Robert R. Selvendran

Determination of aldoses and uronic acid content of vegetable fiber

Abstract The factors affecting the stability, hydrolysis, reduction, acetylation, quantitation, and identification of the neutral sugars from vegetable fiber preparations have been studied critically and optimized. The recommended method offers a consolidation of the recent modifications of the alditol acetate procedure for the estimation of neutral sugars. The recovery of the sugars was tested by glc and ion-exchange chromatography. Also, the modified carbazole method of Bitter and Muir was adapted to make it applicable for the estimation of uronic acid content of fiber because uronic acid cannot be estimated quantitatively by the acetylation procedure. It is emphasized that the proposed method is applicable only to highly purified fiber preparations which are free of coprecipitated intracellular compounds. Also, the levels of pentoses and hexoses in the fiber must be well defined and a suitable correction made for their interference in the assay.

Avoiding oxidative degradation during sodium hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide

Abstract A comparison of the methylation of cellobiitol by the Hakomori method (using potassium methyl-sulfinylmethanide - “potassium dimsyl” - as base) and by a published or a modified sodium hydroxide-mediated procedure is described. Evidence is presented that the modified sodium hydroxide-catalysed procedure is not prone to the oxidative deficiencies of the original and that, given its reduced tendency towards polysaccharide undermethylation, it should now become the method of choice for the methylation of these polymers.

Structure of the acidic extracellular gelling polysaccharide produced by Pseudomonas elodea

Abstract The gelling polysaccharide secreted by the bacterium Pseudomonas elodea contains l -rhamnose, d -glucose, and d -glucuronic acid in the molar ratios 1:2:1. Methylation analysis of native and carboxyl-reduced polysaccharide indicated (1→4)-Rha p , (1→3)-Glc p , (1→4)-Glc p , and (1→4)-Glc p A to be present in the ratios 1:1:1:1. Graded acid hydrolysis gave a series of acidic oligosaccharides that were isolated by ion-exchange chromatography and fractionated by gel-filtration. The purified oligosaccharides were analysed and characterised as their methylated alditol derivatives by e.i.-m.s. and c.i.-m.s., and also examined by fast-atom-bombardment (f.a.b.)-m.s. The tetrasaccharide repeating-unit 1 , excluding acetyl groups, is proposed.

Structural features of cell-wall polysaccharides of onion Allium cepa

Abstract Cell-wall material has been isolated from immature onion tissues and extracted in sequence with cyclohexane- trans -1,2-diaminetetra-acetate (CDTA) at 20°, 0.05 m Na 2 CO 3 at 1°, 0.05 m Na 2 CO 3 at 20°, and 0.5, 1, and 4 m KOH at 20° to leave the α-cellulose residue, which contained a significant amount of pectic material. The polymers isolated from the extracts were fractionated by anion-exchange chromatography and subjected to methylation analysis. This study helped to distinguish between the pectic polysaccharides of the middle lamellae (solubilised by CDTA) and those of primary cell walls (solubilised by dilute alkali); the latter contained more highly branched rhammogalacturonan backbones. All the rhamnogalacturonans were substituted to various degrees with side chains comprising galactans or arabinoglactans which contained mainly (1»4)-linked galactose, lesser amounts of (1»4,1»)- and (1»2,1»6)-linked galactose, and (1»5)-linked arabinose, and small proportions of (1»2)-linked galactose. Most of the branched residues were terminated by galactopyranosyl and arabinofuranosyl groups. The major hemicellulose was a xyloglucans which showed structural features in common with the xyloglucans of dicotyledonous plants. Small amounts of hemicellulosepectic complexes were also isolated.

The wound response of tomato plants can be inhibited by aspirin and related hydroxy-benzoic acids

Abstract The response of tomato plants to injury or to treatment with cell wall fragments has been studied. Wounding, pectic fragments or chitosan causes the systemic accumulation of proteinase inhibitor proteins. This response can be inhibited by pretreatment of the plants by aspirin. The effect of aspirin is rapid and reversible. Related hydroxybenzoic acids are also inhibitory: the structural specificity required for inhibition is very similar to that demonstrated by others to be required for induction of pathogenesis-related proteins in tobacco.

Analysis of cell wall material from plant tissues: Extraction and purification

Abstract The alcohol-insoluble residue (AIR) of immature and mature runner beans contains co-precipitated cytoplasmic proteins, nucleic acids, starch and polyphenols, which contaminate the isolated polysaccharide fractions and their binding is sufficiently tenacious to resist complete extraction with the usual protein solubilizing reagents. Therefore, a method was developed for preparation of “cell wall material” from plant tissues in which the contamination with cytoplasmic constituents was minimal. Alternative solvents for cell disruption and protein extraction have been compared. The method depended for its success on the selective removal of the contaminants from fresh ball-milled tissue by sequential treatments with 1% aq. Na deoxycholate, PhOH-HOAc-H 2 O followed by α-amylase digestion. Ball-milling the tissue ensured almost complete rupture of the cells and organelles and allowed the solvents to penetrate the sample fully and dissolve the cytoplasmic constituents. The purified “cell wall material” has protein contents varying from 2.5 to 5.5% depending on the type and maturity of the tissue. The residual proteins are resistant to pronase, rich in hydroxyproline and have the amino acid composition of purified cell wall proteins, showing that the wall preparations are relatively pure.

Purification and methylation analysis of cell wall material from Solanum tuberosum

Cell wall material (CWM) of potatoes was prepared by sequentially extracting the wet ball-milled tissue with 1 % aq. Na deoxycholate, PhOHHOAcH2O and 90 % (v/v) aq. DMSO. The purity of the CWM (e.g. absence of residual starch) was established by carbohydrate analysis using different acid hydrolysis conditions and by methylation studies. The partially methylated alditol acetates from the CHCl3MeOH soluble fraction (S) of the methylated CWM were separated into 15 main peaks by GLC. Fourteen of these peaks were carbohydrate derivatives and the identity of most of these was established by MS. Reduction of the hydrolysate of S with NaBD4 was used to identify the carbohydrate derivatives present in peaks 7 and 11 above. The occurrence of 4-linked galacturonosyl residues in the methylated polymers was established after reduction of S with LiAlH4 and LiAlD4. The main glycosidic linkages present in the non-cellulosic polysaccharides of the wall in descending order of concentration are: 4-linked galactose, 4-linked galacturonic acid, 5-linked arabinose and 4,6-linked glucose. The major branch points are those through 0–6 of glucose and 0–4 of rhamnose. Arabinose, galactose and xylose residues constituted the non-reducing ends. Graded acid hydrolysis of the CWM made it possible to assess the relative strengths of some of the glycosidic linkages. The general structural features of the CWM are discussed in the light of these results.

Isolation and analysis of cell wall material from beeswing wheat bran (Triticum aestivum)

Abstract Cell wall material (CWM) isolated from beeswing wheat bran contains 66% carbohydrate, 12% Klason lignin, 6% protein and 4% ash. The relative proportions of sugars in the CWM are arabinose 34%, xylose 26%, galactose 2%, glucose 32% and uronic acid 6%. The uronic acid was shown to consist of glucuronic acid and its 4- O -Me analogue in the ratio 1.8:1. Partial acid hydrolysis of the CWM yielded neutral sugars and a uronic acid fraction. The latter was shown to contain Glc p A-(1→2)-Xyl p and Glc p A-(1→2)- O -Xyl p-(1→4)-Xyl p and their 4- O -Me substituted uronic acid analogues. Methylation analysis of the whole CWM and partially degraded methylated CWM revealed the nature of the constituent glycosidic linkages. From the combined evidence we infer that the major structural features of the non-cellulosic polysaccharides are a linear chain of xylopyranose units joined by (1→4)-linkages, and arabinofuranose, xylose, galactose (and uronic acid) end groups, which in at least some of the polysaccharides, are attached directly by (1→2)- and/or (1→3)-linkages to the xylan chain. The CWM has been fractionated by successive extractions with water at 80°, 0.2 M (NH 4 ) 2 C 2 O 4 at 80°, Na chlorite/HOAc at 70°, 0.2 M (NH 4 ) 2 C 2 O 4 at 80°, 1 M and 4 M KOH, and the neutral sugar composition of the fractions determined. It is concluded from these and other experiments that the CWM contains two main types of polysaccharides, the arabinoxylans and cellulosic polymers, and that phenolic ester linkages play a role in holding them together.

Isolation and Analysis of Cell Wall Polymers from Olive Pulp

The olive tree (Olea europaeia) is native to countries having a Mediterranean climate (Fernandez-Diez 1971, 1983). Olive fruit is a drupe, i.e. a meaty stone fruit (Rommani and Jennings 1971), and is usually an elongated spheroid. Depending on the variety, the weight of the fruit may vary from 3 to 10 g. During ripening, the colour changes from green to purple or black. The pulp is very bitter, especially during maturation, and comprises between 70 to 90% of the fruit, the bitter taste is due to the presence of a substantial quantity of phenolic compounds (Amiot et al. 1986; Vlahov 1992). The mature seed is very tough and elongated. The olive fruit is rich in oil which originates from the pulp; most cultivated olives have an oil content of 22% by fresh weight (Kiritsakis and Markakis 1987). The fruit is the raw material for a variety of products including olive oil and table olives. The preparation of table olives is a complex industrial process which involves fermentation and/or alkali treatment (Fernandez-Diez et al. 1985). The pulp of the olive fruit is composed of different tissue types (Fig. 1) and there is a paucity of information on the cell walls of these tissues. A knowledge of the structure and chemical composition of olive cell walls is crucial for a better understanding of the biochemical changes that occur in olives during growth, maturation and processing. This chapter describes methods that can be used for the isolation of relatively pure cell walls from olive pulp and the extraction of cell wall polysaccharides by methods that cause minimum degradation. For detailed structural studies, cell wall material virtually free of intracellular compounds is required. However, for certain practical purposes, an alcohol-insoluble residue (AIR) may suffice, provided the limitations of the preparation are borne in mind. The methods used for the separation and characterization of the polymers are described briefly, with particular reference to some of the more recent developments. The experimental details described below are mainly based on our work on the isolation analysis of cell walls of olive pulp (Coimbra 1993; Coimbra et al. 1994).

Hemicellulosic polymers from the cell walls of beeswing wheat bran: Part I, polymers solubilised by alkali at 2°

Abstract Cell-wall material from beeswing wheat bran was sequentially extracted with 0.05 m NaOH at 2°, m KOH at 2° and 20°, and 4 m KOH at 20° followed by delignification and further extraction with m and 4 m KOH at 20°, to leave the α-cellulose residue which contained a significant amount of arabinoxylan. The hemicellulosic polymers solubilised by m KOH at 2°, which represented ∼20% of the dry weight of the cell walls, were fractionated by graded precipitation with alcohol prior to anion-exchange chromatography and then subjected to methylation analysis. The major polymers were closely related, highly branched arabinoxylans, slightly branched arabinoxylans, and arabinoxylans in close association with xylogucans (arabinoxylan-xyloglucan complexes); the arabinoxylans were acidic and were associated with various amounts of phenolics. The various polymers exhibit heterogeneity, and phenolic ester and phenolic ether cross-links play a major role in the architecture of the cell walls.