Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps

Cellobiohydrolase 1 from Trichoderma reesei ( Tr Cel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor’s mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of Tr Cel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest Tr Cel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated Tr Cel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into Tr Cel7A’s molecular motility mechanism. Cellobiohydrolases are promising tools in biofuel production by hydrolysing cellulose into cellobiose. Here the authors use optical tweezers to show that Cellobiohydrolase 1 from Tricodermia reesei functions processively against moderate load, and likely uses multiple energy sources to fuel each step along the cellulose fibre.