Abstract
Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.
Highlights
Dislocations or slip planes are irregular regions within in the wall of thick-walled plant cells such as fibers and tracheids
One can speculate that compression stress applied in the longitudinal direction of the plant cell gives rise to buckling of the cell wall, but that the cellulose remains piece-wise ordered in segments that follow the buckling
slices of interest (SOIs) were selected based on these profiles, having in mind the consideration discussed in the Microscopy part of the Materials and methods section
Summary
Dislocations or slip planes are irregular regions within in the wall of thick-walled plant cells such as fibers and tracheids. Dislocations have been found to be important for efficient particle size reduction during the initial phase of the enzymatic hydrolysis process, as they are weak regions where fibers break. In this study we look into whether the binding affinities of different cellulase variants vary between dislocations and the surrounding cell wall. Albeit dislocations have been known to science for more than 100 years (Höhnel 1884), their exact structure, formation and role in the living plant remain only partially known. Dislocations have been suggested to be present in both the S1 and S2 layers of the plant cell wall (Hartler 1995; Ander et al 2008). Comparison of scanning electron microscopy and polarized light microscopy results has shown that only larger dislocations are visible on the surface of native fibers (Thygesen et al 2006)
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