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Probing the role of hemicellulase glycosylation in plant cell wall degradation: targeted glycosylation through click chemistry and activity monitoring in complex substrates
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International audience. Protein glycosylation is a major post-translational modification and glycoproteins areinvolved in various functions, including plant cell wall degradation [1]. Our approach aims atdeciphering the role of hemicellulase glycosylation in lignocellulosic biomass deconstruction.Our strategy combines recombinant protein production, chemical glycosynthesis, geneticcode expansion [2] and click chemistry to specifically glycosylate a GH11 xylanase fromNeocallimastix patriciarum at several targeted positions. Model homogeneous glycans and afluorophore are chemically coupled to a clickable platform. Besides, matched clickable noncanonical amino acids are incorporated at targeted positions thanks to a stop codonsuppression system. With this method, the carbohydrate moiety and position in the xylanasecan be varied and their impact on enzyme-substrate affinity and catalytic activity will beinvestigated.In order to study the enzyme on close-to-nature substrates, we use biophysical approaches(surface plasmon resonance -SPR- and Fluorescence Recovery After Photobleaching -FRAPmethods) to monitor its behavior in hemicellulose and cellulose polymer assemblies as thinfilms [3] and gels[4] by measuring affinity constants.By understanding how glycosylation modulates hemicellulase activity, substrate-binding andthe stability of the substrate-enzyme complex during catalysis, our work opens new routes forenzyme engineering with diverse applications, such as lignocellulosic biomass valorization.
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