Interactions between the engineering family 3 carbohydrate-binding module (CBM3) and different types of...

Carbohydrate-binding modules (CBMs) exhibit specific binding affinity for carbohydrate substrates, making them invaluable in the recognition, utilization, and modification of various polysaccharides. Herein, we investigate the nanocellulose-CBM interaction, highlighting a specific engineered family 3 CBM (CBM3). There is a distinct polymorph- and surface morphology-dependency on CBM3's binding preferences, in accordance with the single-molecule force spectroscopy (SMFS) investigation for nanocellulose with different allomorphs (I and II) and in various forms (nanocrystals, CNC, and nanofibrils, CNF). In particular, higher crystallinity and densely packed hydroxyl groups promote CBM3 binding, as indicated by the highest adhesion forces in CNC-I (~4 μN in the gas phase and ~800 pN in the liquid phase). Conversely, CNF-II exhibited the lowest CBM3 binding affinity (~300 nN in the gas phase and ~80 pN in the liquid phase) due to its low crystallinity and flexible fibrillar network. Quartz crystal microbalance (QCM) measurements corroborated these findings in a dynamic adsorption scenario, also showing the most pronounced CBM3 interaction towards CNC-I (frequency drop ~400 Hz). We extend such fundamental understandings to enhance the mechanical properties of macroscopic CNF films by approximately 25%. These findings provide crucial insights into the molecular mechanisms underlying CBM-substrate interactions, paving the way for the rational design of carbohydrate-binding systems in industrial and biotechnological applications.