Abstract
Cellulases are an important class of cell-wall degrading enzymes that biochemically convert cellulosic feedstocks to fermentable sugars. Understanding the molecular basis of cellulase interactions responsible for synergistic behavior observed between cellulases is critical for rationally designed enzyme cocktails. Traditional biochemical techniques that take indirect bulk solution measurements of free protein to determine bound protein concentration are limited in resolving spatial and temporal patterns of enzyme diffusion and binding that are key to understanding synergism. Integrating advanced imaging techniques like epi-fluorescent microscopy with the existing biochemical data will allow us to overcome these limits and offer the potential for extracting greater insight into the molecular mechanisms that govern cellulase synergism. Binding kinetic data for multiple cellulases with different catalytic actions co-localizing on simple and complex cellulose morphologies can be determined with a high degree of spatial and temporal resolution that has not previously been possible; thus, allowing for a more in-depth exploration of the heterogeneous catalytic interactions between synergistic cellulases.Pure fluorescently labeled populations of Thermobidfida fusca endocellulase Cel5A, and exocellulase Cel6B, were applied to immobilized cellulose. Experiments were conducted at room temperature to measure binding without activity, at 45°C to measure binding near peak enzymatic activity and at an intermediate temperature. Binding data, in the form of fluorescent images was recorded using time-lapsed fluorescence microscopy with images taken at defined intervals for a period of four hours to ensure saturating conditions. Kinetic binding curves were established for mixtures of these two cellulases at each temperature to observe the effect of temperature and hydrolytic activity on cellulase binding.
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