed redox reactions for signal generation is proposed by the modulation of electrostatic interactions between a pH-responsive polymer and a redox enzyme to control the off–on transition for electrochemical signal generation. Glassy carbon electrodes are modified with a poly(vinyl)imidazole Os(bipyridine)2Cl redox hydrogel film entrapping urease and PQQ-dependent glucose dehydrogenase, while glucose is present in the solution. The off–on transition is based on the detection of urea as model analyte which is hydrolyzed to ammonia by urease within the hydrogel film concomitantly increasing the local pH value thus invoking deprotonation of the imidazole groups at the polymer backbone. The decrease of positive charges at the polymer decreases electrostatic repulsion between the polymer and the positively charged PQQ-dependent glucose dehydrogenase. Hence, electron transfer rates between polymer-bound Os complexes and PQQ inside the enzyme are enhanced activating electrocatalytic oxidation of glucose. This process generates the electrochemical signal for urea detection.

A wireless transmission system powered by an enzyme biofuel cell implanted in an orange

A biofuel cell composed of catalytic electrodes made of “buckypaper” modified with PQQ-dependent glucose dehydrogenase and FAD-dependent fructose dehydrogenase on the anode and with laccase on the cathode was used to activate a wireless information transmission system. The cathode/anode pair was implanted in orange pulp extracting power from its content (glucose and fructose in the juice). The open circuit voltage, Voc, short circuit current density, jsc, and maximum power produced by the biofuel cell, Pmax, were found as ca. 0.6V, ca. 0.33mA·cm−2 and 670μW, respectively. The voltage produced by the biofuel cell was amplified with an energy harvesting circuit and applied to a wireless transmitter. The present study continues the research line where different implantable biofuel cells are used for the activation of electronic devices. The study emphasizes the biosensor and environmental monitoring applications of implantable biofuel cells harvesting power from natural sources, rather than their biomedical use.

3D-electrode architectures for enhanced direct bioelectrocatalysis of pyrroloquinoline quinone-dependent glucose dehydrogenase.

We report on the fabrication of a complex electrode architecture for efficient direct bioelectrocatalysis. In the developed procedure, the redox enzyme pyrroloquinoline quinone-dependent glucose dehydrogenase entrapped in a sulfonated polyaniline [poly(2-methoxyaniline-5-sulfonic acid)-co-aniline] was immobilized on macroporous indium tin oxide (macroITO) electrodes. The use of the 3D-conducting scaffold with a large surface area in combination with the conductive polymer enables immobilization of large amounts of enzyme and its efficient communication with the electrode, leading to enhanced direct bioelectrocatalysis. In the presence of glucose, the fabricated bioelectrodes show an exceptionally high direct bioelectrocatalytical response without any additional mediator. The catalytic current is increased more than 200-fold compared to planar ITO electrodes. Together with a high long-term stability (the current response is maintained for >90% of the initial value even after 2 weeks of storage), the transparent 3D macroITO structure with a conductive polymer represents a valuable basis for the construction of highly efficient bioelectronic units, which are useful as indicators for processes liberating glucose and allowing optical and electrochemical transduction.

Nano-structured carbon materials for improved biosensing applications

A set of oxidized graphite samples have been newly synthesized using different protocols. Atomic force microscopy, Raman spectroscopy, thermal gravimetric analysis and Brunauer–Emmett–Teller analysis revealed the changes in structure and functionalities of obtained graphite oxidation products (GOPs) compared to pristine graphite. The substances have been tested as electrode materials applicable for bioelectrocatalytic systems using pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). The application of GOPs allowed achieving the direct electron transfer (DET) from active site of PQQ-GDH to the electrode surface. Needless of additional electron transfer (ET) mediating compounds highly improved features of the biosensors. The efficiency of the biosensors has been evaluated for all types of biosensors varied from 32μA/cm2 to 64μA/cm2 using as electrode materials GOP1 and thermally reduced graphite oxide (TRGO), respectively. TRGO containing function groups (according TGA, ∼6% of the weight loss) and smallest particles (average diameter was ∼11nm and the average height was ∼0.5nm) exhibited the higher efficiency for ET acceleration in the biosensor acting on principle of DET.

Sensitive electrochemical measurement of hydroxyl radical generation induced by the xanthine–xanthine oxidase system

A sensitive electrochemical measurement system for hydroxyl radical (OH) was developed using enzyme-catalyzed signal amplification. In the presence of 2,6-xylenol as a trapping agent, glucose as a substrate, and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ–GDH) as a catalyst, the amperometric signal of the trapping adduct 2,6-dimethylhydroquinone (DMHQ) produced by the hydroxylation of 2,6-xylenol was able to be amplified and detected sensitively. The limit of detection (signal/noise [S/N]=3) for DMHQ was 1nM. There was no significant interference from urate and other oxidizable compounds in the reaction mixture at the applied potential of 0V versus Ag/AgCl. This method was employed to observe the OH generation induced by the xanthine–xanthine oxidase (XO) system. The reaction rates of the DMHQ production induced from the xanthine–XO system in the presence and absence of various Fe(III) complexes and proteins were compared. Those with a free coordination site on the Fe atom effectively enhanced the OH generation.

Biofuel cells based on direct enzyme–electrode contacts using PQQ-dependent glucose dehydrogenase/bilirubin oxidase and modified carbon nanotube materials

Two types of carbon nanotube electrodes (1) buckypaper (BP) and (2) vertically aligned carbon nanotubes (vaCNT) have been used for elaboration of glucose/O2 enzymatic fuel cells exploiting direct electron transfer. For the anode pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ)GDH) has been immobilized on [poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid), PABMSA]-modified electrodes. For the cathode bilirubin oxidase (BOD) has been immobilized on PQQ-modified electrodes. PABMSA and PQQ act as promoter for enzyme bioelectrocatalysis. The voltammetric characterization of each electrode shows current densities in the range of 0.7–1.3mA/cm2.The BP-based fuel cell exhibits maximal power density of about 107µW/cm2 (at 490mV). The vaCNT-based fuel cell achieves a maximal power density of 122µW/cm2 (at 540mV). Even after three days and several runs of load a power density over 110µW/cm2 is retained with the second system (10mM glucose). Due to a better power exhibition and an enhanced stability of the vaCNT-based fuel cells they have been studied in human serum samples and a maximal power density of 41µW/cm2 (390mV) can be achieved.

Engineered, highly productive biosynthesis of artificial, lactonized statin side-chain building blocks: The hidden potential of Escherichia coli unleashed

We have recently reported the development of an efficient, whole-cell process for chemoenzymatic production of key chiral intermediates in statin synthesis by employing high-density Escherichia coli culture with the overexpressed deoxyribose-5-phosphate aldolase (DERA). The optically pure, 6-substituted cyclic hemiacetals can be used for the synthesis of atorvastatin, rosuvastatin and pitavastatin using further chemical steps. All of the synthetic routes established to date begin with a regiospecific oxidation of these lactol intermediates into the corresponding lactones, followed by several steps yielding 6-substituted, open-chain or lactonized derivatives which can be coupled by various approaches with the heterocyclic part of the statin molecule. Here we report for the first time the use of PQQ-dependent glucose dehydrogenases for a highly efficient, regioselective oxidation of artificial, derivatized aldohexoses, more specifically, the statin lactol intermediates. First, PQQ-dependent dehydrogenases of both soluble and membrane-bound type were characterized for their activity toward various DERA-derived lactols. Further, we describe a highly productive whole-cell system for oxidation of these 2,4-dideoxyaldopyranoses using a PQQ-dependent glucose dehydrogenase (Gcd) overexpressed in E. coli while taking advantage of the respiratory chain as the mediator of the electron transfer to oxygen. Finally, a two-step artificial biosynthetic pathway was developed by unleashing the intrinsic genetic potential of E. coli. The combined overexpression of the endogenous DERA and the membrane-bound, PQQ-dependent glucose dehydrogenase, the latter being coupled to the respiratory chain, allows direct biosynthesis of 6-substituted lactones in a highly productive, high-yield, cost-effective and industrially scalable process.

Polymers Based on Sulfonated Polyanillines for Direct Electron Transfer with (PQQ)GDH and Application in Carbon Nanostructure-Based Biofuel Cells

Dopant-functionalized anilines with improved electrocatalytic properties are promising building blocks for the construction of bioelectronic devices [1]. The present study is devoted to the use of polyanillines possessing different substitution patterns in the interaction with the enzyme PQQ-GDH (pyrroloquinoline quinone dependent glucose dehydrogenase). The aim is to obtain an electron transfer from the substrate reduced enzyme to the polymer without additional shuttle molecules. This has been first studied in solution and then transferred to a surface in order to build a reagentless enzyme electrode.6 polymers have been prepared from different mixtures of sulfoxy-, methoxy- and carboxy-substituted aniline by chemical synthesis and characterized by UV/VIS, IR and NMR spectroscopy. The substitution pattern influences the reactivity of the different polymers with the enzyme in solution, however when fixed on electrode surfaces the potential can be effectively used to enforce the reaction [2].One of the polymers (poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid) has been applied in modifying carbon nanotube based electrode structures and coupling (PQQ)GDH for construction of a bioanode. The first fuel cell uses MWCNT-based bucky paper as electrode material. The PQQ-GDH has been covalently coupled to the polymer. The glucose oxidation of this electrode achieves a current density of 700μA/cm2. As cathode PQQ modified bucky paper with covalently bound BOD (bilirubin oxidase) is applied. For this electrode a start potential of about 0.5V vs. Ag/AgCl and a maximum current density of 1mA/cm2 can be observed. The biofuel cell resulting from the bucky paper electrodes shows current densities up to about 100μW/cm2 in a 10mM glucose solution.In a second approach vertically aligned carbon nanotubes (vaCNT) have been used for the electrode construction. Both enzymes have been immobilized in a similar way as compared to bucky paper. The vaCNT-based fuel cell achieves a maximum power density of 125µW/cm2. Even after three days and several runs of load a power density of more than 110µW/cm2 is retained (quiescent solution, 10mM glucose, room temperature). Furthermore it can be shown that this biofuel cell operates in human serum samples. [1] Wallace GG, Kane-Maguire LAP. Manipulating and monitoring biomolecular interactions with conducting electroactive polymers. Adv Mater 511 2002;14:953–60. [2] Sarauli D et al. Differently substituted sulfonated polyanilines: The role of polymer compositions in electron transfer with pyrroloquinoline quinone-dependent glucose dehydrogenase Acta Biomater 2013; 9: 8290-8298