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Déshydrogénases et laccases sur des surfaces redox : vers des applications en bioélectronique

Abstract : In order to design a bioanode working with NAD+-dependent-dehydrogenases, we have studied a molecular assembly on carbon electrodes allowing for the immobilization of nicotinic cofactors. An electopolymer of toluidin blue O (TBO) formed at the surface of vitreous carbon electrodes was used as an electrocatalyst to induce NADH oxidation by Mediated Electron Transfer (MET). In an effort to immobilize the nicotinic cofactor on the TBO electropolymer, the 3-aminophenylboronic acid (3AB) was used as a linker molecule. Surface plasmon resonance imaging (SPRi) and electrochemistry measures were carried out on gold electrodes modified by electrografted 3AB previously diazoted. These electrodes are able to immobilize the NAD+ with a molecular density of 0.3 ± 0.03 pmol.mm-2. The linear voltammetry measures have shown that in presence of formate dehydrogenase (FDH) and formate, these electrodes provide a power density of 14 μW.cm-2 at +690 mV (vs.Ag/AgCl). Next, an electrochemistry method enabling the measurement of oxidoreductase activities was developed. This method works with a device able to perform simultaneously intermittent pulse amperometry (IPA) on 96 screen-printed carbon electrodes. This device was used for the NADH oxidation by MET using electrografted redox phenazine mediators. The electrografted TBO had a NADH sensibility of 3586 ± 164 nA.mM-1. This construct was used to screen a library of FDH mutants. A p-value lower than 0.001 (p-value < 0,001, n = 77) showed a high correlation between the results obtained via electrochemical screening and colorimetric screening using PES/NBT couple (Phénazine Ethosulfate/Nitrobleu de tétrazolium). This method was also used to measure the Direct Electron Transfer (DET) with the laccase from Trametes versicolor (TvLac) adsorbed onto 96 carbon electrodes with a coefficient of variation of 22%. This DET can be measured at +100 mV (vs. Ag/AgCl) and at pH 5.4 with a linear range of adsorbed TvLac between 0.5 ng and 75 ng (corresponding to an enzymatic activity of 62 X 10-6 U et 9.37 X 10-3 U) and a sensibility of 3027 μg-1. In addition, this electrochemical device was used to evaluate the inhibitor effect of the DET of fluoride (F-) and azide (N3 -) yielding IC50 values of 280 ± 14 μM et 1.2 ± 0.4 μM. The validation of this study has subsequently allowed to initiate the establishment of a protocol screening for the directed evolution of CotA laccase from Bacillus subtilis
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Submitted on : Wednesday, May 20, 2020 - 4:02:09 PM
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Sofiene Abdellaoui. Déshydrogénases et laccases sur des surfaces redox : vers des applications en bioélectronique. Biochimie, Biologie Moléculaire. Université Claude Bernard - Lyon I, 2013. Français. ⟨NNT : 2013LYO10194⟩. ⟨tel-02614066⟩

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