Abstract
In this study, we developed a method to observe interactions between cellulase and cellulose microfibril by transmission electron microscopy. Although negative staining and low-angle metal shadowing increase image contrast, neither method is sufficient to view enzyme interactions with microfibril. However, we found that the combination of negative staining and low-angle metal shadowing provided better contrast for enzyme-like particles on the microfibril. The lengths of the particles interacting with microfibrils were 7.03 and 5.05 nm, parallel and perpendicular to the fiber direction, respectively. Accounting for the additional thickness owing to metal shadowing, the particle sizes were consistent with that of CBH I from Trichoderma reesei based on a crystalline structural analysis. The combination of these electron staining techniques successfully visualized morphological changes in microfibril as well as enzymes adsorbed on it, thus demonstrating cellulase in action. These results indicate that appropriate staining techniques can be applied to extend the applications of transmission electron microscopy, which may be particularly beneficial for studies on enzymatic behavior.
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Acknowledgments
We would like to express our appreciation to Dr. A. Hirai for providing the sample and Dr. Y. Kobayashi for providing the enzyme. This work was supported by a grant for Mission Research from the Research Institute for Sustainable Humanosphere (RISH) of Kyoto University and the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 15K18723.
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Horikawa, Y., Imai, T. & Sugiyama, J. Visualization of cellulase interactions with cellulose microfibril by transmission electron microscopy. Cellulose 24, 1–9 (2017). https://doi.org/10.1007/s10570-016-1105-9
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DOI: https://doi.org/10.1007/s10570-016-1105-9