Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules
Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules
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DOI：
10.1073/pnas.1912354116
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发表时间：
2019-11-05
影响因子：
11.1
通讯作者：
Saddler, Jack
中科院分区：
文献类型：
--
作者：
Novy, Vera;Aissa, Kevin;Saddler, Jack
两个荧光标记的碳水化合物结合模块（CBM 5），其特异性地结合到结晶（CBM 2a-RRedX）和准结晶（CBM 17-FITC）纤维素，用于区分漂白软木硫酸盐纤维在酶介导的水解期间的超分子纤维素结构。使用共聚焦激光扫描显微镜（CLSM）阐明CBM吸附的差异，并通过各自探针的相对荧光强度定量酶介导的解构过程中发生的结构变化。很明显，高度有序（即，结晶纤维素）发生在纤维素纤维表面，其被较低结构组织和增加的纤维素可及性的区域散布。定量图像分析，支持C-13 NMR，扫描电子显微镜（SEM）成像，和纤维长度分布分析，表明，酶降解占主导地位，在这些区域在反应的初始阶段，导致快速纤维断裂和纤维素表面结晶度的增加。通过应用这种方法来阐明酶介导的解构机制的差异，这项工作进一步表明，干燥降低了酶对这些无序区域的可及性，导致降解和碎片化的延迟发生。使用具有特异性识别位点的荧光标记的CBM 5提供了一种定量的方法来阐明纤维素的超分子亚结构及其对酶可及性的影响。本研究设计了一种定量分析纤维素超微结构和可及性的方法，为纤维素基质的降解机理提供了新的认识。
Two fluorescence-tagged carbohydrate-binding modules (CBM5), which specifically bind to crystalline (CBM2a-RRedX) and para-crystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by C-13 NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBM5 with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.