Assembly of Cellulases from Separate Catalytic Domains and a Cellulose-Binding Module for Understanding Cooperative Crystalline Cellulose Degradation

The biochemical degradation of abundant cellulosic biomass for industrial use and energy production has been extensively researched in recent years. Some elaborate cellulose digestion approaches have been developed based on specialized bacteria, which possess sophisticated mechanisms to efficiently degrade recalcitrant natural carbohydrates. In this study, we assembled catalytic domains from multiple cellulolytic enzymes onto a scaffold along with a cellulose-binding module (CBM), specifically targeting crystalline cellulose. The catalytic domains of endoglucanase and cellobiohydrolase from Acetivibrio thermocellus were linked to a heterotrimeric protein scaffold that assembles in a specific order. The bicatalytic complex failed to show the anticipated synergistic effect in cooperative cellulolysis, presumably because the catalytic domains only serve as weak anchors for each other in binding to the substrate. On the other hand, cellulose digestion was remarkably promoted by incorporating a CBM into a stable complex with a catalytic domain. Interestingly, the reversible association of catalytic domains and excess CBM proved more advantageous than fixed association. This suggests that the dynamic incorporation of CBM units enhances the accessibility of cellulose-degrading catalytic modules to the polysaccharide strand by preventing overly strong binding. This finding could have interdisciplinary applications for enzymes converting polymeric substrates other than cellulose.