Isolation and Characterization of a Xyloglucan from Gobo (Arctium lappa L.)

A xyloglucan was isolated from the 24% KOH extract of gobo (edible burdock, Arctium lappa L.). A methylation analysis and enzymic degradation studies on the polysaccharide showed that gobo-xyloglucan was built up predominantly of repeating-oligosaccharide units of hepta-(Glc: Xyl=4: 3), nona- (Glc: Xyl: Gal: Fuc=4: 3: 1: 1) and deca- (Glc: Xyl: Gal: Fuc=4: 3: 2: 1) saccharides in an approximate molar ratio of 14: 12: 5, which are the typical structural units of dicot xyloglucans.     

Apple-fruit xyloglucans: a comparative study of enzyme digests of whole cell walls and of alkali-extracted xyloglucans.

Apple cell walls or alkali-extracted xyloglucans were digested with an endo-glucanase from Trichoderma viride and the resulting oligosaccharides were isolated by chromatography on Bio-Gel P-4. Three main oligosaccharides were present in similar proportions, and their structures were shown to be [Xyl(Glc)]3-Glc, [Xyl(Glc)]2-(FucGalXyl)Glc-Glc, and XylGlc-(GalXyP)Glc-(FucGalXyl)Glc-Glc. Each non-reducing-end Glc was 6-linked, each reducing-end Glc was 4-substituted, and each other Glc was 4,6-disubstituted. The Xyl was either terminal or 2-substituted, the Fuc was terminal, and the Gal was either terminal or 2-substituted. The 1H-NMR spectra of the oligosaccharides extracted directly from the cell wall showed that they are not acetylated. Other oligosaccharides, notably GalXyl3Glc4, Xyl2Glc4, and Xyl2Glc3, were present in smaller proportions in the digest of the cell walls.     

Fucosylation of exogenous xyloglucans by pea microsomal membranes

Microsomal membrane preparations from growing regions of etiolated pea stems catalyzed the transfer of [14C]fucosyl units from GDP-[U-14C]-L-fucose into exogenously added xyloglucan acceptors, as well as into endogenous xyloglucan. The transfer was more effective using nonfucosylated tamarind seed xyloglucan than with pea wall xyloglucan in which almost all galactose units are already fucosylated. Hydrolysis of products by endo-1,4-beta-D-glucanase yielded in each case radioactive nonasaccharide as the main fucosylated product. UDP-galactose enhanced the fucosylation of endogenous primer but it had little effect on fucosyl transfer to exogenously added xyloglucans. Low-molecular-weight nonfucosylated oligosaccharide fragments up to the octasaccharide Glc4Xyl3Gal (obtained by endoglucanase action on tamarind seed xyloglucan) were ineffectual as fucosyl acceptors but inhibited the fucosylation of endogenous as well as of added xyloglucan. With octasaccharide, the inhibition was competitive in relation to the xyloglucan acceptor (Ki = 70 microM) and noncompetitive in relation to the donor GDP-fucose (Ki = 210 microM). It is concluded that fucosyltransferase acts independently and in a noncoordinated manner from other glycosyltransferases that are required to synthesize xyloglucan. Its active site recognizes a fragment longer than the galactosylated octasaccharide unit before transfucosylation will ensue.     

山豆根木葡聚糖的研究

研究了山豆根中一种木葡聚糖的结构．用１ｍｏｌ／ＬＮａＯＨ提取,ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５离子交换柱层析,Ｆｅｈｌｉｎｇ试剂分级得到山豆根木葡聚糖组分ＳＳｂ－１ＦＡ,用完全酸水解,甲基化分析,部分酸水解,三氧化铬氧化和１Ｈ,１３ＣＮＭＲ等方法对其结构进行研究．结果表明：ＳＳｂ－１ＦＡ的分子量为２．６×１０４,比旋光度［α］２０Ｄ＝＋１０．９°（ｃ０．２２,Ｈ２Ｏ）,由Ｌ－Ｆｕｃ,Ｄ－Ｘｙｌ,Ｄ－Ｇａｌ和Ｄ－Ｇｌｃ组成,摩尔比为：２．９２９．９７．５５９．８．ＳＳｂ－１ＦＡ由１→４连接的β－Ｄ－Ｇｌｃ残基构成主链,分枝有α－Ｄ－Ｘｙｌ（１→,β－Ｄ－Ｇａｌ（１→２）α－Ｄ－Ｘｙｌ（１→等类型,部分非还原末端由Ｌ－Ｆｕｃ（１→构成．     

刺五加果水溶性多糖的研究——AS-2的分离纯化与结构探讨

从刺五加果中抽提出水溶性粗多糖。经酸性乙醇分级及反复冻融得到多糖AS-2。AS-2经Sepharose CL-4B柱层析为单一对称峰,经醋酸纤维素膜电泳为一条带,冻融后高速离心无沉淀可证明其为均一级分。G.C分析表明,AS-2由Ara、Xyl、Rha、Gal、Glc组成,其单糖摩尔比为1.6:1.2:1.8:1.0:3.6。AS-2的分子量约为78kD,比旋光度[α]_D~(25)=+17°,特性粘度[η]=0.068。红外光谱分析含β型糖苷键。部分酸水解、酶解、高碘酸酸化、Smith降解、完全甲基化、G.C,G.C-M.S的分析结果表明,以β(1→3)Glc及β(1→4)Glc构成分子的主链。Glc的C_3上带有分支,约每4个己糖残基带有1个侧链。侧链上,Rha多以1→4苷键相连,部分残基C_2上有分支。Gal存在(1→6)及(1→3)连接方式,多数Glc以(1→6)苷键连结,少数Glc出现在分子非还原末端。位于分子末端的还有Ara与Xyl。     

Biochemical and molecular characterization of secreted α-xylosidase from Aspergillus niger

α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.     

Characterisation of extracellular polysaccharides from suspension cultures of apple (Malus domestica)

The polymers secreted by suspension-cultured apple cells were composed of 85% carbohydrate (76% neutral sugar and 9% uronic acid) and 15% w/w protein. The extracellular polysaccharides (ECPs) contain 23% XG and 59% AGPs. The monosaccharide composition of the ECPs consisted of Gal, Ara, Glc and Xyl, with smaller amounts of Rha, Fuc and Man. Fractionation of the ECPs by anion-exchange chromatography yielded an unbound neutral fraction and a bound acidic fraction. Monosaccharide and linkage compositions of each fraction were determined. The neutral fraction (48% recovered carbohydrate) was composed of xyloglucan (XG; >90 mol%) which was purified by selective precipitation with Fehling’s solution to yield pure XG. The purified XG had a Glc:Xyl:Gal:Fuc ratio of 4.0:2.5:0.8:0.5; the XG was not O-acetylated. The structure of the secreted XG was similar to that extracted from apple-pomace. The acidic fraction (52% recovered carbohydrate) was composed primarily of arabinogalactan-proteins (AGPs) as detected by the β-glucosyl Yariv diffusion test. The AGP had a Gal:Ara ratio of 1.3: 1.0. Minor amounts of arabinan, xylan and mannan were also detected in the ECPs. This study is the first examination of the polysaccharides secreted by apple cells grown in suspension culture.     

A Barley xyloglucan xyloglucosyl transferase covalently links xyloglucan, cellulosic substrates, and (1,3;1,4)-beta-D-glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.     

Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET)

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan-enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0-6.5), Km for the nonasaccharide XLLGol (Gal2.Xyl3.Glc3.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher Km values for XLLGol (70-130 microM) than the four mung bean isoenzymes (16-35 microM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.     

Xyloglucan oligosaccharides promote growth and activate cellulase: evidence for a role of cellulase in cell expansion

Oligosaccharides produced by the action of fungal cellulase on xyloglucans promoted the elongation of etiolated pea (Pisum sativum L.) stem segments in a straight-growth bioassay designed for the determination of auxins. The oligosaccharides were most active at about 1 micromolar. We tested the relative growth-promoting activities of four HPLC-purified oligosaccharides which shared a common glucose₄· xylose₃ (XG7) core. The substituted oligosaccharides XG8 (glucose₄· xylose₃· galactose) and XG9n (glucose₄· xylose₃· galactose₂) were more effective than XG7 itself and XG9 (glucose₄· xylose₃· galactose· fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10⁻⁴ molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of [³H]xyloglucan to ethanol-soluble fragments. This suggest that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products). We suggest that the promotion of midchain xyloglucan cleavage, by loosening the primary cell wall matrix, explains the promotion of growth by the oligosaccharides.     

Biosynthesis of the fucose-containing xyloglucan nonasaccharide by pea microsomal membranes

Pea microsomal membranes catalyze the transfer of [¹⁴C]fucose (Fuc) from GDP-[U-¹⁴C]fucose, with or without added unlabeled UDP-glucose (Glc), UDP-xylose (Xyl) or UDP-galactose (Gal), to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, [¹⁴C] fucose residues occur exclusively in a fragment corresponding in size to the xyloglucan nonasaccharide, Glc₄ Xyl₃ Gal Fuc. This fragment contains a single labeled fucose residue per oligomer, α-linked in a terminal nonreducing position. By comparison, in incubations where GDP-[¹⁴C] fucose is absent and replaced by UDP-[³H]xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products is an octasaccharide. In the presence of both GDP-[¹⁴C]fucose and UDP-[³H]xylose, a nonasaccharide containing the two labels is produced. Fucose and xylose residues are transferred within a few minutes to acceptor molecules of molecular weight up to 300,000. Such products do not elongate detectably over 60 minutes of incubation. The data support the conclusion that the nonasaccharide subunit of xyloglucan may be generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis is dependent on the inclusion of exogenous GDP-fucose.     

STUDIES OF POLYSACCHARIDES FROM THREE EDIBLE SPECIES OF NOSTOC (CYANOBACTERIA) WITH DIFFERENT COLONY MORPHOLOGIES: STRUCTURAL CHARACTERIZATION AND EFFECT ON THE COMPLEMENT SYSTEM OF POLYSACCHARIDES FROM NOSTOC COMMUNE

The cyanobacterium Nostoc commune Vaucher produces quite complex extracellular polysaccharides. The cyanobacterium is nitrogen fixing, and on growing the cyanobacterium in media with and without nitrogen, different types of extracellular polysaccharides were obtained. These were also different from the polysaccharides present in N. commune collected in the field. High pH anion exchange chromatography (HPAEC) of weak acid hydrolysates of the culture-grown material demonstrated that, in this case, HPAEC was useful for comparison of the different polymers. The main differences between the polymers from the field group and the culture-grown samples were the presence of substantial amounts of arabinose, 2- O -methylglucose, and glucuronic acid in the latter. Methylation studies also revealed a difference in the branching points on the glucose units between the field and cultured samples, being 1,4,6 for the first and 1,3,6 for the latter. The field acidic fraction gave, on weak acid hydrolysis and separation on BioGel P2 and HPAEC, 12 oligosaccharide fractions that were isolated and studied by different mass spectroscopy techniques. The structures of the oligosaccharides were determined, and two different series that can originate from two repeating pentamers were identified: GlcA1-4/6GlcM1-4Gal1-4Glc1-4Xyl and GlcA1-4/6Glc1-4Gal1-4Glc1-4Xyl. The difference between these oligosaccharides lies in the methyl substituent on carbon 2 of the glucose unit next to the nonreducing glucuronic acid unit. The polysaccharides from field material were shown to have a strong effect on the complement system.     

Xyloglucan oligosaccharides cause cell wall loosening by enhancing xyloglucan endotransglucosylase/hydrolase activity in azuki bean epicotyls

Addition of xyloglucan-derived oligosaccharides shifted the wall-bound xyloglucans to a lower molecular mass distribution and increased the cell wall extensibility of the native epidermal tissue strips isolated from azuki bean (Vigna angularis) epicotyls. To ascertain the mechanism of oligosaccharide function, we examined the action of a xyloglucan endotransglucosylase/hydrolase (XTH) showing both endotransglucosylase and endohydrolase activities, isolated from azuki bean epicotyl cell walls, in the presence of xyloglucan oligosaccharides. The addition of xyloglucan oligosaccharides enhanced the xyloglucan-degrading activity of XTH against isolated xyloglucan substrates. When the methanol-fixed epidermal tissue strips were incubated with XTH, the molecular mass of wall-bound xyloglucans was decreased and the cell wall extensibility increased markedly in the presence of the oligosaccharides. These results suggest that xyloglucan oligosaccharides stimulate the degradation of xyloglucans by enhancing the XTH activity within the cell wall architecture, thereby increasing the cell wall extensibility in azuki bean epicotyls.     

A beta-D-galactosidase from nasturtium (Tropaeolum majus L.) cotyledons. Purification, properties, and demonstration that xyloglucan is the natural substrate

beta-D-Galactosidase activity has been detected previously in the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds and has been linked to the hydrolysis in vivo of storage xyloglucan (amyloid) (Edwards, M., Dea, I. C. M., Bulpin, P. V., and Reid, J. S. G. (1985) Planta (Berl.) 163, 133-140). The major beta-D-galactosidase present in extracts from the cotyledons of 9-day seedlings has now been purified to apparent homogeneity. The enzyme (Mr 97,000, no subunits) comprised a range of closely related molecular species ranging in isoelectric point from pH 6.6 to 7.1. Further purification to give a single protein band on isoelectric focusing (pI = 7.1) was achieved by chromatofocusing. The pH optimum of the enzyme (mixed molecular species) was 4.0-5.0 (stable from pH 3-10), and the temperature optimum was 50 degrees C (stable to 50 degrees C). It hydrolyzed lactose and beta-D-galactopyranosides but not melibiose and alpha-D-galactopyranosides. It did not release the terminal nonreducing alpha-D-galactopyranosyl residues from seed galactomannans, but catalyzed the rapid removal of terminal nonreducing beta-D-galactopyranosyl residues from xyloglucans. On the basis of the ability of the enzyme to hydrolyze xyloglucans, the kinetics of xyloglucan hydrolysis, and an experimental demonstration of a clear correlation between xyloglucan depletion and the activity in vitro of this enzyme, it is argued that the cell-wall storage xyloglucan of the nasturtium seed is its natural substrate.     

Xyloglucan galactosyl- and fucosyltransferase activities from pea epicotyl microsomes.

Microsomal membranes from growing tissue of pea (Pisum sativum L.) epicotyls were incubated with the substrate UDP-[14C]galactose (Gal) with or without tamarind seed xyloglucan (XG) as a potential galactosyl acceptor. Added tamarind seed XG enhanced incorporation of [14C]Gal into high-molecular-weight products (eluted from columns of Sepharose CL-6B in the void volume) that were trichloroacetic acid-soluble but insoluble in 67% ethanol. These products were hydrolyzed by cellulase to fragments comparable in size to XG subunit oligosaccharides. XG-dependent galactosyltransferase activity could be solubilized, along with XG fucosyltransferase, by the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. When this enzyme was incubated with tamarind (Tamarindus indica L.) seed XG or nasturtium (Tropaeolum majus L.) seed XG that had been partially degalactosylated with an XG-specific beta-galactosidase, the rates of Gal transfer increased and fucose transfer decreased compared with controls with native XG. The reaction products were hydrolyzed by cellulase to 14C fragments that were analyzed by gel-filtration and high-performance liquid chromatography fractionation with pulsed amperometric detection. The major components were XG subunits, namely one of the two possible monogalactosyl octasaccharides (-XXLG-) and digalactosyl nonasaccharide (-XLLG-), whether the predominant octasaccharide in the acceptor was XXLG (as in tamarind seed XG) or XLXG (as in nasturtium seed XG). It is concluded that the first xylosylglucose from the reducing end of the subunits was the Gal acceptor locus preferred by the solubilized pea transferase. These observations are incorporated into a model for the biosynthesis of cell wall XGs.     

草苁蓉根、茎水溶性多糖BRT的结构特征

本文以长白山区珍贵野生药用植物草苁蓉为研究对象 .草苁蓉又名“不老草” ,具有滋补强壮、益寿延年之功及补肾壮阳、润肠止血之效 ,为国家三级重点保护植物[1] .近年的研究发现 ,草苁蓉醇提物不仅可以清除体内的游离基 ,而且还可以显著增强机体的免疫能力 ,同时对草苁蓉化学成分的研究也在逐步深入[2 ] ,但对于草苁蓉多糖的系统研究尚未见报道 .为了更全面地认识和利用草苁蓉这一珍贵的植物资源 ,同时也为探讨多糖的结构与功能的关系 ,本文对草苁蓉根、茎的水溶性多糖ＢＲＴ组分进行了结构测定方面的研究 .1　材料和方法1 1　材料为本研究…     

Xyloglucan from the leaves of Hymenaea courbaril

A fucosylated xyloglucan was isolated from the leaves of Hymenaea courbaril by alkaline extraction, followed by ethanol precipitation and ion-exchange chromatography. The isolated polysaccharide showed Glc:Xyl:Gal:Fuc in molar ratio of 8:5:2.5:1 and (D)(25) +40.5 degrees. Composition and linkage analyses, supported by NMR spectroscopic measurements, showed that the polysaccharide has a glucan backbone which is highly substituted at O-6 with D-xylopyranose residues, about a half of which are substituted at O-2 by D-galactopyranosyl units. Some of the galactose residues are further substituted by L-fucopyranose at O-2. The M(r), as determined by HPSEC, was 49,500.     

Antitumor Protein-containing Polysaccharides from a Chinese Mushroom Fengweigu or Houbitake,Pleurotus sajor-caju (Fr.) Sings

Protein-containing polysaccharides extracted from fruiting bodies of a Chinese fungus named Feng Wei Gu, were fractionated and purified, and their antitumor activities were tested, out of which the following active fractions were obtained.

FI_o-a: A protein-containing xyloglucan, MW 280,000, polysaccharide: protein=76: 24 (w/w), polysaccharide consisting of Man: Gal: Xyl: Glc = 2: 12: 42: 42 (molar ratio). + 25.3°.

FA-2: A protein-containing mannogalactan, MW 120,000, polysaccharide: protein = 76 : 16 (w/w), consisting of Xyl : Man: Gal = 9 : 35 : 56 (molar ratio), + 98.5°.

FII-1: A Protein-containing xylan (62: 21 w/w). MW 200,000, +8.7°.

FIII-1a: A protein-containing glucoxylan (15: 71 w/w), +30.7°, MW 90,000, consisting of Glc : Xyl = 40 : 44 (molar ratio).

FIII-2a: A protein-containing xyloglucan, MW 70,000, polysaccharide: protein = 69: 3 (w /w), polysaccharide consisting of Xyl: Glc = 36 : 62 (molar ratio). + 38.6°.     

Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1→4)-β-d-glucanase from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds

We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(14)--d-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc₄ subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered — 3,— 2, + 2 and + 3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at — 1 and the xylose-substituted glucose at + 1, which never carries a galactosyl substituent. We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific -xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylosesubstitution at glucose residues — 4, — 2, + 1 and + 3 and that xylose substitution at + 2, is a requirement. There may also be a requirement for xylose substitution at — 3. We have demonstrated also that galactosyl substitution of a xylose residue at + 1 prevents, and at — 2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.Abbreviations G unsubstituted glucose residue - X xylose-substituted glucose residue - L galactosylxylose-substituted glucose residue - F fucosyl-galactosylxylose-substituted glucose residue - Gal galactose - Glc glucose - HPAE high-performance anion-exchange chromatography - NXET nasturtium xyloglucan endo-transglycosylase or xyloglucan-specific endo-(14)--d-glucanase - Xyl xylose This work was funded jointly by Unilever UK and the Department of Trade and Industry (UK) via the LINK initiative Agro-Food Quality.     

Xyloglucan mobilisation and purification of a (XLLG/XLXG) specific β-galactosidase from cotyledons of Copaifera langsdorffii

The storage xyloglucan of germinating seeds of Copaifera langsdorffii is degraded by the action of β-galactosidase, endo-β-glucanase, α-xylosidase and β-glucosidase, producing free galactose, glucose and xylose. One of the β-galactosidases from cotyledons of germinating seeds of C. langsdorffii was purified by ion exchange and gel chromatography (Biogel P-60), leading to a single polypeptide (molecular mass 40 kDa). The enzyme has optimum activity at pH 3.2 (stable from pH 2.3 to 6.0) and is active on p-NP-β-gal (K_m 3.5 mM) and lactose but not on o-NP-β-gal or p-NP-β-gal. Small amounts of galactose were released from xyloglucan of seeds of C. langsdorffii, Tamarindus indica and less from Hymenaea courbaril. No galactose was released after incubation with β-1,4-linked galactan from Lupinus angustifolius cotyledons. Much higher activity was observed on oligosaccharides obtained by hydrolysis of C. langsdorffii xyloglucan with Trichoderma viride cellulase. The purified β-galactosidase attacked XLLG and XLXG specifically, producing a mixture of XXXG and XXLG (unsubstituted glucose is assigned G; glucose branched with xylose is assigned X and if galactose is branching xylose, the trisaccharide is assigned L). Considering the recent discovery by Crombie and co-workers that (L) at the non-reducing end of the oligosaccharides prevents β-glucosidase from acting on GLXG or GLLG but not on GXLG or GXXG, the β-galactosidase isolated in this work seems to perform a key role in xyloglucan degradation since it is responsible for the retrieval of a major sterical hindrance (L) for further hydrolysis of the oligosaccharides and therefore essential for completion of xyloglucan mobilisation.