Direct observation of bidirectional motility by the cellobiohydrolase TfCel6B

Abstract

Cellulose, the world’s most abundant biopolymer, is a primary structural component of plants. Its enzymatic breakdown in nature is catalyzed by cellulases, which hold potential to be utilized in bioreactors to convert cellulosic materials such as cultivated grasses, paper waste, and agricultural residues into soluble sugars for fermentation into liquid biofuels. However, crystalline cellulose is highly resistant to hydrolysis and current cellulases lack the efficiency needed to make large-scale biofuel production economically viable. A deeper understanding of the mechanisms by which cellulases degrade cellulose is essential for engineering improved enzymes for industrial applications. The cellulase Thermobifida fusca Cel6B (TfCel6B) is of particular interest due to its retained activity at a wide range of temperatures and pH, yet its hydrolysis mechanism remains poorly understood. This dissertation investigates the molecular-scale activity of TfCel6B to address critical knowledge gaps. Optical tweezers were employed to directly observe individual TfCel6B molecules traversing cellulose microfibrils during processive hydrolysis. Enzyme motility was characterized across multiple cellulose isoforms to examine the influence of cellulose source on enzyme activity. The catalytic core of TfCel6B was also isolated and studied to elucidate the roles of individual domains in cellulose degradation. Single-molecule assays revealed that after binding, TfCel6B frequently reverses its direction of movement—contrary to prior models suggesting unidirectional catalysis. Several hypotheses were proposed to explain this bidirectional motility, with evidence suggesting it is caused by irregularities in the ultrastructure of the cellulose substrate. These findings provide novel insights into cellulase function and pave the way for further mechanistic studies. Optical tweezers-based cellulase motility assays are incredibly sensitive to noise in the form of mechanical drift of the sample plane. To address this, two video-based drift correction methods are employed and their performance is compared. In addition, this dissertation discusses relevant parameters for implementing effective drift correction methodology in an optical tweezers workflow.