(490b) A Single Molecule Investigation of Cellulose Hydrolysis By TrCel7a
Cellobiohydrolase I from Trichoderma reesei (TrCel7a) plays a critical role in the enzymatic hydrolysis of cellulose. Mechanistic understanding of this enzyme’s processive degradation of cellulose substrates, however, is lacking. Single molecule studies have proven to be a powerful tool in discovering mechanistic behavior of molecular motors, such as kinesin, ClpX, myosin, and RNA polymerase. Here we use a single molecule motility assay based on optical tweezers to track TrCel7a with 1nm spatial resolution and piconewton force resolution to probe the mechanistic behavior of TrCel7a on crystalline cellulose substrates. Our work includes the study of a range of cellulose substrates, yielding single molecule motility characteristics such as step size, dwell distributions and translocation velocities.
The reported work also investigates the binding behavior of isolated E. coli expressed TrCel7a binding domain (CBM), yielding binding kinetics of CBM. Together the results of this work provide insight into the relationship between the CBM subdomain and the working mechanism of TrCel7a as a whole.