Elucidation of the chemical fine structure of polysaccharides from soybean and maize kernel cell walls

M.M.H. Huisman

Research output: Thesisinternal PhD, WU

Abstract

<p>The subject of this thesis was the elucidation of the chemical fine structure of polysaccharides from cell walls of soybean and maize kernel. The two species investigated represent different taxonomic groups, soybean belonging to the dicotyledonous and maize to the monocotyledonous plants. Besides representing the most important structures present in cell wall material, these raw materials are of great importance in food and feed industry.<p>The characterisation of the soybean cell wall polysaccharides started with the isolation of the cell wall material as Water-Unextractable Solids (WUS) from soybean meal (chapter 2). The isolation procedure yielded a WUS fraction containing almost all polysaccharides present in the meal and only few other components. WUS was sequentially extracted with chelating agent (Chelating agent Soluble Solids, ChSS), dilute alkali (Dilute Alkali Soluble Solids, DASS), 1 m alkali (1 m Alkali Soluble Solids, 1 MASS) and 4 m alkali (4 m Alkali Soluble Solids, 4 MASS) to leave a cellulose-rich residue (RES). The pectin-rich extracts (ChSS and DASS) were found to have identical sugar compositions and contained predominantly galactose, arabinose, and uronic acid residues. The 1 MASS fraction contained xylose in addition to the former three sugars. The hemicellulose-rich fraction (4 MASS) contained mainly xylose and glucose. No indications were found that ChSS and DASS were structurally different, although it is obvious that their arrangement in the cell wall was not identical.<p>The intact cell wall polysaccharides in the meal and WUS were hardly degradable by enzymes. Once extracted, the polysaccharides from WUS were degraded more easily (chapter 3). The arabinogalactan side chains in the pectin-rich ChSS fraction could to a large extent be removed by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase, and arabinofuranosidase B. The remaining polymer (fraction P) was isolated and represented 30% of the polysaccharides in the ChSS fraction (12% of the polysaccharides in the WUS). This polymer still contained some remaining arabinose and galactose residues, which could not be removed by the enzyme mixture used.<p>The pectic backbone (fraction P) appeared to be resistant to enzymatic degradation by both established (like polygalacturonase) and novel pectic enzymes (like RG-hydrolase). After partial acid hydrolysis of the isolated pectic backbone, one oligomeric and two polymeric populations were obtained by size-exclusion chromatography. Monosaccharide and linkage analyses, enzymatic degradation, and NMR spectroscopy of these two polymeric populations showed that the pectic substances in the original extract (ChSS) contained both rhamnogalacturonan and xylogalacturonan regions, while homogalacturonan was absent (chapter 4). The absence of homogalacturonan distinguishes the pectic substances from soybean from pectic polysaccharides extracted from other sources, which contain homogalacturonan and rhamnogalacturonan regions and can be degraded with polygalacturonase and RG-hydrolase, respectively. Acid hydrolysis of fraction P improves the susceptibility of the remaining polymers for RG hydrolase and exo-galacturonase.<p>The xylogalacturonan present in the ChSS fraction distinguishes itself from xylogalacturonan from other sources known so far. A part of the xylose residues in the xylogalacturonan is substituted with fucose and the xylogalacturonan is resistant to degradation with XGH.<p>The arabinogalactan side chains, which were removed from the ChSS fraction to obtain fraction P, were the subjects of investigation in chapter 5. Fractionation, monosaccharide and linkage analyses, enzymatic degradation, and mass spectrometry of the oligosaccharides in the digest of ChSS after enzymatic digestion with arabinogalactan degrading enzymes indicated the presence of common linear (1,4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1,4)-linked galactose residues bearing an arabino <em>pyranose</em> residue at the non-reducing terminus, and a mixture of linear oligosaccharides constructed of (1,4)-linked galactose residues interspersed with one <em>internal</em> (1,5)-linked arabinofuranose residue. The presence of an internal arabinofuranose residue in a pectic arabinogalactan chain in cell wall polysacchairdes has not been reported previously, not in soybean, nor in other fruit or vegetable cell walls. Another uncommon feature is the presence of arabinopyranose residues in pectic arabinogalactan.<p>The pectic substances form only one network of the plant cell wall, the other is the cellulose/hemicellulose network. The hemicelluloses were solubilised from the residue with 1 and 4 m KOH solutions (chapter 6). The polysaccharides extracted with 1 m KOH were fractionated by ion-exchange chromatography, yielding a neutral and a pectic population. The sugar composition of the neutral population indicated the presence of xyloglucans and possibly xylans. Enzymatic degradation with endo-xylanases and endo-glucanases showed the presence of xyloglucan fragments only. Analysis of the digest formed after incubation of the neutral population with endo-glucanase V showed the formation of the characteristic poly-XXXG xyloglucan oligomers (XXG, XXXG, XXFG, XLXG, and XLFG), so three out of four glucose residues carry a side chain.<p>In chapter 7, the structural features of glucuronoarabinoxylans from maize kernels are described. First of all, maize kernel cell wall material was isolated as Water-Unextractable Solids (WUS). As expected the non-starch polysaccharides (NSP) had concentrated in the WUS (57%). These NSP were composed mainly of glucose, xylose, arabinose, and glucuronic acid. Sequential extractions with a saturated Ba(OH) <sub>2</sub> -solution (BE1 extract), and distilled water (BE2 extract) were used to solubilise glucuronoarabinoxylans from maize WUS. The glycosidic linkage composition of the extracts and their resistance to endo-xylanase treatment indicated that the extracted glucuronoarabinoxylans were highly substituted. In the maize BE1 extract 25% of the xylose was unsubstituted, 38% was monosubstituted and 15% was disubstituted. The glucuronoarabinoxylans in maize BE1 appeared to be resistant to endo-xylanase treatment, but could be degraded by a sub-fraction of Ultraflo, a commercial enzyme preparation from <em>Humicola insolens</em> . The digest contained a number of series of oligomers: pentose <sub>n</sub> , pentose <sub>n</sub> GlcA, pentose <sub>n</sub> hexose, and pentose <sub>n</sub> GlcA <sub>2</sub> . The presence of these glucuronic acid-containing series of oligomers showed that the glucuronic acids in the glucuronoarabinoxylancan can be very close to each other, but are not distributed blockwise. Finally, a new measure for the degree of substitution of glucuronoarabinoxylans was defined. It turned out that the degree of substitution in maize BE1 is much higher (87%) than in sorghum (70%) and wheat flour BE1 (56%). This indicates that the glucuronoarabinoxylans in maize BE1 are more complex than those in sorghum BE1 and explains their resistance to endo-xylanase treatment.<p>From this research, it can be concluded that both soybean and maize kernel cell wall polysaccharides distinguish themselves in a number of respects from other plant cell walls polysaccharides. The absence of homogalacturonan, but also the presence of internal (1,5)-linked arabinofuranose and terminal arabinopyranose in the pectic arabinogalactan side chains from soybean cell walls and the complexity of the glucuronoarabinoxylan from maize kernel cell walls are discussed in chapter 8. In addition, it was shown that techniques like mass spectrometry and NMR spectroscopy are powerfull techniques to be used after (enzymatic) fragmentation, for chemical characterisation of the original polysaccharides.<br/>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Voragen, A.G.J., Promotor, External person
  • Schols, Henk, Promotor
Award date3 Mar 2000
Place of PublicationS.l.
Print ISBNs9789058081872
Publication statusPublished - 2000

Keywords

  • cell walls
  • polysaccharides
  • pectins
  • galactans
  • galacturonic acid
  • zea mays
  • glycine max

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