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Investigating hemicellulases spatial proximity for lignocellulosic biomass valorisation
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International audience. During the last years, research on lignocellulosic biomass (LCB) has been continuouslyin progress due to its characteristic abundance and renewability. It is an environmentallyfriendly source of energy and added value compounds settling the basis for a circularbioeconomy. However, its complete exploitation is mired due to its complex chemicaland structural composition forcing microorganisms to deploy diverse strategies basedon the action of a wide array of enzymatic activities. One intriguing example producedby some anaerobic cellulolytic bacteria is the cellulosome, a multienzyme complex thatis highly efficient for LCB deconstruction. This relies on substrate targeting and enzymeproximity allowing substrate channelling and synergistic action (Artzi et al., 2017).Therefore, suggesting that the spatial organisation of the enzymes in thesenanomachines plays an important role in catalysis. However, cellulosome efficiency isguided by its flexibility (Andrade Pinheiro et al., 2012), making difficult to investigate theeffect of enzymatic spatial proximity during catalysis.Recently, Enjalbert et al. (2020) circumvented this issue by locking the spatial topologybetween two hemicellulases using the Jo-In biomolecular welding system (Bonnet et al.,2017); based on two proteins, Jo and In, which are able to form a covalent bondspontaneously, creating a post translational rigid linker.In this work, we investigated the spatial proximity effect of two hemicellulases from abacteroides xylan utilization loci (XUL) belonging to termite’s microbiota. This XULencodes several hemicellulases constituting an interesting model to explore enzymesynergism and spatial organization of related carbohydrate active enzymes. Exploitingthe antiparallel association between Jo and In, we present the construction, productionin vivo, and initial characterization of two different chimeric enzyme spatial organisationcalled C4 and C12 using a GH11 xylanase and a GH43 xylosidase.The complexes were produced in E. coli and purified after two chromatographic stepsyielding around 35 and 31.5 mg/L for C4 and C12, respectively. The specific activities ofthe pure complexes against p-nitrophenyl-β-D-xylopyranoside, wheat arabinoxylan(WAX) and wheat bran have been assayed and compared to an equimolar amount ofwild type enzymes free in solution. The results show how effectively there is a clear83effect of the complex structure on its capacity to hydrolase the substrate, being theactivity of C4 approximately 1.5-fold higher than the one for C12 on wheat bran.
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