Hybrid Nanocellulosome Design from Cellulase Modules on Nanoparticles: Synergistic Effect of Catalytically Divergent Cellulase Modules on Cellulose Degradation Activity

Cellulosomes, which are assemblies of cellulases with various catalytic functions on a giant scaffoldin protein with a carbohydrate-binding module (CBM), efficiently degrade solid cellulosic biomass by means of synergistically coupled hydrolysis reactions. In this study, we constructed hybrid nanocellulosomes from the biotinylated catalytic domains (CDs) of two catalytically divergent cellulases (an endoglucanase and a processive endoglucanase) and biotinylated CBMs by clustering the domains and modules on streptavidin-conjugated nanoparticles. Nanocellulosomes constructed by separately clustering each type of CD with multiple CBMs on nanoparticles showed 5-fold enhancement in cellulase degradation activity relative to that of the corresponding free CDs, and mixtures of the two types of nanocellulosomes gradually and synergistically enhanced cellulase degradation activity as the CBM valency increased (finally, 2.5 times). Clustering the two types of CD together on the same nanoparticle resulted in a greater synergistic effect that was independent of CBM valency; consequently, nanocellulosomes composed of equal amounts of the endo and endoprocessive CDs clustered on a nanoparticle along with multiple CBMs (CD/CBM = 7:23) showed the best cellulose degradation activity, producing 6.5 and 2.4 times the amount of reducing sugars produced from amorphous and crystalline cellulose, respectively, by the native free CDs and CBMs in the same proportions. Our results demonstrate that hybrid nanocellulosomes constructed from the building blocks of cellulases and cellulosomes modules have the potential to serve as high-performance artificial cellulosomes.