Visualization of cellulase interactions with cellulose microfibril by transmission electron microscopy
Visualization of cellulase interactions with cellulose microfibril by transmission electron microscopy
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DOI：
10.1007/s10570-016-1105-9
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发表时间：
2016
期刊：
影响因子：
5.7
通讯作者：
Y. Horikawa;T. Imai;J. Sugiyama
中科院分区：
文献类型：
--
作者：
Y. Horikawa;T. Imai;J. Sugiyama
在这项研究中，我们开发了一种通过透射电子显微镜观察纤维素酶和纤维素微纤维之间相互作用的方法。尽管负染色和小角度金属阴影增加了图像对比度，但这两种方法都不足以观察酶与微纤维的相互作用。然而，我们发现负染色和小角度金属阴影的结合为微纤维上的酶样颗粒提供了更好的对比度。与微纤维相互作用的颗粒的长度分别为 7.03 和 5.05 nm，平行和垂直于纤维方向。考虑到金属遮蔽导致的额外厚度，基于晶体结构分析，颗粒尺寸与来自里氏木霉的 CBH I 的颗粒尺寸一致。这些电子染色技术的结合成功地可视化了微纤维及其上吸附的酶的形态变化，从而证明了纤维素酶的作用。这些结果表明，可以应用适当的染色技术来扩展透射电子显微镜的应用，这对于酶行为的研究可能特别有益。
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 fromTrichoderma reeseibased 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.