Isolation of Galactoglucomannan from Apple Hemicellulosic Polysaccharides with Binding Capacity to Cellulose

Abstract

The plant cell wall consists of polysaccharides such as pectin, hemicelluloses and cellulose. Hemicellulose components are covalently linked to pectin and hydrogen bonded to cellulose. 1,2) Xyloglucan is a hemicellulosic polysaccharide that is present in the primary cell walls of all higher plants, and is known to interact specifically with cellulose. 36) The interaction between cellulose and xyloglucan is thought to be involved in hydrogen bonding. In our previous works on the cellwall polysaccharides of apple flesh, xyloglucan was isolated from 24% KOH soluble fraction, and then the xyloglucan oligosaccharides were obtained by hydrolysis with Penicillium sp. M451 xyloglucanase. 79) The constituent sugars of the xyloglu-can oligosaccharides were Glc, Xyl, Gal and Fuc. Although Man was found in the 24% KOH soluble fraction from apple flesh cell walls, 79) the Man residue was not recovered in xyloglucan oligosaccharides. In addition, Vinken et al. 10) showed that the neutral fraction of the 4 M KOH extract, which was mainly xyloglucan, was contaminated with Man residues. From these findings, it is likely that a mannosecontaining polysaccharide is present in 24% KOH soluble polysaccharide fraction from apple flesh cell walls. In general, the mannosecontaining poly-saccharides require relatively strong alkali, typically 24%, for their extraction from cell walls. Therefore, it is conceivable that the mannosecontaining polysaccharides also bind to cellulose with hydrogen bonds in cell walls. Glucomannan acts as a preventative of chronic disease 11) and as a weight control agent. 12,13) In softwoods, gluco-mannan has (14) linked Glc and Man residues as main chain with branches through (16)Glc unit. 14,15) In konjac, it was reported that Glc(14)Glc residues , cellobiose, Man and mannotetraose units compose the main chain structure. 16,17) Recently, galactoglucoman-nan was extracted from kiwifruit cell walls, and its chemical structure was characterized. 18,19) In addition, the presence of glucomannan has been described during xyloglu-can purification in persimmon. 20) However, in apple, there is no detailed information for glucomannan or galactoglu-comannan from apple fruit. The polysaccharides extracted with 24% KOH from apple fruit cell wall polysaccharides contained mainly xylo-glucan. Therefore, in this paper, xyloglucan was removed from the 24% KOH extracts by treatment with xylogluca-nase from Geotrichum sp. M128, and then galactogluco-mannan was purified using the difference of their interaction with cellulose. MATERIALS AND METHODS Plant materials. Methanolinsoluble materials of apple flesh were fractionated into five fractions, WS (water soluble polysaccharide), PS (pectic substance), HC (hemi-cellulose)I, HCII and CL (cellulose) fractions, by using successive extraction with water, 0.25% ammonium ox-alate, 4% KOH and 24% KOH, and subsequent dialysis of the individual extracts as described previously. 21) In this study, we used the HCII fraction, which was composed mainly of Xyl, Glc, Ara, Gal and Man residues. Enzymes. Cellulase was purified from a commercial enzyme preparation from Trichoderma vride as follows: Meicelase (Meiji Seika Kaisha, Ltd.) was dissolved in a 20 mM McIlvaine buffer (pH 3.0) and centrifuged. The supernatant was applied to a cellulose column (5.025 cm) preequilibrated with the same buffer. The column was washed with the same buffer (1000 mL), and ad-sorbed materials were eluted with distilled water. Fractions (10.0 mL) were collected and assayed for cellulase Abstract: The hemicellulose fraction which is soluble in 24% KOH solution (HC-II) from the cell-walls of apple flesh contained a small amount of galactoglucomannan in addition to a large amount of xyloglucan. A crude galactoglucomannan was isolated from the HC-II fraction with binding capacity to cellulose microfi-brils. The crude galactoglucomannan fraction was treated with xyloglucanase to remove concomitant xyloglu-can, followed by DEAE-Sephadex A-25 chromatography to give a purified galactoglucomannan fraction. Sugar composition analysis showed that the purified galactoglucomannan fraction consisted of Man, Glc and Gal in the molar ratio of 5.7 : 3.0 : 1.1. Methylation analysis suggested that a galactoglucomannan present in apple flesh had β-(14)-linked Glc and Man residues, some of which were branched at the O-6 position with Gal residues.