Investigating synergism within multimodular glycoside hydrolases during wheat straw cell wall deconstruction
Résumé
In Nature, the plant-based organic carbon contained within plant cell walls is mainly recycled by the action of cellulolytic microorganisms, such as bacteria and fungi, which produce complex arrays of cell wall-degrading enzymes. Many of the enzymes that degrade plant cell wall polysaccharides are modular proteins, which contain single or multiple copies of both catalytic domain(s) and CBM(s). Cellulolytic microorganisms developed a couple of strategies to break down the complex plant cell wall; secrete free enzymes into their surroundings or produce large multi-component cellbound structures, which harbour several enzymes displaying complementary activities. In both case, substrate is attacked by different enzymes acting together at different regions, and much more than sum of individual different enzymatic activities, synergism drives the efficiency of such system. Nevertheless, this complex process is still not completely understood. Despite an abundant literature about the role of CBM as an enhancer of the catalytic activity, we miss information about relationship between enzymes, CBMs and modularity in planta. Here we report an original way to easily associate catalytic domains and CBMs to each other. Jo and In are small proteins (10.6 and 16.7 kDa respectively) from Streptococcus pneumoniae1,2 , which are able to spontaneously form one intradomain isopeptide bond . The xylanase Xyn11A from Neocallimastix patriciarum and the xylosidase Xyl43 from Bacillus halodurans were successfully expressed in E.coli and purified as Jo or In tagged proteins. In addition, cellulose specific CBM3a from Clostridium thermocellum and xylane specific CBM2b-1 from Cellulomonas fimi were also expressed and purified. Various combinations of these proteins were designed as free or solid support associated modular enzymes. Catalytic activities were investigated using chromogenic substrates, beach wood xylan and milled wheat straw. The fine spatial proximity/synergism relationship in multimodular enzymes within the context of intact plant cell wall is presented in this work.
Domaines
Autre
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Loading...