Xylans, acetates extraction

Natural xylan may occur partially esterified with acetic acid. This possibility arises from the observation that most holocellulose preparations contain a small amount of acetic acid. So far, however, it has not been established which polysaccharides are esterified. Treatment of the holocellulose with the alkaline solutions necessary for xylan extraction brings about complete deacetylation. 

The late discovery of acetyl xylan and feruloyl esterases has been partly due to the lack of suitable substrates. Xylans are often isolated by alkaline extraction, in which ester groups are saponified. Treatment of plant materials under mildly acidic conditions, as in steaming or aqueous-phase thermomechanical treatment, leaves most of the ester groups intact. These methods, however, partly hydrolyze xylan to shorter fragments (63,69). Polymeric acetylated xylan can be isolated from delignified materials by dimethyl sulfoxide extraction (70). The choice of substrate is especially important in studies of esterases for deacetylation of xylans. The use of small chromophoric substrates (p-nitrophenyl acetate, a-naphthyl acetate, and methylumbelliferyl acetate) analogously to the assays of disaccharidases may lead to the monitoring of esterases unable to deacetylate xylan (33, 63, 64). 

Before discussing the various derivatives that have been used, it should be remembered that oligosaccharides are often obtained by chromatography on paper or on cellulose columns, and this may cause them to become contaminated by xylan this may be eliminated by extraction of the crude fractions with hot ethanol.474 For similar reasons, glucose that is really extraneous may be detected in fractions separated by electrophoresis on cellulose acetate.475 Gas-liquid chromatography was used to show the presence in pituitary glyco-... 

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

The xylan of hardwoods (O-acetyl-4-O-methylglucuronoxylan) consists of at least 70 p-xylopyranose residues (average degree of polymerization between 150 and 200) linked by P-l,4-glycosidic bonds (Fig. 11.4-4) 139. Every tenth xylose residue carries a 4-O-methylglucuronic acid attached to the C-2 of xylose 131. In addition, hardwood xylans are highly acetylated e. g. birchwood xylan contains more than 1 mol of acetic acid per 2 mols of xylose 140. Acetylation occurs usually at the C-3 rather than the C-2 position of xylose. Acetylation at both positions has also been reported 141, 142. The presence of these acetyl groups is responsible for the partial solubility of xylan in water 133. The alkali extraction of xylan leads to the deacetylation of this substrate 140. ... 

Bird and Ritter isolated, from wood of white oak, a chlorine holocellu-lose which contained all of the 0-acetyl groups present in the wood. Mitchell and Ritter later extracted a chlorine holocellulose from sugar maple with water and obtained a xylan in a yield of 3.4% of the wood. This polysaccharide contained 9.2% of 0-acetyl groups. A xylan which had been obtained in the same way, from aspen, by Wise and Jones, was, on treatment with periodate, oxidized almost to completion. When the wood itself was similarly treated, most of its xylan escaped oxidation. Although it appears evident that all of the xylan in the wood could not possibly have been accessible to the aqueous reagent, it was concluded that the lack of oxidation was most probably due to the fact that the native xylan was partly 0-acetylated. After treatment of wood from Eucalyptus regnans with methanol at 150°, Stewart and coworkers obtained, on extraction with water, a xylan (in a jdeld of 3.7%) which contained 5-6% of acetate... 

In the case of water-soluble O-acetylated xylans and glucomannans, an ammonium acetate solution with a pH of 7 was employed as eluent in connection with the SEC separation step (7). However, for dkali extracted xylans and glucomannans, a more alkaline (pH 13) sodium hydroxide/acetate eluent was required (3). In all cases pretreatment of the SEC fractions by passage through a cation-exchange resin prior to MALDI-MS analysis and/or the use of MALDI probes coated with a Nafion film (2), were necessary in order to minimize the disturbance by buffer ions during the MALDI analysis step. 

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