Modifications chimiques et évolution dirigée de la formiate déshydrogénase de Candida boidinii : vers une compréhension de la relation structure/fonction d’une déshydrogénase en liquide ionique

Abstract : The dehydrogenases are weakly active in the presence of high concentration (> 50% (v/v)) of water-miscible ionic liquids (ILs) and the mechanism of enzyme inactivation in ILs is not fully understood. The structure of the formate dehydrogenase Candida boidinii (FDH) in ILs has been studied by quenching of fluorescence experiments in the presence of iodide or acrylamide. A critical concentration, the CILc (“Critical Ionic liquid concentration"), is defined as the concentration of IL above which the fluorescence is not relevant. In this work, the CILc is comprised between 30 and 40 % (v/v) of the ILs. The ILs have revealed to be denaturing agents which increase the quenching of fluorescence efficiency by 1.4 to 2 times more than in urea. The FDH is chemically modified by analogous cations found in ILs in order to preserve the biocatalyst from the direct interaction with ILs. The enzymes modified by the smaller cations shown 30 45% residual activity at 70% (v/v) of ILs while the native enzyme is fully inactive. In the presence of 30% (v/v) of ILs, the kinetic efficiency (kcat/KM) of the grafted enzymes is improved by a 1.3-3.6 fold factor and the constant of Michaelis (KM) is reduced by a 1.7-4.6 fold factor depending on the grafted cations. In aqueous solution, the half-lives of modified enzymes are 3 to 5 fold higher than the native FDH depending on the size of the cation grafted. Finally, the structure of the grafted enzymes is somehow maintained in the presence of 40% (v/v) of ILs while the native FDH begins to unfold. We also used directed evolution to improve the FDH activity in the presence of ILs. At this stage, 987 mutants have been screened and one mutant (M60) shows 35% of residual activity at 70% (v/v) of ILs. In the presence of 30% (v/v) of ILs, the mutant binds the substrates in a greater extent than the native FDH, the values of KMNAD and the KDN3 (N3 is the azide, a competitive inhibitor of formate) are reduced by a 2 fold factor by comparison to the native enzyme.
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Mourad Bekhouche. Modifications chimiques et évolution dirigée de la formiate déshydrogénase de Candida boidinii : vers une compréhension de la relation structure/fonction d’une déshydrogénase en liquide ionique. Sciences agricoles. Université Claude Bernard - Lyon I, 2011. Français. ⟨NNT : 2011LYO10209⟩. ⟨tel-00834194⟩

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