Nicolas Mano
Centre national de la recherche scientifique
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Featured researches published by Nicolas Mano.
Biosensors and Bioelectronics | 2013
Nicolas Mano; Lise Edembe
Bilirubin oxidases, a sub class of the Multicopper oxidases family, were discovered in 1981 by Tanaka and Murao (Murao and Tanaka, 1981) and first used for the detection of bilirubin. Since 2001 and the pioneering work of Tsujimura, these BODs have attracted a lot of attention for the reduction of O2. Unlike laccases, these BODs are stable in physiological conditions (20mM phosphate buffer, pH 7.4, 0.14 M NaCl, 37 °C) and more than 120 papers have been published in the last 7 years. Here, we will first briefly describe some general features of BODs and then review the use of BODs for bilirubin biosensors and the recent achievements and progress toward the elaboration of efficient O2 reducing cathodes.
Energy and Environmental Science | 2011
Victoria Flexer; Nicolas Brun; Olivier Courjean; Rénal Backov; Nicolas Mano
In the present study we have shown that carbonaceous micro/macrocellular foams can be used for efficient and stable non-specific enzyme entrapment. In this context, Bilirubin Oxidase adsorbed into the porous electrode is able to reduce O2 to water and electrons are transferred directly from the electrode to the enzyme without the need of a redox mediator. The reduction current is stable for several days under continuous operation and therefore we consider the carbonaceous foams are very promising candidates for the construction of 3-dimensional biofuel cell cathodes. Mediator free, the electrode preparation and further enzyme adsorption are extremely simple, low cost and versatile. Most important, the excellent mechanical strength and the synthetic route allow us to design at will the size and external shape of the electrodes, which are of vital importance if we wish to incorporate electrodes into devices. The Integrative Chemistry synthetic route allows accessing hierarchical porosities with the advantage of specific functionalities. Macropores serve to fuel transport and micropores as anchoring sites for enzyme entrapment. Despite the inability to reach the Levich diffusion limited current suggesting that the porosity is not yet fully optimized, the results presented here show that porous carbonaceous foam electrodes allow for an incredible increase in enzyme loading which allows for a 500-fold current enhancement and stabilization of the direct electron transfer current from few hours to several days as compared to conventional flat electrodes.
Journal of Biotechnology | 2011
Olivier Courjean; Nicolas Mano
GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k(cat)/K(M)(glucose)=93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k(cat)/K(M)(glucose)=27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM(ox)) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependent FM(ox) reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k(cat)/K(M)(FM(ox))=27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.
Chemical Reviews | 2017
Nicolas Mano; Anne de Poulpiquet
Catalytic four-electron reduction of O2 to water is one of the most extensively studied electrochemical reactions due to O2 exceptional availability and high O2/H2O redox potential, which may in particular allow highly energetic reactions in fuel cells. To circumvent the use of expensive and inefficient Pt catalysts, multicopper oxidases (MCOs) have been envisioned because they provide efficient O2 reduction with almost no overpotential. MCOs have been used to elaborate enzymatic biofuel cells (EBFCs), a subclass of fuel cells in which enzymes replace the conventional catalysts. A glucose/O2 EBFC, with a glucose oxidizing anode and a O2 reducing MCO cathode, could become the in vivo source of electricity that would power sometimes in the future integrated medical devices. This review covers the challenges and advances in the electrochemistry of MCOs and their use in EBFCs with a particular emphasis on the last 6 years. First basic features of MCOs and EBFCs are presented. Clues provided by electrochemistry to understand these enzymes and how they behave once connected at electrodes are described. Progresses realized in the development of efficient biocathodes for O2 reduction relying both on direct and mediated electron transfer mechanism are then discussed. Some implementations in EBFCs are finally presented.
ChemPhysChem | 2013
Marine Cadet; Xavier Brilland; Sébastien Gounel; Frédéric Louërat; Nicolas Mano
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
Protein Expression and Purification | 2013
Fabien Durand; Sébastien Gounel; Nicolas Mano
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 644Umg(-1). The kinetic parameters, k(cat) and K(M), determined at 37 °C and optimal pH are 1372 s(-1) and 307 μM for ABTS and, 1.29 s(-1) and 10.9 μM, 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.
Energy and Environmental Science | 2017
Ievgen Mazurenko; Karen Monsalve; Pascale Infossi; Marie-Thérèse Giudici-Orticoni; Frederic Topin; Nicolas Mano; Elisabeth Lojou
Using redox enzymes as biocatalysts in fuel cells is an attractive strategy for sustainable energy production. Once hydrogenase for H2 oxidation and bilirubin oxidase (BOD) for O2 reduction have been wired on electrodes, the enzymatic fuel cell (EFC) thus built is expected to provide sufficient energy to power small electronic devices, while overcoming the issues associated with scarcity, price and inhibition of platinum based catalysts. Despite recent improvements, these biodevices suffer from moderate power output and low stability. In this work, we demonstrate how substrate diffusion and enzyme distribution in the bioelectrodes control EFC performance. A new EFC was built by immobilizing two thermostable enzymes in hierarchical carbon felt modified by carbon nanotubes. This device displayed very high power and stability, producing 15.8 mW h of energy after 17 h of continuous operation. Despite the large available electrode porosity, mass transfer was shown to limit the performance. To determine the optimal geometry of the EFC, a numerical model was established, based on a finite element method (FEM). This model allowed an optimal electrode thickness of less than 100 μm to be determined, with a porosity of 60%. Thanks to very efficient enzyme wiring and high enzyme loading, non-catalytic signals for both enzymes were detected and quantified, enabling the electroactive enzyme distribution in the porous electrode to be fully determined for the first time. High total turnover numbers, approaching 107 for BOD and 108 for hydrogenase, were found, as was an impressive massic activity of 1 A mg−1 with respect to the mass of the electroactive enzyme molecules. This strategy, relying on stable enzymes and mesoporous materials, and the model set up may constitute the basis for a larger panel of bioelectrodes and EFCs.
Bioelectrochemistry | 2012
Emmanuel Suraniti; Margot Abintou; Fabien Durand; Nicolas Mano
Here we show that the magnitude of the O(2) reduction current of cathodes based on Bilirubin oxidases (BOD) immobilized into a redox hydrogel strongly depends on the drying conditions such as the curing time and temperature of drying as well as the thermostability of the BOD. To illustrate this effect, we performed experiments with two different BODs: one labile BOD from Trachyderma tsunodae and one highly thermostable BOD from Bacillus pumilus with different preparation protocols. The balance between the kinetics of formation of the hydrogel and the enzyme stability leads to optimal drying conditions of 2h at 25°C for both types of BODs when the most widespread protocol uses 18 hours at ambient temperature. For drying times longer than two hours, the catalytic current decreases because of the instability of T. tsunodae. Finally the optimal conditions for BOD from T. tsunodae lead to a faster preparation of electrodes than with the protocol currently in use (2h vs. 18h) and catalytic currents for oxygen reduction 100% higher (1040μA/cm(2) vs. 517μA/cm(2)).
Biochimie Open | 2017
Claire Stines-Chaumeil; Elodie Roussarie; Nicolas Mano
Multicopper oxidases (MCOs) catalyzed two half reactions (linked by an intramolecular electron transfer) through a Ping-Pong mechanism: the substrate oxidation followed by the O2 reduction. MCOs have been characterized in details in solution or immobilized on electrode surfaces. The nature of the rate-limiting steps, which is controversial in the literature, is discussed in this mini review for both cases. Deciphering such rate-limiting steps is of particular importance to efficiently use MCOs in any applications requiring the reduction of O2 to water.
Journal of Biotechnology | 2016
Sébastien Gounel; Jad Rouhana; Claire Stines-Chaumeil; Marine Cadet; Nicolas Mano
Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expression of multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins due to the lack of copper and/or unavailability of chaperone proteins. The general strategies reported to overcome this issue have been focused on increasing the intracellular copper concentration. Here we report 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 strain from BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on the protein production and specific activity. Our strategy allowed increasing the final amount of enzyme by 858% and its catalytic rate constant by 83%.