Kinetics of enzymatic hydrolysis revealed by video rate AFM single molecule analysis

The rapid growing bio-refining industry is hindered by the slow reaction rate of cellobiohydrolase catalyzed hydrolysis. Analysis of the mechanism of enzyme interaction in the cellobiohydrolase catalyzed hydrolysis is difficult because of the variety factors affecting the enzymatic reaction rate. In this study, we evaluated the processive hydrolysis parameters in the molecular level using a video rate atomic force microscopy (AFM). Both dissociate and association rates (koff and kon) have been calculated by an experimental data fitted Michaelis-Menten model and real-time observation experimental data. The difference in these two association rates suggested that the cellulose substrate governs the hydrolysis rate and that can deepen the understanding of the mechanisms of processive hydrolysis.