Enthalpic Studies of XyloglucanâCellulose Interactions - American

Biomacromolecules 2010, 11, 1417–1428

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Enthalpic Studies of Xyloglucan-Cellulose Interactions Marie Lopez,†,‡ Herve´ Bizot,† Ge´rard Chambat,‡ Marie-France Marais,‡ Agata Zykwinska,† Marie-Christine Ralet,† Hugues Driguez,‡ and Alain Buleón*,† INRA, UR1268 Biopolyme`res Interactions Assemblages, rue de la Ge´raudie`re, BP 71627, F-44316 Nantes, France, and Centre de Recherches sur les Macromolećules Ve´ge´tales UPR-CNRS 5301, BP 53, 38041 Grenoble, France Received September 3, 2009

We report a study of xyloglucan (XG)-cellulose interactions made possible by the preparation of various welldefined cellulosic and xyloglucosidic substrates. Bacterial microcrystalline cellulose (BMCC) as well as cellulose whiskers (CellWhisk) were used as cellulosic substrates. Xyloglucosidic substrates were obtained from Rubus cells and Tamarindus indica seeds. Different primary structure characteristics of XGs such as the backbone length and the nature of the side chains, as well as their repartition, were considered in order to examine the influence of the primary structure on their interaction capacity. Two complementary approaches were carried out: first, the determination of adsorption isotherms and its associated models, and second, an enthalpic study using isothermal titration calorimetry (ITC). This study highlighted that an increase of XG interaction capacity occurred with increasing XG molecular weight. Furthermore, we determined that a minimum of 12 glucosyl residues on the backbone is required to observe significant interactions. Moreover, both the presence of trisaccharidic side chains with fucosyl residues and an increase of unsubstituted glucosyl residues enhanced XG-cellulose interactions. The evolution of adsorption isotherms with temperature and ITC measurements showed that two different processes were occurring, one exothermic and one endothermic, respectively. Although the presence of an exothermic interaction mechanism has long been established, the presence of an endothermic interaction mechanism has never been reported.

1. Introduction Contrary to human cells, plant cells are encapsulated in a cell wall, the most prominent components of which are the polysaccharides: cellulose, heteropolysaccharides and pectins. Xyloglucans (XGs) are abundant hemicellulosic polysaccharides in the primary cell walls of dicots and non graminaceous monocots.1,2 They are considered as load-bearing components as, in primary cell walls, cellulose microfibrils are covered and at least partly interconnected by XGs.3 XGs prevent cellulose microfibrils from self-association and enable the formation of a strong but flexible network,4,5 which secures wall mechanical properties during cell expansion. In vivo studies using microscopic observations showed different locations of XGs in situ, inparticular,XGscoveringandcross-linkingcellulosemicrofibrils.6,7 In addition to this structural role, the presence of xyloglucooligosaccharides (XGOs) in vivo8 was shown to be related to the production of auxin, a key signaling molecule in plants.9,10 Thus, XGs play important roles as structural polysaccharides and as a source of signaling molecules, particularly during cell wall expansion. XGs consist of a (1,4)-β-linked D-glucopyranosyl backbone, where usually three out of every four glucosyl units carry a side chain. The elementary motif or building block of XGs, abbreviates throughout [XGO], was defined as four backbone glucosyl residues, independently of the side chain number and nature.11 This side chain substitution can be either a R-(1,6)D-Xyl, a β-D-Gal-(1,2)-R-D-Xyl, or a R-L-Fuc-(1,2)-β-D-Gal* To whom correspondence should be addressed. Tel.: +33 240675047. Fax: +33 240675043. E-mail: [email protected]. † INRA. ‡ Centre de Recherches sur les Macromolećules Ve´ge´tales, CERMAV, affiliated with University Joseph Fourier, and member of the Institut de Chimie Molećulaire de Grenoble.

(1,2)-R-D-Xyl side chain. XGs can be O-acetylated at specific glycosyl residues that can vary between species.12 This inherent structural complexity, coupled with the added complication of the dynamic architecture of plant cell walls, renders key factors governing XG-cellulose interactions difficult to identify and characterize. Mutants, with modified enzymatic activities, proved to be very helpful for understanding the XG-cellulose network structure in vivo. Two Arabidopsis mutants, mur2 and mur3, were particularly studied. MUR2 encodes a XG-specific fucosyltransferase, which leads to the absence of XG fucosylation in mur2 plants13 and MUR3 encodes a XG galactosyltransferase, which modifies galactosyl residue content of mur3 plant XGs.14 Despite alterations in XG structure, the growth habits of mur2 and mur3 plants appeared visually similar to those of wild-type and only exhibited a subtle collapsed trichome papillae phenotype.13,14 Tensile tests on hypocotyls revealed only minor changes in structural integrity for mur2 but a drastic decrease in stiffness and ultimate stress for mur3.15-17 Recently, single and double mutants affected in xylosyltransferase genes were studied.18 Single mutants had a modest reduction in XG content, whereas in the xxt1 xxt2 double mutant, XG level was appreciably lower. All mutants lacked a significant gross morphological phenotype and exhibited significant reductions in hypocotyls stiffness and ultimate stress,18 but they were nevertheless viable under laboratory conditions. In vitro assemblies of cell wall polysaccharides aiming to mimic cell wall architecture have been widely used to minimize the cell wall complexity. Although not the natural cellular conditions, the use of well characterized cell wall mimicking substrates enables the more precise determination of primary structure influence. In these studies, model XG-cellulose assemblies have been formed by the addition of cellulose

10.1021/bm1002762  2010 American Chemical Society Published on Web 04/30/2010

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microfibrils or microcrystalline cellulose to XG solutions. Binding capacities for given XG concentrations and cellulose amounts or adsorption isotherms were studied.4,19-21 The XG backbone length appeared to be a key factor governing interaction capacity, however, the existence of an optimal XG backbone length has long been controversial. Indeed, results claiming gradually increasing binding capacities for increasing XG molecular weights,22 as well as maximum adsorption capacities for XGs possessing 12-40 backbone glucosyl units19 or improved binding capacities when the molecular weight of XGs is decreased,21 have each been reported. Nonetheless, a minimal length of XG backbone seems necessary, as no efficient adsorption has been observed for oligosaccharides encompassing less than 12 backbone glucosyl residues.19 The influence of XG side chains on binding capacity also remains unclear.21 For example, fucosyl substitution has been claimed to either increase the adsorption affinity22 or to have no influence on binding capacity.21 Vicken et al.19 has suggested that a higher degree, or a specific pattern, of galactosyl substitution could be related to higher XG binding capacity. Cellulose morphology was also proven to be influential in binding capacities, mainly due to the specific surface area. For instance, by decreasing cellulose fiber diameters (i.e., increasing cellulose specific surface area), XG binding capacity has been shown to be a linear function of the specific surface area.4 More recently, in vitro cell wall analogues resulting from the synthesis of bacterial cellulose in the presence of XGs have been implemented for structural studies.23-25 Solid-state NMR, microscopy, and dynamic 2D FT-IR spectroscopy showed that XGs interact strongly with cellulose and induce a marked decrease in cellulose crystallinity. Several XG acting enzymes were tested against cell wall analogues, with results providing direct in vitro evidence for the involvement of XG-specific enzymes in diverse mechanical changes.26 In silico studies have also been carried out to evaluate the role of both the conformation and the nature of XG side chains on XG-cellulose interactions. Early results suggested that fucosyl residues are necessary to induce a twisted conformation of XGs that is favorable to interactions.27 However, recent work has reported no influence of fucosyl residues on interaction capacity and showed cellulose has no specific crystalline plans where XGs can interact preferably.28 Despite the numerous studies dedicated to XG-cellulose interactions, the mechanisms involved and the role of XG side chains still remain uncertain. In the present work, different XG-cellulose models were used to study their interactions as a function of both cellulose and XG characteristics. The use of BMCC and CellWhisk enabled the influence of cellulose morphology and charges present at the crystals surface to be assessed. The impact of XG characteristics, such as the molecular weight and side chain structure, were studied independently using different sources of XGs. A comparative study using ITC and adsorption isotherms at different temperatures was also performed to evaluate enthalpic effects related to interactions. Mechanisms possibly involved in XG-cellulose interactions are discussed.

2. Experimental Section 2.1. Materials. Cellulases from Trichoderma reseei (5 U · mg-1) were purchased from Fluka. β-D-Galactosidase from Aspergillus niger was purchased from Megazyme (Ireland). Cellulase Cel5A (61 U · mg-1) from B. agaradhaerens was a gift from the late Dr. Martin Schu¨lein (NovoZymes, Bagsvaerd, Denmark). Tamarindus indica XGs (3A and 3S class) were provided by Dainippon (Osaka, Japan). 3A and 3S class

Lopez et al. XGs possess a weight average molecular weight (Mw) of 700 and 100 kDa, respectively, and will be abbreviated by XGT700 and XGT100 throughout. 2.2. Substrate Preparations. 2.2.1. Bacterial Microcrystalline Cellulose (BMCC). Acetobacter xylinum (ATCC 53524) was incubated in Hestrin-Schramm medium,29 containing glucose (20 g · L-1) for 5-7 days at 20 °C, under static conditions. The resulting biosynthesized cellulose was then treated with hydrochloric acid (2.5 N) at 110 °C for 1 h, after which the mixture was centrifuged, the pellet retrieved, and hydrolysis repeated using the same conditions for 30 min. The resulting acidic suspension was washed until neutral. 2.2.2. Cellulose Whiskers (CellWhisk). Whatman filters (grade 20Chr) were cut into small pieces (approximately 1 cm2) and mixed with distilled water in a Warring blender. The obtained pulp was filtered through a fritted glass funnel and the residue was suspended in sulfuric acid (65% w/w) at 0 °C. The mixture was further mixed in the Warring blender and the resulting suspension was introduced in a high-pressure homogenizer (Manton-Gaulin, 15MR-8TBA) and pulse homogenized 15 times at 500 bar at a temperature not exceeding 90 °C. Microcrystals were recovered and extensively dialyzed against distilled water to give CellWhisk. To desulfate the CellWhisk, aliquots (4.5 mL at 17 mg · mL-1) were mixed with 10 M trifluoroacetic acid (0.5 mL) in a glass tube with a Teflon joint. The mixture was maintained at a given temperature for 2.5 h. Four temperatures were tested (20, 40, 60, and 80 °C). Samples were then cooled in an ice bath and extensively dialyzed against distilled water to remove all acidic traces. 2.2.3. Tamarind XG Oligosaccharides (XGOs). Tamarind seeds XGs (1.5 g) from Dainippon (3A category) were dissolved in water (150 mL) under stirring (one night at 35 °C followed by 15 min at 60 °C). The XG solution was introduced in an ultrafiltration cell equipped with a polyethersulfone membrane (cutoff 10000 g · mol-1). Cellulase from Trichoderma reseei (5.2 mg at 5 U · mg-1) was added and hydrolysis was performed for 2 days at room temperature. The filtrate was concentrated to reduce volume approximately 15-fold prior to silica gel addition and evaporation to dryness. The resulting powder was introduced on top of a silica gel column (Merck Geduran Silica gel 60, 40-63 µm). After flash chromatography on silica gel (water/ acetonitrile 20/80, 25/75 then 30/70 v/v), three fractions were obtained. These three fractions were a mixture of XGOs constituted of 1, 2, 3 building blocks and are abbreviated throughout [XGO]1 (474 mg), [XGO]2 (407 mg), and [XGO]3 (170 mg), respectively. [XGO]2 and [XGO]3 were used for binding assays. A further XGO fraction was obtained by partial hydrolysis of tamarind seeds XGs followed by water/methanol precipitation. The tamarind seeds XGs were solubilized by stirring in 0.1 M Na-acetate buffer (pH 4.5) (30 min at 50 °C followed by one night at room temperature). Following removal of the insoluble fraction by centrifugation, the supernatant was incubated with endo-glucanase Cel5A (5 µL · 100 mL-1; 61 U · mg-1) for 10 min at 30 °C. After cooling to 4 °C, the hydrolysate was precipitated in MeOH/water (1/2 v/v final proportion). Precipitated XGOs ([XGO]1-2) were recovered by filtration. 2.2.4. Extracellular Rubus XGs (XGREC). Cultured cells from Rubus fruticosus suspension were grown as described by Chambat et al.30 Cells were collected after 24 days of culture. The corresponding culture medium was submitted to a freeze-thawed followed by a thawing sequence and then centrifuged (3000 g; 20 min; 4 °C). The residue was dialyzed and freeze-dried. The supernatant was subjected to tangential ultrafiltration (MINITAN cell, Millipore Corp., U.S.A., fitted with 104 molecular weight cutoff membrane) to reduce volume. The high molecular weight fraction (>104 g · mol-1) was precipitated by 7% CuSO4 and the resulting suspension was centrifuged. The supernatant contained a mixture of XGs and arabinogalactans that were precipitated in ethanol according to Aspinall et al.31 The precipitate was decomplexed from copper by addition of HCl to pH 2-3. The solution was then dialyzed and the XG fraction was purified, as

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Table 1. Characteristics and Abbreviations of Cellulosic Materials cellulosic substrates

origin/treatment

bacterial microcrystalline cellulose cellulose whiskers desulfated cellulose whiskers a

DP ) degree of polymerization.

b

abbreviation

hydrolysis of bacterial cellulose of Acetobacter xylinum acidic treatment of paper cellulose trifluoroacetic acid treatment of cellulose whiskers

BMCC CellWhisk CellWhiskdes

characteristics Mw ) 73 × 10 g · mol-1 DPa ) 450 ds (SO3-)b ) 3.3 × 10-2 gSO3- · 100 gcellulose-1 3

ds (SO3-) ) degree of sulfatation.

described by Chambat et al.,32 on ion-exchange resin (DEAE Sephadex A50) to remove the smallest oligosaccharides before being freeze-dried. 2.2.5. XGs from Rubus Cell Wall (XGRCW). Cultured cells from Rubus fruticosus suspension were grown as described by Chambat et al.30 Cells were collected after 24 days of culture. Dry cell wall material (5 g) was stirred in EDTA solution (2% Na2EDTA; pH ) 5; 500 mL) for 2 h at 70 °C and the resulting suspension filtered through a fritted glass funnel (porosity 2). The solid residue was washed twice with ammonium oxalate (0.5%; pH ) 6.5; 2 × 500 mL) at 80 °C for 2 h. The suspension was filtered and the solid residue treated with sodium borohydride (1% NaBH4; 500 mL) at 20 °C for 15 h with stirring under nitrogen. After filtration, the residue was suspended in a solution of sodium hydroxide (2.5 N; 500 mL) and maintained under stirring during 6 h at 20 °C under nitrogen. After filtration, this last step was repeated in the same conditions during 16 h. The combined filtrates were dialyzed and freeze-dried. XGRCW was purified through Amicon membrane (cutoff 10 × 103 g · mol-1) to remove the smallest oligosaccharides. 2.3. Substrate Characterization. Individual neutral sugars were analyzed as their alditol acetate derivatives by gas chromatography as described by Chambat et al.30 following hydrolysis by 1 M H2SO4 at 100 °C for 6 h. An Agilent 6850 Series GC System equipped with a SP 2380 macrobore column, 30 m × 0.53 mm (Agilent Technologies, Palo Alto, U.S.A.) was used. The degree of sulfation of CellWhisk was determined by conductimetric titration.33 MALDI-TOF mass spectrometry was performed on a BRUKER Autoflex mass spectrometer (Bruker Spectrospin, Wissembourg) equipped with a nitrogen laser operating at 337 nm. Mass spectra were recorded in reflection mode and in positive ion detection using 2,5-dihydroxybenzoic acid (50 mg · mL-1) as matrix. Each of the samples (2 µL, 200 pmole · µL-1) was mixed with 2 µL of matrix and dried under reduced pressure. High-performance size-exclusion chromatography, providing the weight average molecular weight (Mw), was performed at 30 °C on a Waters system constituted of Shodex OH B802 and B803 or B805 and B806 columns mounted in series eluted with 0.1 M NaNO3 containing NaN3 as preservative at a flow rate of 0.5 mL · min-1. A capillary viscometer from Alliance GPC 2000, a differential refractometer and a multiangle laser light scattering DSP-F (Wyatt Technology, U.S.A.) were used as detectors. 2.4. Isothermal Titration Calorimetry (ITC). Calorimetric titrations were performed at 25 °C with a VP-ITC isothermal titration calorimeter from Microcal (Northampton, MA). Aliquots of XGO solutions in water, at a concentration range of 2-75 mg · mL-1, were injected into the ITC cell containing 1.4 mL of 17 mg · mL-1 CellWhisk suspension using a rotating stirrer-syringe. The resulting suspension was stirred in the cell by the rotation of the syringe inside the cell at 300 rpm. The volume of each injection was 10 µL, and the intervals between injections were 300 or 720 s to allow correct equilibration. Usually, the first injection was found to be inaccurate; therefore, a 5 µL injection was conducted first, and the resulting data point was deleted prior to analysis of the remaining data. The heat of dilution was subtracted although this contribution to global heat was very small. Enthalpy changes due to interaction were calculated on the basis of the amount of XGOs injected in the ITC cell (kcal · mg-1). 2.5. Adsorption Isotherms. Adsorption experiments were conducted in distilled water containing 0.02% thimerosal. Wet BMCC (20 mg, 17% dry material) was used as cellulosic material. Solutions of XGs

Figure 1. Transmission electron micrographs of BMCC (A) and CellWhisk (B).

at different concentrations (from 5 to 2000 µg · mL-1) were prepared by dilution of a 2 mg · mL-1 XG mother solution heated at 40 °C under stirring to obtain a clear homogeneous medium. Cellulose aliquots were briefly mixed with 1.5 mL of each XG solution using glass balls, to obtain a homogeneous suspension and improve reproducibility. Mixtures were incubated either in a cold room or in a laboratory oven for 15 h (the incubation time was optimized from kinetic studies, see Supporting Information) at 4, 25, 40, or 60 °C under continuous head-over-tail mixing, using a stirring wheel. Blends were centrifuged (15 min, 20000 g) and the supernatants tested for their total neutral sugar contents by the automated colorimetric orcinol method.34 The amount of adsorbed material was calculated from the difference in sugar content measured for XG mother solutions and supernatants taking into account the amount of sugars released by cellulose control (with no xyloglucosidic substrate introduced). Either two or four independent measurements were performed, depending on the quantity of material available. The average of the measurements was then calculated.

3. Results 3.1. Substrates Characteristics. The choice of model substrates, that are both relevant to plant constituents and wellsuited to the techniques employed, was particularly difficult owing to the diversity of analytical techniques employed requiring various substrate physical properties. Several cellulosic and xyloglucosidic substrates were prepared to assess the influence of a variety of parameters on XG-cellulose interactions, having in mind experimental constraints of each analytical method. The origin, characteristics, and abbreviated names of the cellulosic substrates used are found in Table 1. BMCC was obtained by hydrolysis of bacterial cellulose from Acetobacter xylinum. This cellulosic substrate is homogeneous and free of residual noncellulosic polysaccharides, as assessed by neutral sugar analysis. The absence of bundles and the welldefined structure of the fibrils are highlighted by microscopy (Figure 1). The lateral size of the fibrils was estimated at 0.1 µm. The degree of sulfate substitution of the CellWhisk was quite low (Table 1) but sufficient to allow the formation of colloidal suspensions by charge repulsion. To determine the influence of charge, the quantity of sulfate charges was modified by treatment with acid at a variety of temperatures. Owing to the low sulfate content, the degree of sulfation of the treated whiskers could not be quantified experimentally. Nonetheless, the observation that the medium changed gradually from transparent to opaque

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Table 2. Characteristics and Abbreviation of Xyloglucosidic Substrates substrates

origin/treatment

tamarind xyloglucans

abbreviation Mw (kDa)

commercial dainippon (category 3A) extracted from seeds Rubus xyloglucans cell wall XGs extracellular XGs (extracted from culture medium) extracellular XGs (extracted from culture medium and deacetylated) partially hydrolyzed xyloglucans From XGT700 purified by silica chromatography from XGT700 purified by silica chromatography from XGT700 purified by fractionated precipitation

d

XGT700 XGT100 XGRCW XGREC XGRECdes

700b 100b 110b 30b 20b

[XGO]2 [XGO]3 XGO1-2

2.5c 3.9c 1.7b,c

I pd

Glca

Xyla

Gala Fuca

1.1 1.7 4.1 1.5 1.2

1.17 1.26 1.80 1.40 2.40

1.00 1.00 1.00 1.00 1.00

0.46 0.44 0.36 0.40 0.48

0.00 0.00 0.30 0.20 0.12

a Molar proportions of saccharides by neutral sugar determination. b Mw was determined by SEC experiment. c Mw was determined by mass spectrometry. Ip, polydispersity index, was determined by SEC experiment.

Figure 2. Adsorption isotherms of XGT700, Mw ) 700 kDa (gray circle, gray thin line), XGT100, Mw ) 100 kDa (0, - -), [XGO]3, Mw ) 4 kDa (gray triangle, gray thick line), and [XGO]2, Mw ) 2 kDa ( ×, s) on BMCC at 40 °C.

following acidic treatment indicated an aggregation of the cellulose particles. This aggregation can be attributed to a decrease of charge repulsion and is consistent with the expected reduction in sulfate substitution. Except for the presence of charges, CellWhisk and BMCC exhibited similar morphologies as shown in Figure 1. Xyloglucosidic substrates were obtained from both tamarind and Rubus, leading to oligo- and polysaccharides with varied lengths and degrees of substitution (Table 2). Two tamarind (T) XGs varying in their average molecular weight (Mw ) 700 and 100 kDa) were selected, XGT700 and XGT100, respectively. Both tamarind XGs had similar composition and were not acetylated or fucosylated, as previously reported.35 Rubus (R) extracellular (EC) XGs (XGREC) and cell wall (CW) XGs (XGRCW) were recovered. In contrast to tamarind XGs, Rubus XGs encompass fucosyl units with approximately one trisaccharidic side chain out of every three for XGRCW and one out of every two for XGREC. Extracellular and cell wall Rubus XGs, both fucosylated, contained the same proportion of galactosylated side chains but exhibited different average molecular weights (Mw ) 30 and 110 kDa for XGREC and XGRCW, respectively). XGREC, which was recovered under nonalkaline conditions, was slightly acetylated as visualized by 13 C NMR in contrast to XGRCW. XGREC aliquots were deacetylated under alkaline conditions to give XGRECdes. Removal of the acetyl groups was monitored by 13C NMR (see Supporting Information). In addition to removal of acetyl groups, the deesterification treatment also induced structural degradation of XGREC. Indeed, the molecular weight was lower and the proportion of unsubstituted glucosyl units increased in XGRECdes compared to XGREC.

XGOs were recovered by partial hydrolysis of tamarind XGT700 followed by fractionated precipitation in MeOH/water or flash chromatography on silica gel. XGO fractions recovered by precipitation exhibited an Mw of 1.7 kDa. Mass spectrometry allowed identification of oligosaccharides encompassing four or eight backbone glucosyl residues (see Supporting Information). This XGO fraction was denoted [XGO]1-2, because it includes oligosaccharides consisting of one or two elementary motifs or building blocks of four glucosyl units. Similarly, the two XGO fractions recovered after partial hydrolysis and chromatography were denoted [XGO]2 and [XGO]3 on the basis of mass spectrometry analyses (see Supporting Information) and average molar mass measurements. The prepared xyloglucosidic substrates present significant structural variability, which allowed studying the influence of several parameters on XG-cellulose interactions. 3.2. Influence of XG Structure on Binding Properties. 3.2.1. Molecular Weight. Average molecular weight influence was assessed through adsorption isotherms where the mass of bound material per mass of cellulose (qe) was plotted versus the concentration of free material remaining in solution at equilibrium (Ce) after incubation with BMCC at 40 °C for 15 h. Adsorption isotherms of native tamarind seed XGs (XGT100 and XGT700) and two XGT700-derived oligosaccharidic fractions ([XGO]2 and [XGO]3), with Mw ranging from 2.5 to 700 kDa, were compared. The resulting isotherms are shown in Figure 2. At low concentration, adsorption increases dramatically with increasing molecular weight. A plateau is then reached when saturation is achieved, which corresponds to the maximum adsorption capacity. The maximum adsorption capacity is clearly positively correlated with average molecular weight, in agreement with Hayashi et al.22 For Ce ) 1200 µgXG · mL-1,



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Figure 3. Linearized Langmuir fitting of the adsorption isotherms presented in Figure 2. Table 3. Data Deduced from the Langmuir Model Applied to the Different Adsorption Isotherms entry

XGs/XGOs

Mw(kDa)

fucosyl residue

acetylation

qm ) 1/a1 (µgXG · mgcellulose-1)

b ) 1/a2qm (mL · µgXG-1)c

1 2 3 4 5 6 7

[XGO]31 a [XGO]32 b XGT100 XGT700 XGREC XGRECdes XGRCW

3.9 3.9 100 700 30 20 110

no no no no yes yes yes

no no no no yes no no

43.9 ( 0.2 216.3 ( 27.6 245.3 ( 18.0 267.9 ( 17.9 245.3 ( 18.0 244.1 ( 6.0 357.6 ( 12.8

(3.37 ( 2.31) × 10-2 (0.20 ( 0.01) × 10-2 (3.81 ( 0.09) × 10-2 (4.74 ( 0.09) × 10-2 (0.63 ( 0.05) × 10-2 (0.53 ( 0.03) × 10-2 (3.08 ( 0.96) × 10-2

a Corresponding to the first site (Ce < 100 µgXG · mL-1). b Corresponding to the second site (Ce > 100 µgXG · mL-1). c qm represents the maximum adsorption capacity and b is the adsorption constant.

adsorption capacity increases sharply from [XGO]2 (qe ∼ 15 µgXG · mgcellulose-1) to [XGO]3 (qe ∼ 130 µgXG · mgcellulose-1), then moderately to XGT100 and XGT700 (qe ∼ 240 and 270 µgXG · mgcellulose-1, respectively). These results first suggest that a minimum length of three building blocks (i.e., 12 glucosyl residues) is required to observe significant XG-cellulose interactions, which is in full agreement with previously published data.19 Moreover, gradually increasing binding capacities for increasing XG molecular weights were found, also in agreement with the results of Hayashi et al.36 Isotherm relative to [XGO]3 presented a “break point” at Ce ) 100 µgXG · mL-1, which could indicate an inhomogeneous adsorption mechanism. To quantify this mechanism, a Langmuir model was applied to the experimental data. The Langmuir model37 can be expressed as follows:

qe )

qmbCe 1 + bCe

(1)

where qe is the mass (µg) of absorbed XG per mg of cellulose, qm is the maximum adsorption capacity (µgXG · mgcellulose-1), b is the adsorption constant (mL · µgXG-1), and Ce is the concentration of free polymer remaining in solution at equilibrium (µgXG · mL-1). The experimental binding isotherms data were plotted by the linearized form of Langmuir model with the following equation:

Ce Ce 1 ) + qe bqm qm

(2)

Langmuir plots obtained for [XGO]3, XGT100 and XGT700 exhibiting a significant adsorption level onto cellulose are presented in Figure 3.

For native polymeric tamarind XGs, a linear fitting of data was sufficient. For the tamarind XG oligosaccharides [XGO]3, two lines were necessary for an acceptable fitting, which suggests that BMCC presents two types of interaction sites for XGOs, one for low XGO concentrations (type 1) and one for high XGO concentrations (type 2). Thanks to the Langmuir model, maximum adsorption capacities, calculated from the slopes of the lines (a1) by qm ) 1/a1 and adsorption constants calculated from the intercepts of the lines (a2) by b ) 1/(a2qm), were determined (Table 3). Maximum adsorption capacities (qm) of native XGT100 and XGT700 (entries 3 and 4) were roughly similar (245 and 268 µgXG · mgcellulose-1, respectively). [XGO]3 maximum adsorption capacity on sites of type 2 (entry 2) was only slightly lower (216 µgXGO · mgcellulose-1), while the [XGO]3 maximum adsorption capacity on sites of type 1 (entry 1) was significantly lower (44 µgXG · mgcellulose-1). These results suggest that XGs can be adsorbed on the cellulose site of type 2 irrespective of their molecular weight, whereas the sites of type 1 are only accessible to oligosaccharides. The affinity constants (b) of XGT700, XGT100, and [XGO]3 on sites of type 1 (entries 1, 3, and 4) are roughly similar (4.7, 3.8, and 3.4 × 10-2 mL · µgXG-1, respectively). However, the affinity constant of [XGO]3 for sites of type 2 (entry 2) is much lower (0.2 × 10-2 mL · µgXG-1). In other words, XGOs have a much higher affinity for cellulose sites of type 1 than for sites of type 2. Cellulose sites of type 1 are, however, rapidly saturated, as indicated by the low XGO maximum adsorption capacity. 3.2.2. Side Chains and Degree of Acetylation. The impact of XG side chains and degree of acetylation was investigated by comparing Rubus XGs (XGRCW, XGREC, and XGRECdes) with tamarind XGs. To allow a direct comparison between tamarind and Rubus XGs, isotherms obtained for Rubus XGs are presented in Figure 4 together with the isotherm obtained for

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Figure 4. Adsorption isotherms of XGRCW (gray circle, gray thick line), XGT100 (0, - -), XGREC (gray triangle, gray thin line), and XGRECdes (×, -) BMCC at 40 °C.

tamarind XGT100, which has a similar molecular weight to the XGRCW, for direct comparison. A Langmuir model was applied to the experimental data, with maximum adsorption capacities and adsorption constants summarized in Table 3. For all Rubus XG samples, a linear fitting of data was applicable (squared regression coefficient, R2 > 0.95). Maximum adsorption capacities of XGREC and XGRECdes (entries 5 and 6) were close to that calculated for XGT100 (qm ∼ 240 µgXG · mgcellulose-1), which had yet a higher molecular weight (entry 3). Maximum adsorption capacity of XGRCW (entry 7) was significantly higher (qm ∼ 360 µgXG · mgcellulose-1) than XGT100, with similar molecular weights. Adsorption constant values (b) were quite low for extracellular Rubus XGs (entries 5 and 6), XGREC and XGRECdes (0.63 and 0.53 × 10-2 mL · µgXG-1, respectively), and roughly similar to that of tamarind XGT100 for Rubus XGRCW (entry 7). The origin and fine structure of XGs have a small influence on adsorption constant, although the value obtained for XGRCW is a little lower than that of XGT100, which exhibits roughly the same molecular weight. Affinity would thereby be mostly related to molar mass and weakly dependent on fucosylation. The maximum adsorption capacity values are within the same order of magnitude for both XGREC and XGRECdes (entries 5 and 6, qm ∼ 250 µgXG · mgcellulose-1). It is noteworthy that maximum adsorption capacity values did not differ significantly between XGREC and XGRECdes, suggesting that the degree of acetylation is not likely to be a key factor for interaction at least for weak values such as reported here. The maximum adsorption capacity is significantly enhanced for cell wall Rubus XG, XGRCW, (entry 7, qm ∼ 360 µgXG · mgcellulose-1). This peculiar behavior is most likely attributable to structural specificities. When compared to tamarind XGs of similar average molecular weight (XGT100), cell wall Rubus XGs (XGRCW) exhibit two main structural specificities: (i) a slightly higher proportion of unsubstituted glucosyl residues and (ii) the presence of fucosyl residues (Table 2). The presence of fucosyl residues were previously shown to have a positive influence on adsorption capacity.27 It is most likely that the better adsorption observed with the presence of fucosyl residues, meaning a third saccharide, on the side chain is attributable to a greater degree of flexibility increasing the exposure of the XG backbone to the cellulose surface. The side chain distribution along the XG backbone could also be a key factor for the interactions. Unfortunatly, the XG structural complexity prevents the determination of the position of the side chains along the XG backbone. This increase in adsorption capacity for XGRCW on BMCC compared to XGT700, despite a much lower molecular

weight, highlights the importance of the number, nature, and distribution of side chains on the adsorption capacity. 3.3. Enthalpic Study of XG-Cellulose Interactions. 3.3.1. Adsorption Isotherms. Enthalpic studies can provide information about mechanisms involved in XG-cellulose interactions. The Van’t Hoff equation describes the reaction rate constant as a function of temperature. According to this equation, if the increase of temperature leads to a greater interaction capacity, then the overall process is endothermic, while a lower interaction indicates an exothermic process. Thus, an enthalpic study was first performed by determination of adsorption isotherms of XGT700 and [XGO]3 on BMCC at different temperatures, the resulting isotherms are represented in Figure 5. The curves have a similar shape to what was obtained previously at 40 °C. For [XGO]3, the difference observed in the adsorption at 4 and 70 °C is not significantly important. On the contrary, the adsorption of XGT700 increases significantly with temperature from 4 to 60 °C, whereas isotherms obtained between 25 and 40 °C were very similar (Figure 5A). The change in temperature did not dramatically modify the adsorption capacity of xyloglucosidic substrates, confirming the low enthalpic character of interactions observed previously by de Lima et al.21 However, contrary to other results, such increases observed in binding of both oligo- and polysaccharides with increasing temperature are the sign of endothermic behavior. Moreover, a closer inspection of the XGT700 isotherms at low concentration (Figure 5B) reveals a higher adsorption at 4 °C than at 60 °C, suggesting another interaction mechanism at such concentrations. These observations appear to suggest the presence of two processes involved in XG-cellulose interactions for XGT700, as previously determined for [XGO]3 using the Langmuir model. 3.3.2. ITC Analyses. ITC studies confirmed the presence of at least two interaction mechanisms. This analytical procedure can neither be carried out with BMCC, which does not lead to stable colloidal suspensions, nor with high molecular weight XGs, which generate aggregates and a disturbance in ITC signal. Consequently, a suspension of CellWhisk was titrated against a solution of XGO1-2, which were available in sufficient quantities. Figure 6 shows typical titration data of CellWhisk by XGO1-2 at 25 °C. The raw titration curve, where each heat flow corresponds to one injection of XGO solution, is represented at the top. Integration of this curve and subtraction of the dilution enthalpy leads to the titration isotherm (bottom curve) where enthalpic values are plotted versus the ratio mXGO1-2 · mcellulose-1. Under



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Figure 5. (A) Adsorption isotherms of [XGO]3 and XGT700 on BMCC at different temperatures. (B) Expansion at low Ce values for XGT700 isotherms at 4 and 60 °C.

initial conditions, saturation was achieved at the fourth injection (Figure 6A), preventing a clear observation of the underlying processes. Subsequent ITC studies were conducted using a less concentrated oligosaccharide solution (Figure 6B). Saturation was sufficiently delayed to facilitate curve interpretation with a higher degree of precision. The enthalpic values ranged from -0.25 to 0.13 kcal · mgXGO1-2-1. These results were unexpectedly low, as previously reported by Lima,21 compared to interactions between lectin with carbohydrate, for example, that can achieve 100 kcal · mgcarbohydrate-1.38 The curve clearly revealed two distinct domains. For the first injections, that is, at low mXGO1-2 · mcellulose-1 values, the global enthalpic phenomena gave a negative response (exothermic), whereas this global heat became positive (endothermic) at higher XGO concentrations. These results confirmed the presence of two interaction mechanisms, as previously evidenced by adsorption isotherms. The existence of two interaction mechanisms had never been reported; in particular, the existence of an endothermic process was unknown. The charge of CellWhisk could be a major difference with cellulose found in the cell wall. To evaluate the potential influence of charges on XG-cellulose interactions and the corresponding enthalpic response, CellWhisk desulfated at several temperatures were tested in ITC experiments with XGO1-2 fraction. The effect of the number of charges on CellWhisk on the titration curves is illustrated in Figure 7. The range of charge density was expressed as a function of the temperature used for removing the sulfate groups of the

CellWhisk. Although the sulfation degree of untreated CellWhisk has been determined by titration of sulfate, the charge of the partially desulfated CellWhisk could not been determined due to charge levels that were too low and low quantities of material available. Nevertheless, the modification of charge density was observed by modification of the cellulose suspension turbidity. The four curves corresponding to desulfated CellWhisk (at four different desulfation reaction temperatures) are superimposed to the curve obtained from the untreated whiskers. These results demonstrate that modification of the cellulose charge density has no influence on the enthalpic values and does not modify the XG binding capacity on cellulose. This can be attributable either to a low level of charges present on the CellWhisk, as mentioned above, or a very low contribution of electrostatic effects in the interaction. As no influence of charges was observed, this allowed the comparison of the results obtained on CellWhisk and BMCC, which were shown to already have a similar morphology. Moreover XGT700 presented the same adsorption capacity on both BMCC and CellWhisk (see Supporting Information), which further confirms the validity of this comparison.

4. Discussion This work aimed to achieve a better knowledge of the mechanisms involved in XG-cellulose interactions. A wide and appropriate range of well-defined substrates and complementary techniques such as ITC and adsorption isotherms determination

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Figure 6. Raw data (at the top) and enthalpic curves (at the bottom) of ITC of CellWhisk (17 mg · mL-1) by a solution of XGO1-2, 75 mg · mL-1 (A) and 10 mg · mL-1 (B).

Figure 7. Enthalpic curves obtained from ITC titration of CellWhisk and CellWhiskdes (17 mg · mL-1) by XGO1-2 (10 mg · mL-1) after desulfating CellWhisk at different temperatures, as indicated on the figure.

afforded original results on the nature of the involved interactions. These results have also led to significant improvements in the knowledge of the XG structural parameters having a potential impact on the interaction capacity with cellulose. First, an increase of XG average molecular weight leads to an improved adsorption on BMCC, as previously described on microcrystalline and amorphous cellulose by Hayashi et al.36 However, no optimal molecular weight, where adsorption would be maximal, was determined, contrary to previous work using Avicel microcrystalline cellulose.19 Moreover, in the present work, through the determination of the complete adsorption isotherms, it was possible to determine the maximal adsorption capacity for different XG fractions. XG oligosaccharides [XGO]2 present no specific interactions with BMCC, whereas [XGO]3 corresponds to the minimum structure presenting a noticeable adsorption capacity. The adsorption capacity of [XGO]3 was

only 2-fold lower than XGT700, which had a 175-fold higher molecular weight. Considering the possibility for XGs to form a train, loops, and tails, as mentioned in Vincken’s work,19 and that three building blocks ([XGO]3) are necessary to generate significant interactions, we can propose that the train length on BMCC is 12 glucosyl residues. This hypothesis is reinforced by the relatively small difference observed between the adsorption of native tamarind XGs and [XGO]3 on BMCC. The better adsorption of XGT700 can then be associated with the presence of loops and tails but not to enhanced direct interactions. Indeed, due to the adsorption isotherm determination technique, loops and tails are included in the adsorbed XG quantity, even though these segments are not directly interacting with cellulose. The independence of interactions on charges present at the CellWhisk surface shows that electrostatic effects are weak, in accordance with other works showing a low pH dependence of



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Figure 8. Representation of cellulose glucosyl units made inaccessible by interaction with [XGO]1.

such interactions.19,20 Furthermore, the absence of influence of acetate substitution on interaction capacity can be correlated to this weakness of electrostatic effects. These results suggest that XG-cellulose interactions are governed by several types of interactions, including hydrogen bonding, as previously proposed by Morris et al.,39 based on computational modeling and hydrophobic interactions. ITC and adsorption isotherms at different temperatures clearly demonstrated both exothermic and endothermic processes relative to two XG-cellulose interactions. These results suggested the presence of two types of adsorption sites corresponding respectively to exothermic and endothermic phenomena. Independent of xyloglucosidic substrate length, the interactions on sites of type 1 occurred at low XG (XGO) concentrations, while at higher XG (XGO) concentrations, interactions are localized on sites of type 2. For both XGs and XGOs, the exothermic process, interactions on sites of type 2, was identified as a hydrogen bond creating mechanism between the cellulose surface and the heteropolysaccharides. Indeed, such processes involving hydrogen bond creation were previously described as exothermic.21,39 On the other side, the endothermic mechanism is not necessarily the same for XGs and XGOs. XGs, which have a high molecular weight, are likely to be involved in a self-association process, generating stable self-assembled structures. [XGO]3 could potentially be involved in a similar process; however, due to their low molecular weight, the selfassembled structures would be less stable than with XGs. Moreover, the presence of two different types of binding was also revealed for [XGO]3 by adsorption isotherms and reinforced by the applied Langmuir model, contrary to native tamarind XGs. It can be concluded that some binding sites are only accessible to oligosaccharides. It could be relative to the adsorption of a fraction of XGOs inside BMCC grooves due to their reduced size. Such an adsorption is not likely to occur for XGs, which would be too large to enter in these sites. These endothermic processes are most likely governed by hydrophobic interactions. Furthermore, these interactions are entropy driven due to solvent reorganization, either to enable XGO entry inside

the groove or to permit XGs to adopt a favorable conformation to generate self-association. Indeed, to rearrange their conformation, xyloglucosidic substrates modify their solvation, which generates an increase in the solvent entropy; this solvent reorganization phenomenon has been described in previous enthalpic studies.40,41 To ensure the rationality of our results regarding the proportion of xyloglucosidic versus cellulosic substrates, the surface coverage value was determined. An upper bound of surface coverage for cellulose crystallites by a flat lying branched XG ribbon may be drawn by docking [XGO]1 onto an averaged crystalline lattice. It can be considered that [XGO]1 would occupy or sterically prevent further adsorption of a neighboring XGO over 4 × 3 lattice sites ) 12 AGU units (AGU ) anhydroglucose unit), as sketched on the simplistic picture, Figure 8, where the checkerboard pattern symbolizes the alternate glucose orientation of cellulose. Otherwise stated, each XG backbone glucosyl residue eclipses three cellulose crystal surface AGUs, not counting the dependent tails and loops of the adsorbed XG and the corrugated crystallite surfaces. Thereby, a 1/1 ratio of moles of saccharide units between XGs and crystallite surface chains provides a reasonable order of magnitude for surface coverage. The fraction of surface chains for nanocrystalline cellulose can be evaluated geometrically considering a square cross section.43-45 Typically bacterial microcrystalline cellulose with a rectangular cross section 7 × 40 nm would be saturated at about 0.18 mgXG · mgcellulose-1. For adsorption of both XGs and XGOs on BMCC, the maximum adsorption capacity (qm ∼ 0.25 mgXG · mgcellulose-1) is higher than the estimated surface coverage, which is consistent with the presence of self-assembly or adsorption in BMCC grooves. The originality of these results lies in the observation of both exo- and endothermic mechanisms. Previous ITC studies conducted on tamarind and bean XGs showed only a single exothermic interaction21 with no adsorption isotherms described on a temperature range as wide as in this study. Moreover, Avicel type microcrystalline cellulose, used in previous works,

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Figure 9. Scatchard model applied to the adsorption of native XGs (A) and [XGO]3 (B) on BMCC.

has a very different morphology when compared to BMCC or CellWhisk, including smaller length, lower crystallinity, and a higher level of aggregation that can strongly modify the nature of interactions involved. Interaction mechanisms can be investigated more thoroughly by applying the Scatchard model42 to adsorption isotherms. Figure 9 represents graphs of the Scatchard model for native XGs and [XGO]3 on BMCC. According to the applied Scatchard model, the presence of a maximum shows the cooperative character of interactions. Consequently, adsorption of native XGs appears to be cooperative in contrast to [XGO]3. A cooperative mechanism was previously demonstrated for adsorption of cellulase-like CBH on microcrystalline cellulose using the Hill equation.46 From our results it can be concluded that three building block XGs create an anchor on the cellulose surface, which initiates the formation of interactions along the XG chain. Apart from XG length, the nature and distribution of side chains also play an important role in the interaction capacity. As previously reported by Levy et al.,27 using computational modeling, fucosylation seems to favor maximum adsorption capacity when comparing XGRCW and XGT100, which have similar molecular weights. However, the trisaccharidic nature of the fucose bearing side chain is more likely to be a key factor than the nature of this saccharide itself. In this case, our results can be considered to be more consistent with the results of Hanus et al.28 who established the absence of influence of fucosyl residues on adsorption. However, the improvement of an adsorption capacity of Rubus XGs, compared to tamarind XGs, could also be the

consequence of a differing side chain distribution or of the increase of free glucosyl residues. It could then be suggested that interactions could take place between the backbone of XGs and the cellulose surface. Consequently, glucosyl residues without side chains could favor interactions as well as trisaccharidic side chains, which enable XGs to change conformation to make the XG backbone more accessible.

5. Conclusion Adsorption isotherms at different temperatures and isothermal titration calorimetry (ITC) were used to determine the mechanisms involved between cellulose and xyloglucans (XGs) with well-defined average molecular weight, side chain, and acetylation contents. This work has highlighted a minimum chain length required for adsorption of XGs on cellulose and that maximum adsorption capacity increases with XG molecular weight. Thanks to adsorption isotherms, two mechanisms, respectively, exothermic in the first stage of adsorption at low XG concentration and endothermic in a second stage for higher XG concentration, were identified. Such an endothermic adsorption was previously unknown and was confirmed by ITC. It was concluded that exothermic adsorption involved a hydrogen bond creating mechanism, while the endothermic process was governed by hydrophobic interactions and entropy driven due to solvent reorganization. The independence of interactions on charges present on the cellulose surface or in the presence of acetyl groups on XG side chains reveal a very low contribution of electrostatic interactions. This work could be further improved


through the design and synthesis of xyloglucan oligosaccharides, with an optimized number of building blocks and a very welldefined number and location of side chains. Work is in progress using a chemo-enzymatic approach. Acknowledgment. We would like to thank the late Dr. M. Schu¨lein from Novozymes A/S (Denmark) for the generous gift of Cellulase Cel5A. The assistance and the work of Joe¨lle Davy, Marie-Jeanne Cre´peau, and Stephanie Boulanger were very helpful for ITC studies, determination of total neutral sugar contents, and mass spectrometry, respectively. The work at Centre National de Recherche sur les Macromolećules ve´ge´tales (CERMAV) was supported in part by the Centre de la Recherche Scientifique (CNRS) and the Institut National de la Recherche Agronomique (INRA). This work was part of the Groupement de Recherche-Assemblages des Molećules Ve´ge´tales (GDRAMV). Supporting Information Available. Kinetic studies (XGT700 and [XGO]3) for incubation time optimization. MALDI-TOF spectras of [XGO]1, [XGO]2, [XGO]3, and XGO1-2. 13C NMR spectras of XGREC and XGRECdes. Comparison of adsorption isotherms of XGT700 on BMCC and CellWhisk. SEC experiment elution curves of XGT100, XGT700, and XGREC. This material is available free of charge via the Internet at http://pubs.acs.org.


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References and Notes (1) Fry, S. C. The Structure and Functions of Xyloglucan. J. Exp. Bot. 1989, 40 (1), 1–11. (2) Hayashi, T. Xyloglucans in the Primary Cell Wall. Ann. ReV. Plant Phys. Plant Mol. Biol. 1989, 40 (1), 139–168. (3) Carpita, N. C.; Gibeaut, D. M. Structural Models of Primary Cell Walls in Flowering Plants: Consistency of Molecular Structure with the Physical Properties of the Walls during Growth. Plant J. 1993, 3 (1), 1–30. (4) Hayashi, T.; Marsden, M. P. F.; Delmer, D. P. Pea Xyloglucan and Cellulose: VI. Xyloglucan-Cellulose Interactions In Vitro and In Vivo. Plant Physiol. 1987, 83 (2), 384–389. (5) Whitney, S. E. C.; Gothard, M. G. E.; Mitchell, J. T.; Gidley, M. J. Roles of Cellulose and Xyloglucan in Determining the Mechanical Properties of Primary Plant Cell Walls. Plant Physiol. 1999, 121 (2), 657–663. (6) Hayashi, T.; Maclachlan, G. Pea Xyloglucan and Cellulose: I. Macromolecular Organization. Plant Physiol. 1984, 75 (3), 596–604. (7) Fujino, T.; Sone, Y.; Mitsuishi, Y.; Itoh, T. Characterisation of crosslinks between cellulose microfibrils and their occurence during elongation growth of pea epicotyl. Plant Cell Physiol. 2000, 41, 486– 494. (8) Fry, S. C. Cross-Linking of Matrix Polymers in the Growing Cell Walls of Angiosperms. Ann. ReV. Plant Physiol. 1986, 37 (1), 165– 186. (9) McDougall, G. J.; Fry, S. C. Anti-Auxin Activity of Xyloglucan Oligosaccharides: the Role of Groups other than the Terminal R-LFucose Residue. J. Exp. Bot. 1989, 40 (2), 233–238. (10) McDougall, G. J.; Fry, S. C. Xyloglucan Oligosaccharides Promote Growth and Activate Cellulase: Evidence for a Role of Cellulase in Cell Expansion. Plant Physiol. 1990, 93 (3), 1042–8. (11) Fry, S. C.; York, W. S.; Albersheim, P.; Darvill, A.; Hayashi, T.; Joseleau, J. P.; Kato, Y.; Lorences, E. P.; Maclachlan, G. A.; McNeil, M.; Mort, A. J.; Reid, J. S. G.; Seitz, H. U.; Selvendran, R. R.; Voragen, A. G. J.; White, A. R. an Unambiguous Nomenclature for Xyloglucan-Derived Oligosaccharides. Physiol. Plant. 1993, 89 (1), 1–3. (12) Lerouxel, O.; Choo, T. S.; Seveno, M.; Usadel, B.; Faye, L.; Lerouge, P.; Pauly, M. Rapid Structural Phenotyping of Plant Cell Wall Mutants by Enzymatic Oligosaccharide Fingerprinting. Plant Physiol. 2002, 130 (4), 1754–1763. (13) Vanzin, G. F.; Madson, M.; Carpita, N. C.; Raikhel, N. V.; Keegstra, K.; Reiter, W.-D. The mur2 Mutant of Arabidopsis thaliana Lacks Fucosylated Xyloglucan because of a Lesion in Fucosyltransferase AtFUT1. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (5), 3340–3345. (14) Madson, M.; Dunand, C.; Li, X.; Verma, R.; Vanzin, G. F.; Caplan, J.; Shoue, D. A.; Carpita, N. C.; Reiter, W.-D. The MUR3 Gene of

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