Xyloglucan oligosaccharide alpha-L-fucosidase activity from growing pea stems and germinating nasturtium seeds.

[14C]Fucose-labelled xyloglucan (XG) was synthesized from tamarind seed XG by incubating it with GDP-[14C]fucose plus solubilized pea fucosyltransferase, and [14C]fucose-labelled XG nonasaccharide was prepared from the parent hemicellulose by partial hydrolysis with fungal cellulase. alpha-L-Fucosidase activity was readily detected in crude enzyme extracts of growing regions of etiolated pea stems (Pisum sativum) and in cotyledons of germinating nasturtium seedlings (Tropaeolum majus) using the fucosylated XG-nonasaccharide as substrate. Both enzymes showed little activity against intact fucosylated XG and they were totally inactive against p-nitrophenyl-alpha-L-fucoside. Auxin treatment of pea stems, which greatly increased the activity of endo-1,4-beta-glucanases that hydrolyse XG in apical growing regions, failed to result in a similar increase in XG-nonasaccharide alpha-fucosidase activity. However, germination of nasturtium seed, which resulted in a large increase in endo-1,4-beta-glucanase (XG-ase) activity in the cotyledons, was accompanied by comparable increases in XG-alpha-fucosidase activity.     

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.     

Xyloglucan sidechains modulate binding to cellulose during in vitro binding assays as predicted by conformational dynamics simulations

Cross-links between cellulose microfibrils and xyloglucan (XG) molecules play a major role in defining the structural properties of plant cell walls and the regulation of growth and development of dicotyledonous plants. How these cross-links are established and how they are regulated has yet to be determined. In a previous study, preliminary data were presented which suggested that the different sidechains of XG may play a role in controlling cellulose microfibril-XG interactions. In this study, this question is addressed directly by analyzing to what extent the different sidechains of pea cell wall XG and nasturtium seed storage XG affect their binding to cellulose microfibrils. Of particular importance to this study are the chemical data indicating that pea XG possesses a trisaccharide sidechain, which is not found in nasturtium XG. To this end, conformational dynamic simulations have been used to predict whether oligosaccharides representative of pea and nasturtium XG can adopt a hypothesized cellulose-binding conformation and which of these XGs exhibits a preferential ability to bind cellulose. Extensive analysis of the conformational forms populated during 300 K and high-temperature Monte Carlo simulations established that a planar, sterically accessible, glucan backbone is essential for optimal cellulose-binding. For the trisaccharide sidechain-containing oligosaccharide as found in pea XG, sidechain orientation appeared to regulate the gradual acquisition of this hypothesized cellulose binding conformation. Thus, conformational forms were identified that included the twisted backbone (non-planar) putative solution form of XG, forms in which the trisaccharide sidechain orientation enables increased backbone planarity and steric accessibility, and finally a planar, sterically accessible, backbone. By applying these conformational requirements for cellulose binding, it has been determined that pea XG possesses a two- to threefold occurrence of the cellulose binding conformation than nasturtium XG. Based on this finding, it was predicted that pea XG would bind to cellulose at a higher rate than nasturtium XG. In vitro binding assays showed that pea XG-avicel binding does indeed occur at a twofold higher rate than nasturtium XG-avicel binding. The enhanced ability of pea cell wall XG over nasturtium seed storage XG to associate with cellulose is consistent with a structural role of the former during epicotyl growth where efficient association with cellulose is a requirement. In contrast, the relatively low ability of nasturtium XG to bind cellulose is consistent with the need to enhance the accessibility of this polymer to glycanases during germination. These findings suggest potential roles for XG sidechain substitution, enabling XG to function in a variety of different biological contexts.     

Ping-pong character of nasturtium-seed xyloglucan endotransglycosylase (XET) reaction.

Plant xyloglucan endotransglycosylase (XET, EC 2.4.1.207) degrades its substrate by a transglycosylation mechanism while endo-cleaving xyloglucan (XG) molecules at their beta-1,4-linked polyglucosyl main chain and transferring the newly generated reducing chain ends to hydroxyls at C-4 of non-reducing glucosyl ends of the main chains of other XG molecules or of low-Mr XG-fragments (OS). Kinetic data obtained with purified nasturtium seed (Tropaeolum majus, L.) XET while using high-Mr xyloglucan and 3H-labeled XGOS alditols (DP 7-9) as substrates could be best fitted to the model for Ping-Pong Bi Bi reaction mechanism. Such mechanism is typical for transglycosylases operating with retention of the anomeric configuration of the formed glycosidic bond and involving the formation of a covalent glycosyl-enzyme reaction intermediate.     

Purification of xyloglucan endotransglycosylase based on affinity sorption of the active glycosyl-enzyme intermediate complex to cellulose.

Xyloglucan endotransglycosylase (XET) catalyzes the cleavage of xyloglucan (XG) molecules by a transglycosylation mechanism involving two steps: (a) endocleavage of the beta-(1,4)-linked polyglucosyl backbone of the xyloglucan molecule with formation of a glycosyl-enzyme intermediate; (b) transfer of the glycosyl residue from the intermediate to the C-4 position of the nonreducing end glucosyl unit of another molecule of XG or an XG-derived oligosaccharide with liberation of the enzyme (Z. Sulová et al., 1998, Biochem. J. 330, 1475-1480). The formation of a relatively stable active complex of XET with XG and the tendency of xyloglucan to bind tightly via hydrogen bonds to cellulose were exploited in the present method of purification of XET. Crude extracts from nasturtium (Tropaeolum majus) cotyledons and other plant sources containing the enzyme were mixed with XG in order to form the XET:XG complex, which was applied onto cellulose. Unadsorbed proteins were removed by washing and the XET was released from the adsorbed XET:XG complex by transglycosylation of its glycosyl moiety to added XG-derived oligosaccharides. The described procedure resulted in an over 100-fold increase in specific activity of XET in a single step. Further purification of the enzyme to homogeneity was achieved by gel-permeation chromatography on Bio-Gel P30. Similar procedure could be used for purification of XET from other plant sources, such as lentil (Lens culinaris) seeds, pea (Pisum sativum) epicotyls, and supernatant of suspension-cultured Catharanthus roseus cells.     

Characterization of a new xyloglucan endotransglucosylase/hydrolase (XTH) from ripening tomato fruit and implications for the diverse modes of enzymic action

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall-modifying enzymes that align within three or four distinct phylogenetic subgroups. One explanation for this grouping is association with different enzymic modes of action, as XTHs can have xyloglucan endotransglucosylase (XET) or endohydrolase (XEH) activities. While Group 1 and 2 XTHs predominantly exhibit XET activity, to date the activity of only one member of Group 3 has been reported: nasturtium TmXH1, which has a highly specialized function and hydrolyses seed-storage xyloglucan rather than modifying cell wall structure. Tomato fruit ripening was selected as a model to test the hypothesis that preferential XEH activity might be a defining characteristic of Group 3 XTHs, which would be expressed during processes where net xyloglucan depolymerization occurs. Database searches identified 25 tomato XTHs, and one gene (SlXTH5) was of particular interest as it aligned within Group 3 and was expressed abundantly during ripening. Recombinant SlXTH5 protein acted primarily as a transglucosylase in vitro and depolymerized xyloglucan more rapidly in the presence than in the absence of xyloglucan oligosaccharides (XGOs), indicative of XET activity. Thus, there is no correlation between the XTH phylogenetic grouping and the preferential enzymic activities (XET or XEH) of the proteins in those groups. Similar analyses of SlXTH2, a Group 2 tomato XTH, and nasturtium seed TmXTH1 revealed a spectrum of modes of action, suggesting that all XTHs have the capacity to function in both modes. The biomechanical properties of plant walls were unaffected by incubation with SlXTH5, with or without XGOs, suggesting that XTHs do not represent primary cell wall-loosening agents. The possible roles of SlXTH5 in vivo are discussed.     

Acceptor requirements for GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase activity solubilized from pea epicotyl membranes.

GDP-fucose:xyloglucan (XG) fucosyltransferase from growing Pisum epicotyl tissue was solubilized in detergent and used to examine the capacity of intact XG from Tamarindus seeds, and its partial hydrolysis products, to act as fucose acceptors with GDP-[14C]fucose as donor. Native seed XG (Mr greater than 10(6) Da) was partially depolymerized by incubation with Trichoderma cellulase for various periods of time. Cellulase was inactivated and reaction mixtures were incubated with GDP-[14C]fucose plus solubilized pea fucosyltransferase and then fractionated on columns of Sepharose CL-6B or Bio-Gel P4. Specific activities (Bq/microgram carbohydrate) of fragments with Mr ranging from 10(6) to 10(4) Da were constant throughout the size ranges, indicating that all stretches of the XG chains were available for fucosylation. More complete cellulase hydrolysis yielded subunit oligosaccharides that chromatographed in a cluster of hepta-, octa-, and nonasaccharides, none of which acted as fucosyl acceptors when incubated with pea fucosyltransferase. However, a substantial amount (up to half of hydrolysate) of larger transient oligosaccharides was also formed with a size equivalent to three of the oligosaccharide subunits. Octasaccharide subunits in this trimer were readily fucosylated. This fucosyltransfer was inhibited by uncombined (free) subunit oligosaccharides, which implies that the latter could bind to the transferase and displace at least part of the trimer, even though they could not themselves be fucosylated. Reduction of the trimer oligosaccharide with NaB3H4, followed by further hydrolysis with cellulase, resulted in tritiated nonasaccharide and unlabeled octasaccharide in a concentration ratio of 1:2. The tamarind XG trimer which accepts fucose is therefore composed mainly of the subunit sequence: octa-octa-nonasaccharide (reducing). One of the terminal oligosaccharide subunits in this trimer, probably the nonasaccharide, appears to be required as a recognition (binding) site in fucosyltransferase in order for adjacent octasaccharide(s) to be fucosylated by the active (catalytic) enzyme site.     

Screening for hetero-transglycosylating activities in extracts from nasturtium (Tropaeolum majus)

Using combinations of different polysaccharides as glycosyl donors and of oligosaccharides fluorescently labeled by sulforhodamine (SR) as glycosyl acceptors, we screened for the presence of transglycosylating activities in extracts from nasturtium (Tropaeolum majus). Besides xyloglucan endotransglycosylase/hydrolase (XTH/XET, EC 2.4.1.207) activity, which transfers xyloglucanosyl residues from xyloglucan (XG) to XG-derived oligosaccharides (XGOs), a glycosyl transfer from XG to SR-labeled cellooligosaccharides and laminarioligosaccharides has been detected. The XGOs also served as acceptors for the glycosyl transfer from soluble cellulose derivatives carboxymethyl cellulose and hydroxyethylcellulose. The effectivity of these polysaccharides as glycosyl donors for transfer to XG-derived octasaccharide [1-3H]XXLGol decreased in the order XG > HEC > CMC. Isoelectric focusing in polyacrylamide gels showed that bands corresponding to hetero-transglycosylase activities coincided with zones corresponding to XTH/XET. These results can be explained as due either to substrate non-specificity of certain isoenzymes of XTH/XET or to existence of enzymes catalyzing a hetero-transfer, that is the formation of covalent linkages between different types of carbohydrate polymers.     

Graft copolymers of xyloglucan and methyl methacrylate

Xyloglucan, a water-soluble food grade polysaccharide, was reported as a substrate for graft copolymerization of methyl methacrylate (MMA). Grafting PMMA (polymethyl methacrylate) with xyloglucan (XG) makes a new material with improved thermal stability and shelf life without affecting its hydrophilicity. XG was isolated from tamarind seed mucilage by aqueous extraction. Grafting of MMA was initiated by ceric ion in aqueous medium under N₂ atmosphere and the progress of the reaction was monitored gravimetrically by varying different reaction parameters. Grafting of MMA onto XG was confirmed by FTIR spectroscopy, NMR spectroscopy, differential scanning calorimetric (DSC) studies, thermal gravimetric analysis (TGA) studies and scanning electron micrographs (SEMs). This material might find potential to be used in drug delivery systems.     

Further studies of the ability of xyloglucan oligosaccharides to inhibit auxin-stimulated growth

The structural features required for xyloglucan oligosaccharides to inhibit 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stem segments have been investigated. A nonasaccharide (XG9) containing one fucosyl-galactosyl side chain and an undecasaccharide (XG11) containing two fucosyl-galactosyl side chains were purified from endo-β-1,4-glucanase-treated xyloglucan, which had been isolated from soluble extracellular polysaccharides of suspension-cultured sycamore (Acerpseudoplatanus) cells and tested in the pea stem bioassay. A novel octasaccharide (XG8′) was prepared by treatment of XG9 with a xyloglucan oligosaccharide-specific α-xylosidase from pea seedlings. XG8′ was characterized and tested for its ability to inhibit auxin-induced growth. All three oligosaccharides, at a concentration of 0.1 microgram per milliliter, inhibited 2,4-dichlorophenoxyacetic acid-stimulated growth of pea stem segments. XG11 inhibited the growth to a greater extent than did XG9. Chemically synthesized nona- and pentasaccharides (XG9, XG5) inhibited 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stems to the same extent as the same oligosaccharides isolated from xyloglucan. A chemically synthesized structurally related heptasaccharide that lacked a fucosyl-galactosyl side chain did not, unlike the identical heptasaccharide isolated from xyloglucan, significantly inhibit 2,4-dichlorophenoxyacetic acid-stimulated growth.     

Action of a pure xyloglucan endo-transglycosylase (formerly called xyloglucan-specific endo-(1-4)-β-d-glucanase) from the cotyledons of germinated nasturtium seeds

The action on tamarind seed xyloglucan of the pure, xyloglucan-specific endo-(1→4)-β-D-glucanase from nasturtium (Tropaeolum majus L.) cotyledons has been compared with that of a pure endo-(1→4)-β-D-glucanase (‘cellulase’) of fungal origin. The fungal enzyme hydrolysed the polysaccharide almost completely to a mixture of the four xyloglucan oligosaccharides: Exhaustive digestion with the nasturtium enzyme gave the same four oligosaccharides plus large amounts of higher oligosaccharides and higher-polymeric material. Five of the product oligosaccharides (D,E,F,G,H) were purified and shown to be dimers of oligosaccharides A to C. D (glc₈xyl₆) had the structure A→A, H (glc₈xyl₆gal₄) was C→C, whereas E (glc₈xyl₆gal), F (glc₈xyl₆gal₂) and G (glc₈xyl₆gal₃) were mixtures of structural isomers with the appropriate composition. For example, F contained B₂→B₂ (30%), A→C (30%), C→A (20%), B₂B₁ (15%) and others (about 5%). At moderate concentration (about 3 mM) oligosaccharides D to H were not further hydrolysed by the nasturtium enzyme, but underwent transglycosylation to give oligosaccharides from the group A, B, C, plus higher oligomeric structures. At lower substrate concentrations, hydrolysis was observed. Similarly, tamarind seed xyloglucan was hydrolysed to a greater extent at lower concentrations. It is concluded that the xyloglucan-specific nasturtium-seed endo-(1→4)-β-D-glucanase has a powerful xyloglucan-xyloglucan endo-transglycosylase activity in addition to its known xyloglucan-specific hydrolytic action. It would be more appropriately classified as a xyloglucan endo-transglycosylase. The action and specificity of the nasturtium enzyme are discussed in the context of xyloglucan metabolism in the cell walls of seeds and in other plant tissues.     

Fucosyltransferase and the Biosynthesis of Storage and Structural Xyloglucan in Developing Nasturtium Fruits

Young, developing fruits of nasturtium (Tropaeolum majus L.) accumulate large deposits of nonfucosylated xyloglucan (XG) in periplasmic spaces of cotyledon cells. This “storage” XG can be fucosylated by a nasturtium transferase in vitro, but this does not happen in vivo, even as a transitory signal for secretion. The only XG that is clearly fucosylated in these fruits is the structural fraction (approximately 1% total) that is bound to cellulose in growing primary walls. The two fucosylated subunits that are formed in vitro are identical to those found in structural XG in vivo. The yield of XG-fucosyltransferase activity from membrane fractions is highest per unit fresh weight in the youngest fruits, especially in dissected cotyledons, but declines when storage XG is forming. A block appears to develop in the secretory machinery of young cotyledon cells between sites that galactosylate and those that fucosylate nascent XG. After extensive galactosylation, XG traffic is diverted to the periplasm without fucosylation. The primary walls buried beneath accretions of storage XG eventually swell and lose cohesion, probably because they continue to extend without incorporating components such as fucosylated XG that are needed to maintain wall integrity.     

A new type of endo-xyloglucan transferase devoted to xyloglucan hydrolysis in the cell wall of azuki bean epicotyls

A new type of xyloglucan-degrading enzyme was isolated from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls and its characteristics were determined. The enzyme was purified to apparent homogeneity by Concanavalin A (Con A)-Sepharose, cation exchange, and gel filtration columns from a cell wall protein fraction extracted with 1 M sodium chloride. The purified enzyme gave a single protein band of 33 kDa on SDS-PAGE. The enzyme specifically cleaved xyloglucans and showed maximum activity at pH 5.0 when assayed by the iodine-staining method. An increase in reducing power in xyloglucan solution was clearly detected after treatment with the purified enzyme. Xyloglucans with molecular masses of 500 and 25 kDa were gradually hydrolyzed to 5 kDa for 96 h without production of any oligo- or monosaccharide with the purified enzyme. The purified enzyme did not show an endo-type transglycosylation reaction, even in the presence of xyloglucan oligosaccharides. Partial amino acid sequences of the enzyme shared an identity with endo-xyloglucan transferase (EXGT) family, especially with xyloglucan endotransglycosylase (XET) from nasturtium. These results suggest that the enzyme is a new member of EXGT devoted solely to xyloglucan hydrolysis.     

In-vivo formation of xyloglucan nonasaccharide: A possible biologically active cell-wall fragment

The in-vivo formation of a specific nonasaccharide of xyloglucan was investigated. This nonasaccharide has been reported to have biological activity, inhibiting auxin-induced growth in pea stem segments. Cell-suspension cultures of spinach were grown in the presence of [³H]arabinose and [³H]fucose, and the culture-filtrates were examined for oligosaccharides by gelpermeation chromatography and by paper chromatography. Sixteen [³H]pentose-containing oligosaccharides were found, including twelve that contained the sequence [³H]xylosyl-(16)-glucose, which is diagnostic of xyloglucan. In addition, [³H]fucose-containing oligosaccharides of at least three sizes were found. Radiochemical evidence is presented that one of these oligosaccharides was labelled with both [³H]fucose and with [³H]pentose, and was identical with the major xyloglucan-derived nonasaccharide associated with anti-auxin activity. It was largely present in the form of acylated (possibly acetylated) derivatives. It accumulated extracellularly to a steady-state concentration of about 4.3·10^-7M. This is the first report of the production of a biologically-active oligosaccharide by living plant cells.Abbreviations BAB butanone/acetic acid/H₃BO₃-saturated water (9:1:1) - BAW butan-1-ol/acetic acid/water (12:3:5) - BPW butan-1-ol/pyridine/water/(4:3:4) - DP degree of polymerisation - FAW ethyl acetate/acetic acid/water (10:5:6) - EPW ethyl acetate/pyridine/water (8:2:1) - k _av elution volume relative to Blue Dextran (k _av.=0.0) and glucose (k _av.=1.0) - XG7 XG9 minus the fucose and galactose residues - XG9 the particular xyloglucan nonasaccharide illustrated in Fig. 1 - W water-saturated phenol     

黄原胶寡糖生物活性的研究

利用黄原胶降解菌Cellulom onassp.XT11生产的黄原胶降解酶,对黄原胶进行生物降解,生产具有不同粘度/还原末端比的黄原胶寡糖,并研究了黄原胶寡糖在清除羟基自由基、植物防卫反应中激活因子活性和对植物病原菌抑制能力等方面的生物活性,结果表明黄原胶寡糖具有清除羟基自由基能力,并能激活植物防卫系统以抵御病原菌的侵染,同时对野油菜黄单孢菌也具有抑菌活性。     

Solubilization and properties of GDP-fucose: xyloglucan 1,2-alpha-L-fucosyltransferase from pea epicotyl membranes

GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase from pea (Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% (w/v) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona- and decasaccharide subunits. The apparent Km for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 microM GDP-fucose. This was 10 times the apparent Km for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at -70 degrees C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% (w/v) tamarind XG, and its activity located by incubation in GDP-[14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.     

Application of xyloglucan to improve the gluten membrane on breadmaking

Effects of xyloglucan (XG) on the physical properties of dough and bread quality were studied. XG was fractionated to water-soluble (WS-XG) by enzymatic hydrolysis with cellulase, and the four kinds of WS-XG: WS-XG-A (average degree of polymerization; 17), WS-XG-B (32), WS-XG-C (78) and WS-XG-D (223) were obtained. XG without enzymatic hydrolysis was termed water-insoluble xyloglucan (WI-XG). Additions of WS-XG-A (3%), WS-XG-D (1–5%) and WI-XG (3%) increased the stability of the dough and improved the loaf and softness of bread samples. Especially, the WS-XG-D (1–5%) significantly improved the various factors of the final products, such as loaf volume, storage properties and good appearances with fine distribution of small size gas cells, and its addition of low level (1%) still showed improving effects, as compared with other additives. The more viscous gluten matrix could be observed in the mixed doughs with WS-XG-D, than the control sample without XG. WS-XG-D increased the water activity of the dough, and therefore the gluten matrix of dough became strong and uniform. Since the WS-XG-D had the higher degree of polymerization, it might be polymerized during mixing or fermentation, followed by the formation of new insoluble-XG after baking. Appropriate amount of the new WI-XG formed from WS-XG-D was considered to improve the storage properties with higher water holding property than WS-XG-D alone.     

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.     

A xyloglucan oligosaccharide‐active, transglycosylating‐D‐glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings – purification, properties and characterization of a cDNA clone

A -D-glucosidase has been purified to apparent homogeneity from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seedlings during the mobilization of the xyloglucan stored in the cotyledonary cell walls. The purified protein (M_r 76 000; a glycoprotein; pI > 9.5; apparent pH optimum 4.5; temperature optimum 30°C) catalysed the hydrolysis of p-nitrophenyl--D-glucopyranoside, cello-oligosaccharides, -linked glucose disaccharides, and certain xyloglucan oligosaccharides. Glucose disaccharides with different linkages were hydrolysed at different rates [(1ν3) > (1ν4) > (1ν2) > (1ν6)] with significant transglycosylation occurring in the early stages of the reaction. Cello-oligosaccharide hydrolysis was also accompanied by extensive transglycosylation to give transitory accumulations of higher oligosaccharides. At least some of the glycosyl linkages formed during transglycosylation were (1ν6)-. Xyloglucan oligosaccharides xylose-substituted at the non-reducing terminal glucose residue (XXXG, XXLG, XLXG and XLLG, where G is an unsubstituted glucose residue, X is a xylose-substituted glucose residue, and L is a galactosylxylose-substituted glucose residue) were not hydrolysed. Some xyloglucan oligosaccharides with an unsubstituted non-reducing terminal glucose residue (GXXG, GXLG and GXG) were hydrolysed, but others (GLXG and GLLG) were not. This indicated steric hindrance by L but not X substitution at the glucose residue next to the one at the non-reducing end of the oligosaccharide. Hydrolysis of xyloglucan oligosaccharides was not accompanied by transglycosylation. Natural xyloglucan subunit oligosaccharides (XXXG, XXLG, XLXG, XLLG) were totally degraded to their monosaccharide components when treated with nasturtium -D-galactosidase ( Edwards et al. (1988 ) J. Biol. Chem. 263, 4333–4337), followed by alternations of nasturtium xyloglucan-specific α-xylosidase ( Fanutti et al. (1991 ) Planta 184, 137–147) and this enzyme. Several extensively overlapping cDNA clones were obtained by RT–PCR and by screening cDNA libraries. A composite, full-length DNA had an open reading frame of 1962 bp, encoding a polypeptide of 654 amino acids, including all N-terminal and internal sequences obtained from the purified -glucosidase protein, and a motif resembling plant signal sequences thought to direct proteins to the cell wall. Database searches revealed homology with -glucosidases from several sources (plant, bacteria, yeast), notably with glycosylhydrolases of ‘Family 3’, according to the classification of Henrissat ( Henrissat (1991) Biochem. J. 280, 309–316). There was strong sequence homology with a -glucan exo-hydrolase from barley ( Hrmova et al. (1996 ) J. Biol. Chem. 271, 5277–5286). The nasturtium -glucosidase is ascribed a role in xyloglucan mobilization, and its interaction with the α-xylosidase and the -galactosidase is modelled.