Research on lignocellulosic biomass (LCB) has been continuously in progress due to its characteristic abundance and renewability. It is an environmentally friendly source of energy and added valued compounds settling the basis for a circular bioeconomy. However, its complete exploitation is mired due to its complex chemical and structural composition forcing microorganisms to deploy diverse strategies based on the action of a wide array of enzymatic activities to achieve its complete hydrolysis. One intriguing example produced by some anaerobic cellulolytic bacteria is the cellulosome, a multienzyme complex that is highly efficient for LCB deconstruction. This relies on substrate targeting and enzyme proximity allowing substrate channelling and synergistic action (Artzi et al., 2017) ). Therefore suggesting that the spatial organisation of the enzymes in this nanomachines plays an important role in catalysis. However, cellulosome efficiency is mainly guided by its flexibility (Andrade Pinheiro et al., 2012), making difficult to investigate the effect of enzymatic spatial proximity during catalysis.
Recently, Enjalbert et al. (2020) circumvented this issue by locking the spatial topology between 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 bond spontaneously, creating a post translational rigid linker.
In this work, we investigated the spatial proximity effect of two hemicellulases from a bacteroides xylan utilization loci (XUL) belonging to termite’s microbiote . This XUL encodes several hemicellulases constituting an interesting model to explore enzyme synergism and spatial organization of related carbohydrate active enzymes. Exploiting the antiparallel association between Jo and In, we achieved the construction, production in vivo, and initial characterization of two different chimeric enzyme spatial organisation called C4 and C12 using a GH11 xylanase and a GH43 xylosidase.
The complexes were produced in E. coli and purified after two chromatographic steps yielding around 35 and 31.5 mg/L for C4 and C12, respectively. The specific activities of the pure complexes against p-nitrophenyl-β-D-xylopyranoside, wheat arabinoxylan (WAX) and wheat bran have been assayed and compared to an equimolar amount of wild type enzymes free in solution. The results show how effectively there is a clear effect of the complex structure on its capacity to hydrolase the substrate, being the activity of C4 approximately 1.5-fold higher than the one for C12 on wheat bran. In parallel, the complexes were analysed by cryoelectron microscopy to gain information on structural organisation, this work provided initial conditions for freezing prior to cryoEM.
The results have been presented in the Lignobiotech research conference, in Vancouver August 2022, and spatial organisation study were used to publish a review in a special issue on carbohydrate modifying enzymes in Essays in biochemistry.
