(235b) A Single Molecule Study of Cellobiohydrolase 1 From Trichoderma Reesei Conference: AIChE Annual MeetingYear: 2013Proceeding: 2013 AIChE Annual MeetingGroup: Sustainable Engineering ForumSession: Biological Conversions and Processes for Renewable Feedstocks I Time: Tuesday, November 5, 2013 - 8:55am-9:20am Authors: Brady, S., Vanderbilt University Best, K., Princeton University Lang, M., Vanderbilt University TrCel7a is a cellulase motor that processively degrades crystalline cellulose into cellobiose units. This and other similar enzymes have been of great interest in the biofuels and paper industries. TrCel7a is known to be the most abundant cellulase produced by Trichoderma reesei  and is thought to be powered by the energy released in the hydrolysis of glycosidic bonds . While much work has been done to characterize and understand the mechanisms that drive a spectrum of molecular motors that move on tracks such as microtubules, actin filaments and DNA, relatively little is known about the mechanism of cellobiohydrolase motors that move along a crystalline cellulose based track. Thus, a clear understanding of the enzyme’s mechanistic action at the single molecule level has yet to be developed. We designed a single molecule motility assay based on optical tweezers that allows for precision tracking of TrCel7a with individual step resolution. Our work includes filter paper, cotton, and algae based substrates. The processive TrCel7a walks slowly, exhibiting both distinct run and pause states with a step distribution, between pauses, that is non-uniform. At low to moderate loads, the enzyme does not appear to be mechanically limited. Through these observations and characterization of the enzyme’s kinetics, we gain insight into the mechanistic action of TrCel7a at a level that is unattainable in solution studies, and are provided a molecular level picture of potential mechanisms for motility of TrCel7a. 1. Sandgren, Mats. “Structural and Functional Studies of Glycoside Hydrolase Family 12 Enzymes from Trichoderma reesei and other Cellulolytic Microorganisms [dissertation],” Uppsala, Sweden: Uppsala University (2003). 2. Igarashi, Kiyohiko, et al. “Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface,” Science , 333 pp. 1279-1282 (September 2011).