Structural Profiling of Xyloglucans from Food Plants by High-Performance Anion-Exchange Chromatography with Parallel Pulsed Amperometric and Mass Spectrometric Detection.

Xyloglucans are the dominant hemicelluloses in the primary cell wall of dicotyledonous plants, fulfilling numerous functions. However, routine methods of cell wall analytical chemistry such as methylation analysis are time-consuming and often not adequate to capture the structural diversity of xyloglucans. Here, a xyloglucan profiling method based on the enzymatic release of xyloglucan oligosaccharides by a xyloglucan-specific endo-ß-(1→4)-glucanase and subsequent analysis of these oligosaccharides by high-performance anion-exchange chromatography (HPAEC) with parallel pulsed amperometric and mass spectrometric detection was developed. For this purpose, a set of 23 authentic xyloglucan oligosaccharides was generated, structurally characterized by mass spectrometry and NMR spectroscopy, and established as analytical standard compounds. Coupling of HPAEC with parallel electrochemical and MS detection was demonstrated to be an excellent tool to analyze xyloglucan-derived oligosaccharides. The applicability of the method was demonstrated by characterizing the xyloglucan architecture from a set of nine economically relevant food plants from the botanical orders Caryophyllales (rhubarb, buckwheat, amaranth, and quinoa), Cucurbitales (Hokkaido squash), Laurales (avocado), Myrtales (pomegranate), and Sapindales (mango and orange) for the first time. In future studies, this method can ideally be used to monitor structural alterations of xyloglucans as a result of genetic engineering, plant/tissue maturation, and processing of plant material.

Products Released from Structurally Different Dextrans by Bacterial and Fungal Dextranases.

Dextran hydrolysis by dextranases is applied in the sugar industry and the medical sector, but it also has a high potential for use in structural analysis of dextrans. However, dextranases are produced by several organisms and thus differ in their properties. The aim of this study was to comparatively investigate the product patterns obtained from the incubation of linear as well as O3- and O4-branched dextrans with different dextranases. For this purpose, genes encoding for dextranases from Bacteroides thetaiotaomicron and Streptococcus salivarius were cloned and heterologously expressed in Escherichia coli. The two recombinant enzymes as well as two commercial dextranases from Chaetomium sp. and Penicillium sp. were subsequently used to hydrolyze structurally different dextrans. The hydrolysis products were investigated in detail by HPAEC-PAD. For dextranases from Chaetomium sp., Penicillium sp., and Bacteroides thetaiotaomicron, isomaltose was the end product of the hydrolysis from linear dextrans, whereas Penicillium sp. dextranase led to isomaltose and isomaltotetraose. In addition, the latter enzyme also catalyzed a disproportionation reaction when incubated with isomaltotriose. For O3- and O4-branched dextrans, the fungal dextranases yielded significantly different oligosaccharide patterns than the bacterial enzymes. Overall, the product patterns can be adjusted by choosing the correct enzyme as well as a defined enzyme activity.