Cellulose nanocrystals formation leveraging synergy between lytic polysaccharide monooxygenase and endoglucanase

Cellulose nanocrystals (CNCs) are crystalline fragments released from cellulose via selective disruption of the fibrillar structure. This study investigated CNC formation by leveraging synergy between a C1/C4-active lytic polysaccharide monooxygenase (LPMO) TaAA9A and glycoside hydrolase family 45 endoglucanase (EG) NsGH45. The LPMO TaAA9A alone could not liberate CNCs from the cellulose substrate, and the endoglucanase NsGH45 alone yielded limited CNC release; their co-incubation, however, exhibited strong synergy, increasing CNC yields by up to 81% relative to additive enzyme action. Varying enzyme ratios in the reaction revealed distinct LPMO: EG ratios to achieve maxima for CNC formation versus soluble sugar release, indicating that the reaction can be tuned to favor CNC production over sugar solubilization. Further, LPMO activity was shown to increase productive cellobiohydrolase binding capacity of residual cellulose, supporting a mechanism whereby LPMO activity increases endoglucanase access to previously inaccessible regions of the cellulose fibrils. Our findings demonstrate that LPMO-endoglucanase synergy can be harnessed and tuned in cellulose deconstruction pathways towards CNC production.