背景:在植物中,大量的多糖包括细胞壁。各主要类型的植物细胞壁多糖,包括纤维素,半纤维素,还有果胶,具有独特的结构和功能,有助于墙体力学并影响植物形态发生。近年来,果胶的价值化因其在生物质解构中的作用日益扩大而备受关注,食品和材料科学,和环境修复。然而,果胶的利用受到我们对其结构不完全了解的限制。在这里,我们提出了一个工作流程的原则相关的多糖一级结构的表征使用性质的最复杂的多糖,鼠李糖半乳糖醛酸-II(RG-II),作为一个模型。
结果:我们概述了如何使用化学或酶处理以及大小排阻色谱从芹菜和浮萍细胞壁以及红葡萄酒中分离RG-II。从那里,我们应用了基于质谱(MS)的技术来确定完整RG-II和衍生寡糖的糖基残基和连接组成,包括对不稳定单糖的特殊考虑。在这样做的时候,我们证明,在浮萍狼尾草中,侧链B的阿拉伯吡喃(Arap)残基在O-2处被鼠李糖取代。我们使用电喷雾MS技术来鉴定非糖基修饰,包括甲基醚,甲酯,和RG-II衍生的寡糖上的乙酰酯。然后,我们展示了质子核磁共振波谱(1H-NMR)可用于研究完整RG-II的结构并补充使用尺寸排阻色谱法进行的RG-II二聚化研究。
结论:果胶多糖结构的复杂性阻碍了旨在使其增值的努力。在这项工作中,我们使用RG-II作为模型来证明使用色谱分离和表征多糖所必需的步骤,MS,和NMR技术。这些原理可应用于其他糖结构的表征,并将帮助研究人员了解未来糖结构如何与功能特性相关。
BACKGROUND: In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature\'s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model.
RESULTS: We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography.
CONCLUSIONS: The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.