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Variants of the highly active [NiFeSe] hydrogenase from D. vulgaris Hildenborough that exhibit enhanced O2 tolerance were used as H2‐oxidation catalysts in H2/O2 biofuel cells. Two [NiFeSe] variants were electrically wired by means of low‐potential viologen‐modified redox polymers and evaluated with respect to H2‐oxidation and stability against O2 in the immobilized state. The two variants showed maximum current densities of (450±84) μA cm−2 for G491A and (476±172) μA cm−2 for variant G941S on glassy carbon electrodes and a higher O2 tolerance than the wild type. In addition, the polymer protected the enzyme from O2 damage and high‐potential inactivation, establishing a triple protection for the bioanode. The use of gas‐diffusion bioanodes provided current densities for H2‐oxidation of up to 6.3 mA cm−2. Combination of the gas‐diffusion bioanode with a bilirubin oxidase‐based gas‐diffusion O2‐reducing biocathode in a membrane‐free biofuel cell under anode‐limiting conditions showed unprecedented benchmark power densities of 4.4 mW cm−2 at 0.7 V and an open‐circuit voltage of 1.14 V even at moderate catalyst loadings, outperforming the previously reported system obtained with the [NiFeSe] wild type and the [NiFe] hydrogenase from D. vulgaris Miyazaki F., Triple protection: A stable, high‐current‐density‐based H2‐oxidation bioanode is presented. It is equipped with [NiFeSe] variants that show enhanced O2 tolerance, which are immobilized and wired to electrode surfaces with a low‐potential viologen‐modified polymer. The polymer acts simultaneously as a high‐potential and O2 shield. The triply protected bioanodes are incorporated in membrane‐free biofuel cells, which reveal benchmark performances at moderate catalyst loading.

This work is dedicated to a multi-scale modelling of coupled diffusion and reaction in a porous microelectrode operating in the Direct Electron Transfer mode. The pore-scale physico-electrochemical unsteady model is developed considering the oxygen reduction, catalyzed by an enzyme coating the pores of the electrode, coupled to the diffusion of oxygen and mass balance of enzymes. This model is formally upscaled to obtain an original closed unsteady macroscopic model operating at the electrode scale, together with the associated closure providing the effective diffusivity tensor. A validation of this model is carried out from a comparison with the solution of the initial 3D pore-scale governing equations considering the bilirubin oxydase as the catalyst. The relevance and accuracy of the macroscale model are proved allowing a considerable simulation speedup. It is further employed to successfully predict experimental voltammetry results obtained with porous gold electrodes functionnalized with a bilirubin oxidase mutant (BOD S362C). This model represents a breakthrough by providing an operational simple way of understanding and further optimizing porous electrodes functioning in DET mode.

International audience; High-potential multicopper oxidases (MCOs) are excellent catalysts able to perform the oxygen reduction reaction (ORR) at remarkably low overpotentials. Moreover, MCOs are able to interact directly with the electrode surfaces via direct electron transfer (DET), that makes them the most commonly used electrocatalysts for oxygen reduction in biofuel cells. The central question in MCO electrocatalysis is whether the type 1 (T1) Cu is the primary electron acceptor site from the electrode, or whether electrons can be transferred directly to the trinuclear copper cluster (TNC), bypassing the rate-limiting intramolecular electron transfer step from the T1 site. Here, using sitedirected mutagenesis and electrochemical methods combined with data modeling of electrode kinetics we have found that there is no preferential superexchange pathway for DET to the T1 site. However, due to the high reorganization energy of the fully oxidized TNC, electron transfer from the electrode to the TNC does occur primarily through the T1 site. We have further demonstrated that the lower reorganization energy of the TNC in its two-electron reduced, alternative resting, form enables DET to the TNC, but this only occurs in the first turnover. This study provides insight into the factors that control the kinetics of electrocatalysis by the MCOs and a guide for the design of more efficient biocathodes for the ORR.

International audience; Enzymatic reactions involving glucose hold the potential for building implantable biosensors and embedded power generators for various medical applications. While Biofuel cells (BFCs) such as enzymatic glucose/O2 are ensured to benefit from abundant chemical resources that can be harvested in the immediate environment of the human body, the highly critical in vivo kinetics of biofuel cell is not yet fully understood. Unfortunately, existing solutions for real-time monitoring of the reaction on rodents are not possible today, or too bulky, which has a biasing impact on the animal behavior. This work presents a light, battery-less, and wireless strategy to continuously monitor a BFC implanted in a laboratory rat using a Frequency Identification (RFID) link. An extremely lightweight and flexible tag antenna of footprint lower than 10 cm² is presented with communication capability above 60 cm in field environment. The operational capabilities are demonstrated with a 24-hour continuous monitoring of an enzymatic glucose/O2 reaction, both in vitro and in vivo.

International audience; Biofuel cells (BFCs) are electrochemical devices that rely on the transformation of chemical energy into electricity through biochemical pathways, however delivering only moderate or low power and voltage. This intrinsic limitation narrows their potential applications for driving electronics and thermodynamic systems with higher energy demands than what can be delivered by the BFCs alone. Nevertheless, coupling BFCs to electronic circuits, able to raise their voltage, allows circumventing these drawbacks. In this proof-of-concept study, we demonstrate an unconventional way of achieving highly efficient electrochemical corrosion protection of an iron surface in a chloride rich medium. The required protecting cathodic potential is generated by a self-powered bioelectronic system, consisting of a BFC, which can, despite its low voltage output (0.3 V), completely prevent interfacial corrosion if it is combined with an electronic boost converter.

We present a new heterogeneous biocatalyst based on the grafting of Bilirubin Oxidase from Bacillus pumilus into macrocellular Si(HIPE) materials dedicated to water treatment. Due to the host intrinsic high porosity and monolithic character, on-line catalytic process is reached. We thus used this biocatalyst toward uni-axial flux decolorizations of Congo Red and Remazol Brilliant Blue (RBBR) at two different pH (4 and 8.2), both in presence or absence of redox mediator. In absence of redox mediators, 40% decolorization efficiency was reached within 24 h at pH 4 for Congo Red and 80% for RBBR at pH 8.2 in 24 h. In presence of 10μM ABTS, it respectively attained 100% efficiency after 2h and 12h. We have also demonstrated that non-toxic species were generated upon dyes decolorization. These results show that unlike laccases, this new biocatalyst exhibits excellent decolorization properties over a wide range of pH. Beyond, this enzymatic-based heterogeneous catalyst can be reused during two months being simply stored at room temperature while maintaining its decolorization efficiency. This study shows that this biocatalyst is a promising and robust candidate toward wastewater treatments, both in acidic and alkaline conditions where in the latter efficient decolorization strategies were still missing.

International audience; The immobilization of bilirubin oxidase (BOD) on macroporous gold electrodes for the optimization of bioelectrocatalytic activity is described. A bilirubin oxidase mutant S362C (cys-BOD) engineered with a cysteine residue located on purpose at the enzyme surface close to the T1 active center was used. It allows the attachment in one-step of a self-assembled monolayer of the enzyme to gold through a reaction between the thiol group of the cysteine residue and the metal surface. BOD immobilization of wild type and S362C mutant in macroporous gold electrodes allowed high retention of activity and perfect control of the overall BOD loading due to the fine-tuning of the macroporous structure. The macroporous arrangement together with the use of cys-BOD makes these rationally designed enzyme-modified electrodes very promising candidates for high-performance bioelectrocatalytic devices with improved activity and stability.

International audience; We report the fabrication of a polymer/enzyme based biosupercapacitor (BSC)/biofuel cell (BFC) hybrid device with optimized cell voltage that can be switched on demand from energy conversion to energy storage mode. The redox polymer matrices used for the immobilization of the biocatalyst at the bioanode and biocathode act simultaneously as electron relays between the integrated redox enzymes and the electrode surface (BFC) and as pseudo-capacitive charge storing elements (BSC). Moreover, due to the self-charging effect based on the continuously proceeding enzy-matic reaction, a Nernstian-shift in the pseudo-capacitive elements, i.e. in the redox polymers, at the individual bioelectrodes leads to a maximized open circuit voltage of the device in both operating modes. Comparison with a conventional fuel cell design, i.e. using redox mediators with redox potentials that are close to the potentials of the used redox proteins, indicates that the novel hybrid device shows a similar voltage output. Moreover, our results demonstrate that the conventional design criteria commonly used for the development of redox polymers for the use in biofuel cells have to be extended by considering the effect of a Nernstian-shift towards the potentials of the used biocatalysts in those pseudo-capacitive elements.

In this work we extended the generic approach for the site-directed immobilization of enzymes based on maleimide\thiol coupling of engineered enzymes to the oriented immobilization of variants of bilirubin oxidase from Magnaporthe oryzae (MoBOD) to electrodes. We show that this approach leads to the stable attachment of the enzyme to the electrode surface and that the immobilised MoBOD variants are active for bioelectrocatalytic reduction of di-oxygen through direct (unmediated) electron transfer (DET) from the electrode. For the three MoBOD variants studied significant differences are observed in the kinetics of DET that relate to the orientation of the enzyme and the distance of the T1 site from the electrode surface. The stability of the immobilized enzymes allows us to compare the DET and mediated electron transfer (MET) pathways and to investigate the effects of pH and Cl‾. Our studies show a change in the slope of pH dependence at pH 6.0 and highlight the effect of Cl‾ on the direct oxygen reduction by MoBOD as a function of pH for the immobilized enzyme and the interconversion of the resting oxidized (RO) form of the immobilized enzyme and the alternative resting (AR) state formed in the presence of Cl‾.

Chemical systems do not allow the coupling of energy from several simple reactions to drive a subsequent reaction, which takes place in the same medium and leads to a product with a higher energy than the one released during the first reaction. Gibbs energy considerations thus are not favorable to drive e.g., water splitting by the direct oxidation of glucose as a model reaction. Here, we show that it is nevertheless possible to carry out such an energetically uphill reaction, if the electrons released in the oxidation reaction are temporarily stored in an electromagnetic system, which is then used to raise the electrons’ potential energy so that they can power the electrolysis of water in a second step. We thereby demonstrate the general concept that lower energy delivering chemical reactions can be used to enable the formation of higher energy consuming reaction products in a closed system., Most chemical reactions proceed downhill without external energy input. Here, authors employ an electronic flyback and a boost converter to store energy and spontaneously drive an uphill reaction. The concept is exhibited by an enzymatic biofuel cell, driving water splitting in a single compartment.

Bilirubin oxidases (BODs) belong to the multi-copper oxidase (MCO) family and efficiently reduce O2 at neutral pH and in physiological conditions where chloride concentrations are over 100 mM. BODs were consequently considered to be Cl− resistant contrary to laccases. However, there has not been a detailed study on the related effect of chloride and pH on the redox state of immobilized BODs. Here, we investigate by electrochemistry the catalytic mechanism of O2 reduction by the thermostable Bacillus pumilus BOD immobilized on carbon nanofibers in the presence of NaCl. The addition of chloride results in the formation of a redox state of the enzyme, previously observed for different BODs and laccases, which is only active after a reductive step. This behavior has not been previously investigated. We show for the first time that the kinetics of formation of this state is strongly dependent on pH, temperature, Cl− concentration and on the applied redox potential. UV-visible spectroscopy allows us to correlate the inhibition process by chloride with the formation of the alternative resting form of the enzyme. We demonstrate that O2 is not required for its formation and show that the application of an oxidative potential is sufficient. In addition, our results suggest that the reactivation may proceed thought the T3 β.

Malvidin-3-O-galactoside, a widely occurring anthocyanin, was incubated with laccases (LCCs) from the basidiomycetes Pleurotus pulmonarius (Ppu) and Funalia trogii (Ftr), and with bilirubin oxidases (BODs) from Bacillus pumilus (Bpu) and Magnaporthe oryzae (Mor). LC-MS and LC-MS2 analyses identified 2,6-dimethoxy-1,4-benzoquinone (DMBQ) and tentatively identified the corresponding coumarin galactoside fragment (m/z 355) resulting from the cleavage of the substrate. The enzymes cleaved malvidin-3-O-galactoside within a few minutes under acidic and neutral conditions and in the absence of a mediator. The fungal LCCs were more active at pH 4 and 5.5, while the BODs worked best in the neutral range. The initial cleavage product coumarin galactoside was further oxidised, as indicated by a continuous decline of concentration and concurrent formation of a brown precipitate. Based on the structure of the products and the general LCC mechanism, a reaction sequence was proposed starting with a stable tertiary carbocation, attack of molecular oxygen and formation of a 1,2-dioxetane intermediate between B and C rings which is finally split into the two carbonyl products. Similar reactions may be catalysed by plant oxidases in anthocyanin-rich tissues resulting in the formation of the strong antibacterial and cytotoxic DMBQ.

Herein, the direct electrochemistry of bilirubin oxidase from Magnaporthe orizae (MoBOD) was studied on CNTs functionalized by electrografting several types of diazonium salts. The functionalization induces favorable or unfavorable orientation of MoBOD, the latter being compared to the well-known BOD from Myrothecium verrucaria (MvBOD). On the same nanostructured electrodes, MoBOD can surpass MvBOD in terms of both current densities and minimal overpotentials. Added to the fact that MoBOD is also highly active at the gas-diffusion electrode (GDE), these findings make MoBOD one of the MCOs with the highest catalytic activity towards the oxygen reduction reaction (ORR).

International audience; A biocathode was designed by the modification of a carbon nanotube (CNT) gas-diffusion electrode with bilirubin oxidase from Bacillus pumilus, achieving high current densities up to 3 mA cm−2 for the reduction of O2 from air. A membraneless air-breathing hydrogen biofuel cell was designed by combination of this cathode with a functionalized CNT-based hydrogenase anode.

6 pages; International audience; A new laccase from the filamentous fungus Podospora anserina has been isolated and identified. The 73 kDa protein containing 4 coppers, truncated from its first 31 amino acids, was successfully overexpressed in Pichia pastoris and purified in one step with a yield of 48% and a specific activity of 644 U mg-1. The kinetic parameters, kcat and KM, determined at 37 -C and optimal pH are 1372 s-1 and 307 M for ABTS and, 1.29 s-1 and 10.9 lM, for syringaldazine (SGZ). Unlike other laccases, the new protein displays a better thermostability, with a half life > 400 min at 37 °C, is less sensitive to chloride and more stable at pH 7. Even though, the new 566 amino-acid enzyme displays a large homology with Bilirubin oxidase (BOD) from Myrothecium verrucaria (58%) and exhibits the four histidine rich domains consensus sequences of BODs, the new enzyme is not able to oxidize neither conjugated nor unconjugated bilirubin.

The O2 reactivity of flavoproteins and particularly, glucose oxidase, is a limitation for numerous biotechnological applications, and this has been the subject of intense research. In the present study, we performed site-directed mutations on Val464 of GOx from Penicilium amagasakiense and combined steady-state, rapid kinetics and electrochemistry to investigate the effect of such mutations on the O2 reactivity. We propose that replacing the non-polar Valine with a polar Serine permits to decrease the diffusion/stabilization of O2 near the active redox center without altering the oxidation of glucose. When incorporated in osmium hydrogels, the resulting electrodes were insensitive to O2 in the 0.5–60 mM glucose concentration range and were 4.7 times more stable under continuous operation than similar electrodes made with the native GOx. To the best of our knowledge, this is the first example in which O2 sensitivity in GOx can be significantly decrease without altering the electrochemical efficiency for glucose oxidation.

Controlling the interface between biological tissues and electrodes remains an important challenge for the development of implantable devices in terms of electroactivity, biocompatibility, and long-term stability. To engineer such a biocompatible interface a low molecular weight gel (LMWG) based on a glycosylated nucleoside fluorocarbon amphiphile (GNF) was employed for the first time to wrap gold electrodes via a noncovalent anchoring strategy, that is, self-assembly of GNF at the electrode surface. Scanning electron microscopy (SEM) studies indicate that the gold surface is coated with the GNF hydrogels. Electrochemical measurements using cyclic voltammetry (CV) clearly show that the electrode properties are not affected by the presence of the hydrogel. This coating layer of 1 to 2 μm does not significantly slow down the mass transport through the hydrogel. Voltammetry experiments with gel coated macroporous enzyme electrodes reveal that during continuous use their current is improved by 100% compared to the noncoated electrode. This demonstrates that the supramolecular hydrogel dramatically increases the stability of the bioelectrochemical interface. Therefore, such hybrid electrodes are promising candidates that will both offer the biocompatibility and stability needed for the development of more efficient biosensors and biofuel cells.

4 pages; International audience; Fungi for the better: The so far highest known current density (1.37 mA cm−2) for the enzymatic O2 reduction under physiological conditions is reported. This is achieved by the design of a new redox polymer with an increased catalytic site density and by using a new bilirubin oxidase (BOD) from Magnaporthe oryzae

Enzymatic biofuel cells (BFCs) may power implanted medical devices and will rely on the use of glucose and O2 available in human bodily fluids. Other than well-established experiments in aqueous buffer, little work has been performed in whole human blood because it contains numerous inhibiting molecules. Here, we tested our BFCs in 30 anonymized, random and disease-free whole human blood samples. We show that by designing our anodic and cathodic bioelectrocatalysts with osmium based redox polymers and home-made enzymes we could reach a high selectivity and biofunctionnality. After optimization, BFCs generate power densities directly proportional to the glycaemia of human blood and reached a maximum power density of 129µWcm(-2) at 0.38V vs. Ag/AgCl at 8.22mM glucose. This is to our knowledge the highest power density attained so far in human blood and open the way for the powering of integrated medical feedback loops.

International audience; Here we detail an optimized transparent capacitive glucose oxidizing bioanode, capable of supplying current densities of 10 mAcm¢2 at applied potentials of 0.1 V–0.2V versus saturated calomel electrode, when continuously performing in a simple phosphate buffer, pH 7.4 and artificial human tears, both with a glucose concentration of 0.05 mM only. When operating in pulsemode, the bioanode was able to deliver current densities as high as 21 mAcm¢2 at the beginning of the pulse with 571 mCcm¢2 total charges stored. The biogenic part of the enzymatic device was a recombinant glucose oxidase mutant from Penicillium amagasakiense with high catalytic efficiency towards glucose, up to 14.5·104 M¢1 s¢1. The non-biogenic part of the anodic system was based on a poly(3,4-ethylenedioxythiophene)-graphene nanocomposite, as a highly capacitive component with a capacitance density in the 1 mFcm¢2 range, multi-walled carbon nanotubes, as an additional nanostructuring element, anda conductive organic complex, as an electron shuttle between the redox enzyme and the electrode surface. The bioanode could potentially serve as a prototype of a double-function enzymatic anode for hybrid electric power biodevices, energizing smart contact lenses.

4 pages; International audience; While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu−Cu distance, while the alternative resting form shows unusually small A∥ and high g∥ EPR features, lack of 330 nm absorption intensity, and a long T3 Cu−Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis.

A new generation of mediatorless H2/O2 biofuel cells was designed based on a hyperthermophilic O2-tolerant hydrogenase and a thermostable bilirubin oxidase both immobilized on carbon nanofibers. A power density up to 1.5 ± 0.2 mW·cm−2 at 60 °C was reached. This first demonstration of a H2/O2 biofuel cell able to deliver electricity over a wide range of temperatures, from 30 °C up to 80 °C, and over a large pH window, allows considering this device as an alternative power supply for small portable applications in various environments, including extreme ones. Keywords: Enzymatic H2/O2 biofuel cell, Hydrogenase, Bilirubin oxidase, Thermostable enzymes, Direct electron transfer, Carbon nanofiber

International audience; Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expressionof multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins dueto the lack of copper and/or unavailability of chaperone proteins. The general strategies reported toovercome this issue have been focused on increasing the intracellular copper concentration. Here wereport a complementary method to optimize the expression in E. coli of a promising Bilirubin oxidase(BOD) isolated from Bacillus pumilus. First, as this BOD has a disulfide bridge, we switched E.coli strainfrom BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterialcytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on theprotein production and specific activity. Our strategy allowed increasing the final amount of enzyme by858% and its catalytic rate constant by 83%

Multicopper Oxidases (MCOs) utilize an electron shuttling Type 1 Cu (T1) site in conjunction with a mononuclear Type 2 (T2) and a binuclear Type 3 (T3) site, arranged in a trinuclear copper cluster (TNC), to reduce O2 to H2O. Reduction of O2 occurs with limited overpotential indicating that all the coppers in the active site can be reduced via high-potential electron donors. Two forms of the resting enzyme have been observed in MCOs: the Alternative Resting form (AR), where only one of the three TNC Cu’s is oxidized, and the Resting Oxidized form (RO), where all three TNC Cu’s are oxidized. In contrast to the AR form, we show that in the RO form of a high-potential MCO, the binuclear T3 Cu(II) site can be reduced via the 700 mV T1 Cu. Systematic spectroscopic evaluation reveals that this proceeds by a two-electron process, where delivery of the first electron, forming a high energy, meta-stable half reduced T3 state, is followed by the rapid delivery of a second energetically favorable electron to fully reduce the T3 site. Alternatively, when this fully reduced binuclear T3 site is oxidized via the T1 Cu, a different thermodynamically favored half oxidized T3 form, i.e. the AR site, is generated. This behavior is evaluated by DFT calculations, which reveal that the protein backbone plays a significant role in controlling the environment of the active site coppers. This allows for the formation of the meta-stable, half reduced state and thus the complete reductive activation of the enzyme for catalysis.

3 pages; International audience; Here we report, for the first time, the efficient reduction of O2 in serum using a glassy carbon electrode modified with a new bilirubin oxidase from Bacillus pumilus and a polycationic redox polymer, without coating the electrode with any protective membrane. After 3.3 h of continuous operation in serum, the new cathode only lost 9% of its current density while an electrode made with Trachyderma tsunodae lost more than 38% within the same period of time. This result, in combination with the high thermostability, low sensitivity to chloride anions and high activity, makes the new BOD a promising candidate for the development of cathodes for implanted biofuel cells.

International audience; Carbon-based monoliths have been designed using a simple synthetic pathway based on using high internal phase emulsion (HIPE) as a soft template to confine the polymerization and hydrothermal carbonization of saccharide derivatives (furfural) and phenolic compounds (phloroglucinol). Monosaccharides can be isolated from the cellulosic fraction of lignocellulosic biomass and phloroglucinol can be extracted from the bark of fruit trees; however, this approach constitutes an interesting sustainable synthetic route. The macroscopic characteristics can be easily modulated; a high macroporosity and total pore volume of up to 98 % and 18 cm3 g−1 have been obtained, respectively. After further thermal treatment under inert atmosphere, the as-synthesized macroporous carbonized HIPEs (carbo-HIPEs) have shaping capabilities relating to interesting mechanical properties as well as a high electrical conductivity of up to 300 S m−1. These conductive foams exhibit a hierarchical structure associated with the presence of both meso- and micropores that exhibit specific Brunauer-Emmett-Teller (BET) surface areas and DFT total pore volumes up to 730 m2 g−1 and 0.313 cm3 g−1, respectively. Because of their attractive structural characteristics and intrinsic properties, these macroporous monoliths have been incorporated as a proof of principle within electrochemical devices as modified thin carbon disc electrodes. A promising two-fold improvement in the catalytic current is observed for the electrooxidation of glucose after the immobilization of a glucose oxidase-based biocatalytic mixture onto the carbo-HIPE electrodes compared to that observed if using commercial glassy carbon electrodes.

3 pages; International audience; Here we uncover a key amino acid at the entrance of the active site of glucose oxidase from Penicilium amagasakiense and we successfully redesign it by nonactive site mutations, leading to enzymatic anodes with 2.4 fold higher current densities. The increase in current density could be correlated with a better interaction between the negatively charged amino acid located in position 424 and the positively charged redox mediator. We also demonstrate that this increase in current was not specific to Osmium and that further introduction of negative charges around the active site of glucose oxidase lead to a decrease in current density, illustrating the uniqueness of this amino acid in position 424

7 pages; International audience; A CotA multicopper oxidase (MCO) from Bacillus pumilus, previously identified as a laccase, has been studied and characterized as a new bacterial bilirubin oxidase (BOD). The 59 kDa protein containing four coppers, was successfully over-expressed in Escherichia coli and purified to homogeneity in one step. This 509 amino-acid enzyme, having 67% and 26% sequence identity with CotA from Bacillus subtilis and BOD from Myrothecium verrucaria, respectively, shows higher turnover activity towards bilirubin compared to other bacterial MCOs. The current density for O2 reduction, when immobilized in a redox hydrogel, is only 12% smaller than the current obtained with Trachyderma tsunodae BOD. Under continuous electrocatalysis, an electrode modified with the new BOD is more stable, and has a higher tolerance towards NaCl, than a T. tsunodae BOD modified electrode. This makes BOD from B. pumilus an attractive new candidate for application in biofuel cells (BFCs) and biosensors.

2 pages; International audience; We report a two-step protocol for the synthesis of 7,8-dichlororiboflavin with a 2.5-fold increase in yield compared to previous five-step protocol methods.

10 pages; International audience; A novel bilirubin oxidase (BOD), from the rice blast fungus Magnaporthe oryzae, has been identified and isolated. The 64-kDa protein containing four coppers was successfully overexpressed in Pichia pastoris and purified to homogeneity in one step. Protein yield is more than 100 mg for 2 L culture, twice that of Myrothecium verrucaria. The kcat/Km ratio for conjugated bilirubin (1,513 mM−1 s−1) is higher than that obtained for the BOD from M. verrucaria expressed in native fungus (980 mM−1 s−1), with the lowest Km measured for any BOD highly desirable for detection of bilirubin in medical samples. In addition, this protein exhibits a half-life for deactivation >300 min at 37 °C, high stability at pH7, and high tolerance towards urea,making it an ideal candidate for the elaboration of biofuel cells, powering implantable medical devices. Finally, this new BOD is efficient in decolorizing textile dyes such as Remazol brilliant Blue R, making it useful for environmentally friendly industrial applications.