endo-1,4-β-Galactanase (Aspergillus niger)

The elucidation of the structural characteristics of polysaccharides from natural sources is generally difficult owing to their structural complexity and heterogeneity. In our previous study, an immuno-stimulatory polysaccharide (RGP-AP-I) was isolated from Korean red ginseng (Panax ginseng C.A. Meyer). The present study aims to elucidate the structural characteristics of RGP-AP-I. Sequential enzyme hydrolysis was performed using four specific glycosylases, and chemical cleavage via β-elimination was carried out to determine the fine structure of RGP-AP-I. The degraded fragments were chemically identified using various chromatographic and spectrometric analyses, including HPLC-UVD, GC–MS, and tandem mass spectrometry. The results indicated that RGP-AP-I comprises a rhamnogalacturonan I (RG-I) backbone with repeating disaccharide units [→2)-Rhap-(1 → 4)-GalAp-(1→] and three side chains substituted at the C(O)4 position of the rhamnose residue in the backbone. The three side chains were identified as a highly branched α-(1 → 5)-arabinan, a branched β-(1 → 4)-galactan, and an arabino-β-3,6-galactan. Our results represent the first findings regarding the fine structure of the immuno-stimulatory polysaccharide RG-AP-I isolated from red ginseng.

The aim of the present study was chemical clarification of in vitro Peyer's patch-immunomodulating polysaccharides in sugar cane molasses, and evaluation of in vivo modulating activity on immune function of T lymphocytes in Peyer's patches and on microenvironment of hemopoietic system. Five kinds of glucans, comprising of dextranase-sensitive and activity-related d-glucosyl moieties, were purified as in vitro Peyer's patch-immunomodulating polysaccharides from the molasses. Oral administration of a glucan-enriched subfraction induced IL-2 and GM-CSF-producing T lymphocytes in Peyer's patches, resulting in enhancement of IL-6 production in a hemopoietic microenvironment to boost neutrophil numbers in the peripheral blood stream. Oral administration of purified glucan or glucan-enrich sub-fraction of sugar cane reduced the number of Plasmodium berghei- or P. yoelii-infected erythrocytes in a murine infection model, using polysaccharide alone or via co-administration with the antimalarial drug, artesunate. These results suggested that Peyer's patch-immunomodulating glucans enhanced protective immunity through axis of Peyer's patches-hemopoietic system.

Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched β-1, 4-galactan. Plants in which all three genes were inactivated had no detectable β-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.

Tomato fruit texture depends on histology and cell wall architecture, both under genetic and developmental controls. If ripening related cell wall modifications have been well documented with regard to softening, little is known about cell wall construction during early fruit development. Identification of key events and their kinetics with regard to tissue architecture and cell wall development can provide new insights on early phases of texture elaboration. In this study, changes in pectin and hemicellulose chemical characteristics and location were investigated in the pericarp tissue of tomato (Solanum lycopersicon var Levovil) at four stages of development (7, 14 and 21 day after anthesis (DPA) and mature green stages). Analysis of cell wall composition and polysaccharide structure revealed that both are continuously modified during fruit development. At early stages, the relative high rhamnose content in cell walls indicates a high synthesis of rhamnogalacturonan I next to homogalacturonan. Fine tuning of rhamnogalacturonan I side chains appears to occur from the cell expansion phase until prior to the mature green stage. Cell wall polysaccharide remodelling also concerns xyloglucans and (galacto)glucomannans, the major hemicelluloses in tomato cell walls. In situ localization of cell wall polysaccharides in pericarp tissue revealed non-ramified RG-I rich pectin and XyG at cellular junctions and in the middle lamella of young fruit. Blocks of non-methyl esterified homogalacturonan are detected as soon as 14 DPA in the mesocarp and remained restricted to cell corner and middle lamella whatever the stages. These results point to new questions about the role of pectin RGI and XyG in cell adhesion and its maintenance during cell expansion.

Cell wall composition affects the texture of plant-based foods. In addition, the main components of plant cell walls are dietary fiber constituents and are responsible for potential physiological effects that are largely affected by the structural composition of the cell walls. Radish (Raphanus sativus L. var. sativus) is known to develop a woody and firm texture during maturation and ripening, most likely due to changes in the cell wall composition. To describe these changes chemically, radish was cultivated and harvested at different time points, followed by detailed chemical analysis of insoluble fiber polysaccharides and lignin. During maturation, changes in polysaccharide profiles were observed, with a decrease in the portion of neutral pectic side chains and an increase in the xylan portion being predominant. Radish lignin was characterized by unexpectedly high incorporation of p-coumaryl alcohol into the polymer. Maturation dependent increases in lignin contents were accompanied by compositional changes of the lignin polymers with sinapyl alcohol being preferentially incorporated.

Galactose-rich oligosaccharides/oligomers (oligo-RG I) were produced by the enzymatic treatment of potato galactan-rich rhamnogalacturonan I (RG I) with endo-β-1,4-galactanase and Depol 670L multi-enzymatic preparation. The digestibility study revealed that 81.6 and 79.3% of RG I and its corresponding oligomers remained unhydrolyzed, respectively. The prebiotic properties of RG I and its hydrolysates were investigated using a continuous culture system inoculated with immobilized fecal microbiota. Both RG I and oligo-RG I have stimulated the growth of Bifidobacterium spp. and Lactobacillus spp., with oligo-RG I hydrolysates being more selectively fermented by these beneficial bacteria. Furthermore, none of RG I nor its hydrolysates increased the populations of Bacteroidetes and Clostridium leptum. Total amounts of short chain fatty acids, generated upon the fermentation of oligo-RG I, were higher than those obtained with its parent RG I and the positive control (fructooligosaccharides). The overall study contributes to the understandings of the prebiotic properties of potato RG I and its corresponding oligosaccharides/oligomers.

Xylogenesis, a process by which woody tissues are formed, entails qualitative and quantitative changes in the cell wall. However, the molecular events that underlie these changes are not completely understood. Previously, we have isolated two monoclonal antibodies, referred to as XD3 and XD27, by subtractive screening of a phage-display library of antibodies raised against a wall fraction of Zinnia elegans xylogenic culture cells. Here we report the biochemical and immunohistochemical characterization of those antibodies. The antibody XD3 recognized (1→4)-β-D-galactan in pectin fraction. During xylogenesis, the XD3 epitope was localized to the primary wall of tracheary-element precursor cells, which undergo substantial cell elongation, and was absent from mature tracheary elements. XD27 recognized an arabinogalactan protein that was bound strongly to a germin-like protein. The XD27 epitope was localized to pre-lignified secondary walls of tracheary elements. Thus these cell-wall-directed monoclonal antibodies revealed two molecular events during xylogenesis. The biological significance of these events is discussed in relation to current views of the plant cell wall.