Sébastien Gounel

Bilirubin oxidase from Bacillus pumilus: A promising enzyme for the elaboration of efficient cathodes in biofuel cells

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 O(2) 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.

Emulsion-Templated Macroporous Carbons Synthesized By Hydrothermal Carbonization and their Application for the Enzymatic Oxidation of Glucose

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 cm(3)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 Sm(-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 m(2)g(-1) and 0.313 cm(3)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.

An enzymatic glucose/O2 biofuel cell operating in human blood.

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.

Design of a Highly Efficient O2 Cathode Based on Bilirubin Oxidase from Magnaporthe oryzae

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

Purification and characterization of a new laccase from the filamentous fungus Podospora anserina.

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.

Two-electron Reduction versus One-electron Oxidation of the Type 3 Pair in the Multicopper Oxidases

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 Cus is oxidized, and the resting oxidized form (RO), where all three TNC Cus 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, metastable 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 metastable, half reduced state and thus the complete reductive activation of the enzyme for catalysis.

Mechanism of Chloride Inhibition of Bilirubin Oxidases and Its Dependence on Potential and pH

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.

Low-Molecular-Weight Hydrogels as New Supramolecular Materials for Bioelectrochemical Interfaces

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.

Increasing the catalytic activity of Bilirubin oxidase from Bacillus pumilus: Importance of host strain and chaperones proteins

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%.

Direct Electrochemistry of Bilirubin Oxidase from Magnaporthe orizae on Covalently-Functionalized MWCNT for the Design of High-Performance Oxygen-Reducing Biocathodes

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).