Lignocellulose-active enzymes play a central role in the deconstruction of plant cell wall materials. But their activity is often restricted by sterical hindrances that limit their accessibility to substrate and by non-specific interactions that stick enzymes far from their substrate. Generally, the biochemical characterization of enzymes is performed with pure isolated polymers or synthetic molecules, which do not reflect the chemical and structural complexity of plant cell walls. Thus there is a need to better assess the interactions of enzymes with relevant materials.To achieve this purpose, we have first designed bioinspired model assemblies that contain some of the polymers and covalent interactions found in plant cell walls. These assemblies contain feruloylated arabinoxylans (FAXs), cellulose nano-crystals (CNCs) and model lignin (DHP) at various concentrations in solution (free polymers) or in gels (cross-linked polymers) [1]. Then, starting from a set of modulated endoglucanases belonging to glycoside hydrolase family 45, appended or not to one CBM or five CBMs from family 1 [2], we have grafted these proteins to a fluorophore so that they become fluorescent probes. Each probe has been directly embedded into the different assemblies and their mobility evaluated by confocal microscopy using the FRAP technique [3], to determine their affinity [4]. Overall, results show that the different controlled features related to the assemblies (solution or gel, polymer concentration, water content) and to the probes (type, size) have different impact on affinity, varying in this order: probe size &gt; DHP concentration &gt; FAX concentration. More importantly, the GH45 endoglucanase interacts with DHP through some surface motifs that are partially masked when the enzyme is appended to at least one CBM, making the enzyme alone more sensitive to interactions with lignin. Bioinspired lignocellulosic materials are therefore relevant tools for identifying and hierarchizing features influencing enzymes affinity. 1. Paës G and Chabbert B (2012). Biomacromolecules 13, 206-214 2. Couturier M et al. (2011). Microb. Cell. Fact. 10 3. Paës G et al. (2013). Biomacromolecules 14, 2196-2205 4. Paës G et al. (2015). Soft Matter 11, 6586-6594

Bioinspired lignocellulosic materials as sensors for unravelling interaction features of enzymes

251st National Meeting (2016)

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