Interchain Heterogeneity of Enzymatically Deesterified Lime Pectins

Biomacromolecules 2002, 3, 917-925

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Interchain Heterogeneity of Enzymatically Deesterified Lime Pectins Marie-Christine Ralet* and Jean-Franc¸ ois Thibault Unite´ de Recherche sur les Polysaccharides, leurs Organisations et Interactions, Institut National de la Recherche Agronomique, rue de la Ge´ raudie` re, B.P. 71627, F-44316 Nantes Cedex 3, France Received April 29, 2002; Revised Manuscript Received May 21, 2002

Two series of pectins with different levels and patterns of methyl esterification were produced by treatment of a very highly methylated lime pectin with a fungus- or plant-pectin methylesterase. The interchain distribution of free carboxyl groups was investigated by size exclusion and ion exchange chromatography. “Homogeneous” populations with respect to molar mass or charge density were thereby obtained, and their composition, molar mass, and calcium binding properties were investigated. The composition varies from one size exclusion chromatography fraction to another, the highest molar mass fraction being richer in rhamnogalacturonic sequences and exhibiting a slightly higher degree of methylation (DM). Separation of pectins by ion exchange chromatography revealed a narrow charge density distribution for pectins deesterified by fungus-pectin methylesterase, in agreement with a multichain mechanism. Conversely, pectins deesterified by plant-pectin methylesterase exhibited a very large charge density distribution suggesting a processive mechanism. The interchain polydispersity with regard to DM was however shown to have no impact on calcium binding properties of the different fractions. The progressive dimerization through calcium ions with decreasing DM of pectins deesterified by plant-pectin methylesterase seems to be the result of a peculiar intrachain pattern of methyl esterification that can be attributed to a multiple attack mechanism. Introduction Pectins are complex polysaccharides that represent around 30% of the primary plant cell walls and play a key role in the control of cell wall ionic status, cell expansion, cell to cell adhesion, and cell separation.1 Their dominant feature is a linear chain of R-(1f4)-linked D-galacturonic acid units that can be methyl esterified at position 6 and acetylated at position 2 and/or 3. These homogalacturonans are interrupted by rhamnogalacturonic regions in which galacturonic acid residues are interspersed with R-(1f2)-linked l-rhamnopyranosyl residues carrying neutral sugar side chains. The galacturonic acid units in rhamnogalacturonans can be methyl esterified and acetylated as in homogalacturonans2 (Figure 1). In addition to the complex structure of a single molecule, pectins are characterized by a high degree of heterogeneity with respect to composition, molar mass, and degree of substitution (methyl, acetyl, ...).3,4 Pectins exhibit gelling capacity that makes them a very important additive in the food industry.5 They are commercially extracted from citrus peels or apple pomace with hot dilute mineral acid.2 The resulting pectins exhibit a high degree of methylation (DM) and gel in an acidic medium on addition of sucrose. Pectins of lower ester content can be prepared, generally by controlled acid or alkaline deesterification in ethanol or 2-propanol, leading to a random repartition of nonesterified carboxyl groups. The ability of these low methoxyl pectins to cross-link via calcium bridges is the basis for several applications.2,5 * Corresponding author (telephone, +33 (0)240675064; fax, +33 (0)240675066; e-mail, [email protected]).

Within the cell wall, it is commonly assumed that part of the pectin is cross-linked through calcium ions.6-8 The ability of chelating agents to extract a fraction of wall pectins is often claimed to be in favor of a significant functional role of calcium-pectin interactions in the organization of the cell wall network.9 Pectin methylesterases (PMEs) catalyze the demethoxylation of pectins. They have been isolated from various sources. Acidic microbial (Aspergillus japonicus, Aspergillus niger, Aspergillus foetidus) PMEs are claimed to lead to pectins with a random distribution of free carboxyl groups.10-14 Pectins deesterified by fungus-PMEs are able to cross-link via calcium bridges for DM below 30-35%.14 The action of alkaline PMEs from higher plants (tomato, orange, alfalfa, apple) and from fungi (Trichoderma reesei) is thought to result in a blockwise arrangement of free carboxyl groups in the pectin molecules.12-16 The exact action patterns of plant-PMEs on their substrate are however not yet fully explained as published results are in disagreement. It is likely that the substrate characteristics, the presence of different enzyme isoforms, and the pH value may play a role in the demethoxylation process.15-17 Pectins deesterified by plant-PMEs seem to adopt a calcium-induced dimeric conformation up to a DM of 40%.18,19 Kohn et al. however have shown that pectins deesterified by plant-PMEs exhibited calcium binding properties close to that of polygalacturonic acid up to a DM of at least 60%.12 The number of contiguous unesterified galacturonic acid residues needed to form stable junction zones in vivo is presently unknown. In vitro, an uninterrupted sequence of at least 9,18 14,20 or 18-2821 galacturonic acid residues seems necessary to stabilize the dimers.

10.1021/bm020055o CCC: $22.00 © 2002 American Chemical Society Published on Web 07/11/2002

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Biomacromolecules, Vol. 3, No. 5, 2002

Ralet and Thibault

Figure 1. Schematic representation of a pectin chain.

The three mechanisms that are classically proposed for enzymes acting on polysaccharides22 have been considered for the action pattern of PMEs:15-17 (1) a single-chain mechanism, where the binding of the enzyme is followed by a conversion of all contiguous substrate sites on the homogalacturonan; (2) a multichain mechanism, where the enzyme-substrate complex dissociates after each reaction resulting in deesterification of only one residue for each attack; (3) a multiple attack mechanism, where the enzyme catalyses the deesterification of a limited number of residues for every active enzyme-complex formed. The average number of units deesterified per productive enzymesubstrate association has been defined as the degree of multiple attack. Dene`s et al. recently proposed a new classification taking into account both the intra- and interchain levels.16 Both single-chain and multichain mechanisms can occur according to a multiple or a single attack process. In a previous work, three series of pectins were produced by deesterifying the same mother pectin by alkali, fungusPME, or plant-PME.23 Although alkali and fungus-PME processes are thought to lead both to a random distribution of nonesterified galacturonic acid residues,10,12 a slight but significant difference in the calcium binding properties was evidenced.14 With enzymatic degradations, a more “ordered” random deesterification process was hypothesized for fungusPME.23 However, all these studies were carried out on crude (unfractionated) pectins. Beside the intrachain pattern of methyl esterification, interchain polydispersity with regard to DM might play an important role in calcium binding properties of pectin samples. For pectins deesterified by the plant-PME, it has been suggested that dimerization though calcium ions could take place progressively with decreasing DM.14 However, the progressive dimerization with decreasing DM observed could here again be mainly due to interchain disparity. The purpose of this work was to obtain “homogeneous” populations, with respect to molar mass or charge density, by fractionating pectins deesterified by fungus- and plantPMEs by size exclusion or ion exchange chromatography. The interchain distribution of sugars and methoxyl groups

was studied in the different fractions and related to calcium binding properties. The different action patterns of the fungus- and plant-PMEs used are discussed on the basis of these results. Experimental Section Synthesis of Model Pectins. A commercial pectin (L72) from Mexican lime peel (Citrus aurantifolia) with a DM of 72% was esterified in acid-methanol medium to give a pectin (E81) with a DM of 81%, and two series of pectins with defined DM were prepared by enzymatic treatment of E81 as described elsewhere.23 F69, F58, and F43 (DM 69%, 58%, and 43%, respectively) were prepared using a fungusPME from Aspergillus niger purified from Pektolase (Danisco Ingredients, Braband). The enzymatic deesterification was performed at pH 4.5 and 40 °C until the desired DM was achieved. P70, P60, and P41 (DM 70%, 60%, and 41%, respectively) were prepared using a plant-PME purified from orange peels as described by Christensen et al.24 This enzyme is a 36-kDa protein with an isoelectric point >9 and a pH optimum at 7. The enzymatic deesterification was performed at pH 7.0 and 40 °C until the desired DM was achieved. After the deesterification was stopped and the enzyme inactivated, pectins were recovered by isopropyl alcohol precipitation. Analytical. All values were calculated on a moisture-free basis. Galacturonic acid and neutral sugars (expressed as galactose) were quantified colorimetrically by the automated m-phenylphenol25 and orcinol26 methods, respectively, the latter being corrected for interfering galacturonic acid. Except for chromatographic fractions, galacturonic acid was quantified after saponification of the pectin samples (0.05 M NaOH, 30 min, room temperature) and neutralization (0.05 M HCl). Pectins were hydrolyzed in 2 M trifluoroacetic acid (2 h, 121 °C). The individual sugars were reduced, acetylated, and analyzed by gas-liquid chromatography (GLC).27 Pectins were first recovered in their acidic form (cf. Physicochemical Characterization). Free carboxylic functions were quantified at the neutralization point by conductometric titrations with a base of known molarity and total carboxylic functions by colorimetry on the same solutions after saponi-

Biomacromolecules, Vol. 3, No. 5, 2002 919

Enzymatically Deesterified Lime Pectins

fication.14 DM was calculated as DM ) 100 × (total carboxylic functions free carboxylic functions)/total carboxylic functions Chromatography. Size Exclusion Chromatography. Size exclusion chromatography was performed on a column (92 × 5 cm) of Sephacryl S-500 equilibrated with 0.1 M sodium succinate, pH 4.5. Thimerosal (0.02%) was added in the buffer as a preservative. Solutions (50 mL at 5 mg/mL) were loaded onto the column and eluted by upward elution at 250 mL/h. Fractions (15 mL) were collected and analyzed. Ion-Exchange Chromatography. Chromatography on DEAE-Sepharose CL-6B was performed on a column (31 × 2.6 cm) equilibrated with 0.05 M sodium succinate, pH 4.5, at a flow rate of 100 mL/h. Thimerosal (0.02%) was added in the buffer as a preservative. Samples (50 mL of a solution at 4 mg/mL) were loaded onto the column, and the gel was washed with 500 mL of buffer. The bound material was eluted with a linear NaCl gradient (0-0.4 M, 2 L); 15 mL fractions were collected and analyzed. Physicochemical Characterization. Molar Mass Determination. Pectin samples were gently dispersed into ultrapure water (∼3 mg/mL) and left for dissolution overnight under magnetic stirring; NaNO3 was then added (0.05 M final). The solution was centrifuged in a benchtop centrifuge and filtered on 0.45 µm Minisart RC15 Sartorius membranes. Sample was then injected on a high-performance size exclusion chromatography (HPSEC) system consisting of a Shodex OH SB-G precolumn followed by two Shodex OHpack 804 and 805 columns used in series. The elution was performed at room temperature with 0.05 M NaNO3, containing 0.02% NaN3 as preservative, at a constant 42 mL/h flow rate. On-line intrinsic viscosity and molar mass determinations were performed using a differential viscosimeter (T-50A, Viscotek) and a differential refractometer (ERC 7517 A) (dn/dc ) 0.146 mL/g). Intrinsic viscosity [η] and molar mass (Mw, weight-average molar mass; Mn, number-average molar mass) were calculated using TriSEC software, version 3.0 (Viscotek). Calcium Transport Parameter and Calcium ActiVity Coefficient. Transport parameters were determined using conductometric measurements as already described.13,14 All conductometric measurements were carried out at 25.0 ( 0.2 °C with a CDM 83 conductimeter (Radiometer Analytical S.A.) equipped with a double platinum electrode CDC 241U (Radiometer Analytical S.A.). The cell constant was determined with 0.01 M KCl before each set of measurements. The titrations were performed on pectin samples in the acidic form (Cp ∼ 1 mequiv/L) with freshly prepared 10 mequiv/L solutions of KOH, LiOH, and Ca(OH)2. The limiting law for the equivalent conductivity of polyelectrolyte without external salts is given by Λ ) f (λp + λc)

(1)

with Λ the equivalent conductivity (S‚cm2/equiv) of the salts in solution, λp the equivalent conductivity of the active monomer carried by the polyelectrolyte, λc the equivalent conductivity of the isolated counterion in pure solvent at

infinite dilution at 25 °C, and f the transport parameter. By measurement of the conductivity of three ionic forms of the polyelectrolyte (Li , K, and Ca forms) and by consideration of transport parameter independent of the nature of the monovalent counterion, the transport parameters for monovalent (fLi+, K+) and calcium (fCa2+) cations and the equivalent conductivity of the polyelectrolyte (λp), can be calculated. The calcium activity coefficients at the neutralization point (γCa2+) were determined by means of a dual-wavelength spectrophotometric method using tetramethylmurexide (TMMX) as an activity probe for calcium ions.14 A calibration curve was obtained using CaCl2 solutions. Values reported correspond to the ratio of the activity of calcium ions in the presence of pectins to the activity of calcium ions in ideal CaCl2 solutions at the same ionic concentrations. The transport parameter values and activity coefficients were compared with theoretical predictions from Manning’s model28-30 zξh < 1

f)1-

0.55(|z|ξh)2 |z|ξh + π

γ ) e-|z|ξh/2 zξh g 1

f)1γ)

e-1/2 |z|ξh

(2) (3)

0.87 |z|ξh

(4)

(5)

where z is the charge of the counterion and ξh the structural charge density of pectins.31 Results E81, three fungus-PME deesterified samples (F69, F58, and F43; DM 69%, 58%, and 43%, respectively), and three plant-PME deesterified samples (P70, P60, and P43; DM 70%, 60%, and 41%, respectively) were chosen for the present study. These crude pectins have been characterized in a previous work.14 They exhibited a high galacturonic acid content (856-914 mg/g) and a low amount of galactose (2644 mg/g), rhamnose (11-14 mg/g), and arabinose (3 mg/ g). Molar masses of 124400, 116500, 116800, and 109200 mol/g were observed for E81, F69, F58, and F43, respectively. Samples deesterified by the plant-PME were characterized by lower molar masses (96300, 94000, and 88500 mol/g for P70, P60, and P41, respectively). All samples were quite heterogeneous as revealed by the polydispersity index values around 2.2.14 Fractionation by Preparative Size Exclusion Chromatography. The elution patterns obtained by fractionating E81, fungus- and plant-PME deesterified pectins on Sephacryl S-500 are shown in Figure 2. The recoveries were close to 100% for all the samples studied. The elution patterns revealed that all pectic samples exhibit large hydrodynamic volumes and wide size distribution, in agreement with their high polydispersity index.14 Five populations were recovered and their yield and chemical and physicochemical characteristics are summarized in Table 1.

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Figure 2. Elution profiles of E81, fungus-PME, and plant-PME deesterified pectins on size-exclusion chromatography (Sephacryl S-500): (b) galacturonic acid; (O) neutral sugars.

Some global tendencies with respect to acidic and neutral sugars contents were observed along size exclusion chromatography fractionation whatever the pectin studied (Table 1). The highest galacturonic acid contents and the lowest neutral sugars contents were obtained for intermediate size fractions which represent the bulk of the samples. As already pointed out,4,32 the highest molar mass fractions exhibited a higher amount of both rhamnose and galactose, the low galacturonic acid/rhamnose ratio (18-43 mol/mol) indicating the presence of long or numerous rhamnogalacturonic sequences. The fractions of lower molar mass exhibited a higher content in galactose but not in rhamnose, suggesting the presence of either free neutral polysaccharides or longer or more numerous neutral sugars side chains attached to the pectic backbone. The galacturonic acid/rhamnose ratio was high (37-97 mol/mol). A trend of moderately decreasing DM along fractionations was observed, in agreement with previously reported data.4,32 The higher DM observed for the higher molar mass fractions might be attributed to the

presence of longer or more numerous rhamnogalacturonic sequences which are known to be very highly methoxylated.33 As expected, the size exclusion chromatography fractions exhibited decreasing average molar mass with increasing elution volume (Table 1). It was previously shown for P60 that fraction 1 is representative of the high molar mass population observed in unfractionated pectins.34 Fraction 2 also contained part of this high molar mass population, while fractions 3, 4, and 5 were devoid of it.34 Similar conclusions can be drawn for E81, fungus-PME, and plant-PME deesterified pectins. Except for several fractions 1, which remain heterogeneous with regard to molar mass, the mass heterogeneity of size exclusion chromatography fractions was significantly reduced compared to unfractionated pectins. Fractionation by Preparative Ion Exchange Chromatography. Ion exchange chromatography on DEAE-Sepharose CL-6B was used to fractionate pectins according to their charge density (Figure 3). Galacturonic acid and neutral sugars recoveries were >95% for all samples. E81 exhibited



Table 1. Composition and Physicochemical Properties of Fractions Obtained after Chromatography on Sephacryl S-500 sample E81

F69

F58

F43

P70

P60

P41

sugar content (mg/g)

DEAEfraction

yield (%)

GalA

Rha

Ara

Gal

DM (%)

Mw (1000 g/mol)

I (Mn/Mw)

expt

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

12.5 19.2 29.3 24.3 14.8 12.7 21.3 28.5 26.7 10.8 13.5 23.2 31.2 22.3 9.9 9.2 14.5 26.0 32.1 18.4 9.3 20.8 32.1 28.0 9.9 6.9 21.4 41.5 24.3 5.9 6.9 13.9 28.8 24.8 25.6

731 868 901 862 811 727 843 917 883 853 720 870 854 853 842 662 852 850 806 776 668 826 855 837 809 763 896 896 908 746 728 866 931 936 910

14 11 10 10 11 19 12 12 11 12 27 15 15 11 11 30 19 14 16 15 29 15 15 14 9 31 17 13 15 14 25 14 11 8 9

3 3 2 2 3 3 2 2 2 3 3 3 3 3 3 5 2 3 4 3 4 4 3 4 4 4 2 3 3 4 4 3 3 3 3

30 23 27 33 46 35 18 25 24 29 40 21 27 30 43 41 21 23 35 47 52 28 41 48 37 45 21 25 53 71 50 24 28 25 37

85 83 81 81 80 70 67 68 68 69 61 59 57 58 57 46 45 45 45 41 71 70 68 67 65 68 60 58 61 60 42 39 38 35 38

259 130 101 86 63 268 133 108 89 81 300 152 104 69 54 279 158 109 82 59 207 90 90 81 63 280 117 67 50 36 315 139 89 73 68

2.6 1.7 1.7 1.6 1.6 1.9 1.5 1.6 1.5 1.4 1.6 1.6 1.7 1.3 1.4 1.6 1.5 1.7 1.5 1.4 2.1 1.7 1.6 1.4 1.3 1.6 1.5 1.4 1.3 1.3 2.2 1.7 1.5 1.4 1.4

0.90 0.83 0.88 0.88 0.81 0.76 0.84 0.90 0.82 0.89 0.78 0.77 0.77 0.77 0.79 0.67 0.75 0.76 0.73 0.77 0.73 0.73 0.71 0.71 0.77 0.76 0.74 0.72 0.72 0.76 0.70 0.71 0.72 0.72 0.73

a broad elution pattern while fungus-PME deesterified samples eluted as single peaks, progressively more symmetrical and thin, and eluting at increasing ionic strength when the DM lowered. This is in favor of a fairly narrow distribution of DM in fungus-PME-treated pectin samples, in agreement with a multichain enzymatic mechanism.16 P70 and P60 exhibited a broad elution pattern indicating that pectins are certainly distributed over a wide range of DM. On the opposite, P41 was eluted as a fairly thin peak. This behavior cannot be ascribed to a single-chain mechanism known to lead to the production of two populations of pectins, one being not modified and the other being totally demethoxylated.16 A peculiar multiple attack mechanism, different from the multichain mechanism observed for fungus-PME, can be proposed. These results agree with those of Schols et al. who have shown that contrary to fungusPME deesterified pectins, plant-PME deesterified pectins elute in large peaks on ion exchange chromatography.35 The yield and the characteristics of each fraction recovered by ion exchange chromatography were determined (Table 2). As expected, DM decreased along the fractionations. Average DM values recalculated from the contribution of each of their constituting fractions were in good agreement with average DM values determined on whole samples. E81

fLi+,K+ theory 0.99 0.99 0.99 0.99 0.98 0.96 0.96 0.96 0.96 0.96 0.94 0.94 0.93 0.93 0.93 0.90 0.89 0.89 0.89 0.88 0.97 0.96 0.96 0.96 0.95 0.96 0.94 0.93 0.94 0.94 0.88 0.87 0.87 0.84 0.87

expt 0.66 0.63 0.68 0.66 0.58 0.44 0.46 0.50 0.46 0.50 0.39 0.41 0.38 0.40 0.41 0.26 0.30 0.30 0.29 0.30 0.47 0.46 0.42 0.39 0.43 0.38 0.34 0.34 0.34 0.34 0.22 0.22 0.21 0.20 0.23

fCa2+ theory 0.97 0.96 0.94 0.95 0.94 0.87 0.83 0.84 0.83 0.87 0.69 0.66 0.63 0.64 0.62 0.50 0.49 0.49 0.49 0.46 0.89 0.87 0.84 0.81 0.78 0.84 0.69 0.64 0.68 0.67 0.46 0.44 0.44 0.42 0.43

λp 21.9 28.2 26.4 25.9 30.4 40.4 33.9 29.4 33.0 31.4 40.3 31.4 39.6 35.7 33.4 50.0 44.5 47.4 51.7 51.6 36.5 34.6 39.8 45.5 33.3 31.4 39.5 39.8 43.1 35.2 42.8 42.8 46.4 46.4 43.7

was rather homogeneous with respect to DM (67-90%). F58 and F43 were also quite homogeneous with regard to DM, which range from 49 to 61% and 33 to 50% for F58 and F43, respectively. F69 was eluted as a large peak and exhibited a wider charge density distribution with DM ranging from 50 to 74%. Figure 4 shows the course of the ionic strength of the eluant and of Debye length (i.e., eluant ionic atmosphere thickness) versus DM for ion exchange chromatography fractions. A linear correlation (R2 ) 0.90) between DM and the ionic strength of the eluant was shown. Furthermore, no difference between pectins demethoxylated by fungus- or plant-PME was evident, suggesting that elution could be mainly governed by charge density independently of charge distribution patterns. The presence of numerous but fairly short blocks of deesterified galacturonic acid units in pectins deesterified by plant-PME may also explain the similarity of elution between the two pectin series, in agreement with the multiple attack mechanism proposed above. These results differ with those of Schols et al. and of Kravtchenko et al., who showed that a blockwise distribution of free carboxyl groups result in zones of higher charge density able to bind strongly to the ion exchanger.35,36 DEAE-recovered fractions contained galacturonic acid and neutral sugars in varying proportions. Fractions 1 were

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Figure 3. Elution profiles of E81, fungus-PME, and plant-PME deesterified pectins on ion-exchange chromatography (DEAE-Sepharose CL6B): (b) galacturonic acid; (O) neutral sugars; (s) NaCl molarity.

characterized by a fairly low content in galacturonic acid and a high content in rhamnose and galactose, suggesting the presence of longer or more numerous rhamnogalacturonic sequences (galacturonic acid-to-rhamnose ratio 12-24 mol/ mol) compared to the other fractions (galacturonic acid-torhamnose ratio 43-124 mol/mol). A trend of slightly decreasing neutral sugars content from fraction 2 to the end of the fractionation was evidenced for all fungus- and plantPME deesterified samples. These results are in good agreement with previously published data.36 The fractions obtained by ion exchange chromatography have been rechromatographed on HPSEC, and the average molar mass of each fraction was estimated by viscosimetry using a universal calibration curve (Table 2). These fractions did not appear homogeneous on HPSEC, especially those at the beginning and at the end of the elution patterns (data not shown). Fractions 1 contained a mixture of low and high molar mass molecules. Intermediate fractions, which were shown to be chemically quite similar, exhibited also similar average molar masses, close to those determined for unfractionated samples.14 The last fractions consisted of a mixture

of low and high molar mass molecules. Kravtchenko et al. also noticed that ion exchange chromatography fractions did not appear homogeneous on size exclusion chromatography, although no values of molar mass were reported.36 Interaction of Pectins with Cations. Interactions of pectic fractions with monovalent cations and calcium were quantified by conductimetry, and experimental values were compared to those calculated from Manning’s theory28-30 (Tables 1 and 2), which is proposed for infinitely dilute solutions of rodlike polyelectrolytes. The transport parameter values of monovalent counterions for size exclusion and ion exchange chromatography fractions were in the range 0.67-0.90 and decreased regularly with decreasing DM in agreement with previously reported data on unfractionated samples.13,14 As already pointed out,13,14 experimental values were in fairly good agreement with Manning’s theoretical ones, showing that the interactions between monovalent ions and pectins are close to a “classical electrostatic” binding. As previously observed on unfractionated pectins,14 no difference could be seen between fungus-PME and plant-PME deesterified samples.



Table 2. Composition and Physicochemical Properties of Fractions Obtained after Chromatography on DEAE-Sepharose CL-6B sample E81

F69

F58

F43

P70

P60

P41

sugar content (mg/g)

fLi+,K+

DEAEfraction

yield (%)

GalA

Rha

Ara

Gal

DM (%)

Mw (1000 g/mol)

expt

theory

expt

1 2 3 4 1 2 3 4 5 1 2 3 4 1 2 3 1 2 3 4 5 1 2 3 4 5 6 7 1 2 3

15.8 39.5 27.6 13.1 2.7 10.5 41.0 34.1 10.5 14.2 43.0 32.9 9.8 20.4 64.2 15.5 6.5 18.1 36.1 19.4 20.0 1.5 3.6 9.8 19.9 27.5 25.7 11.9 17.4 31.4 51.1

792 878 934 908 495 898 902 881 836 770 905 912 809 738 840 735 447 859 914 843 669 532 776 823 832 861 841 839 710 851 900

12 6 8 10 33 8 9 7 8 30 9 7 7 25 7 7 26 11 9 9 8 26 14 16 13 9 9 8 29 10 6

2 2 2 2 6 3 3 3 3 5 3 3 3 3 2 3 8 3 3 2 2 6 4 5 3 3 2 3 5 2 2

20 11 14 15 61 12 13 9 10 53 14 10 10 37 10 11 222 17 15 13 10 63 27 24 21 13 12 10 63 18 9

>90 81 79 67 n.d. 74 71 64 50 61 60 53 49 50 41 33 n.d. 79 75 68 54 n.d. n.d. 72 68 61 48 34 65 49 31

68 87 122 338 + 58 (30% + 70%) 345 + 33 (49% + 51%) 78 86 90 281 + 57 (25% + 75%) 496 + 50 (25% + 75%) 95 102 338 + 64 (18% + 82%) 452 + 50 (23% + 77%) 90 328 + 64 (23% + 77%) 31 55 72 434 + 42 (23% + 77%) 243 + 51 (20% + 80%) 28 27 504 + 36 (7% + 93%) 434 + 42 (8% + 92%) 62 61 261 + 50 (14% + 86%) 459 + 47 (12% + 88%) 73 81

n.d. 0.85 0.83 0.77 n.d. 0.79 0.88 0.82 0.74 0.72 0.74 0.78 0.70 0.78 0.74 0.72 n.d. 0.85 0.83 0.81 0.72 n.d. n.d. n.d. 0.83 0.81 0.74 0.75 0.81 0.77 0.68

0.99 0.98 0.96 0.97 0.97 0.95 0.91 0.94 0.94 0.92 0.91 0.91 0.88 0.81 0.98 0.98 0.96 0.92 0.97 0.96 0.94 0.90 0.82 0.95 0.91 0.78

n.d. 0.67 0.59 0.43 n.d. 0.58 0.54 0.44 0.34 0.43 0.42 0.37 0.30 0.36 0.28 0.25 n.d. 0.61 0.54 0.44 0.29 n.d. n.d. n.d. 0.44 0.38 0.27 0.21 0.43 0.28 0.18

Figure 4. Ionic strength of the eluant (I), at the elution volume of each DEAE-recovered fraction, as a function of their degree of methylation: (O) E81 and fungus-PME deesterified pectins; (0) plantPME deesterified pectins. Debye length (Ie-1/2), at the elution volume of each DEAE-recovered fraction, as a function of their degree of methylation: (b) E81 and fungus-PME deesterified pectins; (9) plantPME deesterified pectins.

Calcium transport parameter values of E81 and fungusPME size exclusion and ion exchange chromatography fractions decreased also with decreasing DM (Figure 5a). The calcium transport parameter versus DM course of fractionated pectins is close to that of unfractionated ones, except for high-molar mass fractions (fraction 1 from Sephacryl S-500 patterns) which exhibited significantly lower

fCa2+ theory 0.95 0.94 0.81 0.90 0.88 0.75 0.54 0.69 0.67 0.58 0.53 0.54 0.45 0.41 0.93 0.91 0.85 0.59 0.89 0.84 0.69 0.52 0.41 0.76 0.53 0.39

λp n.d. 29.8 28.4 42.0 n.d. 33.9 32.1 38.5 43.3 45.9 41.7 44.3 46.7 43.3 48.7 48.6 n.d. 31.8 35.5 39.4 44.1 n.d. n.d. n.d. 30.5 39.8 46.2 31.6 42.3 43.1 52.8

values. As said above, these fractions were particularly rich in rhamnogalacturonic sequences which are known to be virtually fully methoxylated.37 Calcium binding properties are controlled by intrinsic parameters such as the length of homogalacturonans and the level and pattern of methyl esterification. DMs observed for rhamnogalacturonan-rich fractions might therefore be higher than those of their constitutive homogalacturonan sequences explaining the shift in the calcium transport parameter values. A transition zone around DM 30-35%, which was attributed to the dimerization of pectic molecules through calcium ions, was demonstrated for alkali- and fungus-PME deesterified pectins.10,13,14 In the range 35% < DM < 80%, experimental-to-theoretical ratio remained roughly constant around 0.60-0.65 for chemically and fungus-PME deesterified pectins.13,14 The sudden drop in the ratio of experimental to theoretical values around DM 35% was explained by an intermolecular binding of the calcium ions to carboxyl groups of two molecules leading to the formation of dimers.13,38 For chromatographic fractions from the F-series (except fractions 1 from size exclusion chromatography), a constant ratio of experimental to theoretical values of 0.610 ((0.033) can be calculated (Tables 1 and 2) showing that no dimerization of pectic chains through calcium ions above a DM of 30-35% could be evidenced also for these “homogeneous” fractions. This confirms a “true” random repartition of all the free carboxyl groups in pectins deesterified by fungus-PME. Similarly,

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Ralet and Thibault

Figure 6. Variations of the ratio of experimental to theoretical values of calcium transport parameter (fCa2+) with the degree of methylation (DM): (s) unfractionated alkali and fungus-PME deesterified pectins;14 (- - -) unfractionated plant-pectin methylesterase deesterified pectins;14 (4) P41 DEAE-fractions; (0) P60 DEAE-fractions; (O) P70 DEAE-fractions. Variations of the ratio of experimental to theoretical values of calcium activity coefficient (γCa2+) with the degree of methylation (DM): (b) pectins deesterified by Asp. foetidus PME (values from ref 12); (2) pectins deesterified by tomato PME (values from ref 12).

Figure 5. (a) Variations of values of calcium transport parameter (fCa2+) with the degree of methylation (DM): (- - -) unfractionated fungus-PME deesterified pectins; (- - -) unfractionated plant-PME deesterified pectins; (]) F43 DEAE-fractions; (4) F58 DEAE-fractions; (O) F69 DEAE-fractions; (3) E81 DEAE-fractions; ([) F43 S500fractions; (2) F58 S500-fractions; (b) F69 S500-fractions; (1) E81 S500-fractions. (b) Variations of values of calcium transport parameter (fCa2+) with the degree of methylation (DM): (- - -) unfractionated fungus-PME deesterified pectins; (- - -) unfractionated plant-PME deesterified pectins: (]) P41 DEAE-fractions; (4) P60 DEAEfractions; (O) P70 DEAE-fractions; (3) E81 DEAE-fractions; ([) P41 S500-fractions; (2) P60 S500-fractions; (b) P70 S500-fractions; (1) E81 S500-fractions.

fractionated pectins from the P-series exhibited the same global behavior as unfractionated ones. In the range 20% < DM < 75%, pectins from the P-series exhibited lower calcium transport parameter values than chemically or fungus-PME deesterified samples, indicating a stronger binding of calcium ions (Figure 5b). As observed for unfractionated pectins from the P-series,14 a progressive decrease of the ratio of experimental to theoretical values was evidenced for these homogeneous samples (Figure 6). Calcium binding properties, as calcium activity coefficients, were also determined by a metallochromic indicator method14 (data not shown). Results obtained by this method were very similar to those obtained by conductimetry. Discussion The interchain heterogeneity with regard to DM, although particularly important for plant-PME deesterified samples, was shown to have no impact on calcium binding properties

of fungus- and plant-PME deesterified fractions. Pectins demethoxylated by plant-PME at pH 7.0 were shown to be highly heterogeneous with regard to DM. However, even the low DM fractions were not able to fully dimerize (Figure 6), suggesting that short sequences of demethoxylated galacturonic acid residues are generated, together with longer ones able to form “egg boxes”. Long demethoxylated sequences are all the more numerous when the DM decreases. A “single chain multiple attack” mechanism has been reported by Dene`s et al. for plant-PME at pH 7.0.16 This peculiar mechanism leads to a blockwise distribution of demethoxylated galacturonic acids on a part of the pectin molecules, the remaining pectins being not affected.16 Catoire et al. have shown that different isoforms of plant-PME operate by a multiple attack mechanism at neutral pH.17 Grasladen et al. provided also some experimental evidence for a multiple attack mechanism for plant-PME at neutral pH.15 Our results support that plant-PME operates by a multiple attack mechanism with a large range in the degree of multiple attack. Kohn et al. reported very low calcium activity coefficient values for pectins deesterified by a tomato pectin methylesterase.12 In this case, demethoxylated pectins could fully dimerize up to DM 66% (Figure 6). The differences observed between Kohn’s results and those reported here could be due to variations in the substrate used. The “mother” pectin used by these authors had a very high DM (94.7%). The degree of multiple attack might be related to the amount of demethoxylated galacturonic acid units. The type of isoform present in the PME preparation used might also be involved. The methoxyl group distribution in pectins incubated with various PME isoforms from V. radiata was indeed shown to be significantly different.17 Acknowledgment. We thank Mrs Sylviane Daniel for skillful technical assistance and Danisco-Cultor (Denmark) for providing the pectin samples. Financial support from the European Community within the EU-Biotechnology Program, Contract Number ERBIO4CT960685, is gratefully acknowledged.


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Biomacromolecules, Vol. 3, No. 5, 2002 925 (19) Liners, F.; Thibault, J.-F.; Van Cutsem, P. Plant Physiol. 1992, 99, 1099-1104. (20) Powell, D. A.; Morris, E. R.; Gidley, M. J.; Rees, D. A. J. Mol. Biol. 1982, 155 , 516-531. (21) Kohn, R. Pure Appl. Chem. 1975, 42, 371-397. (22) Greenwood, C. T.; Milne, E. A. AdV. Carbohydr. Chem. Biochem. 1968, 23, 281-366. (23) Limberg, G.; Ko¨rner, R.; Buchholt, H. C.; Christensen, T. M. I. E.; Roepstorff, P.; Mikkelsen, J. D. Carbohydr. Res. 2000, 327, 293307. (24) Christensen, T. M. I. E.; Nielsen, J. E.; Kreiberg, J. D.; Rasmussen, P. J. Mikkelsen, D. Planta 1998, 206, 493-503. (25) Thibault, J.-F. Lebensm.-Wiss. -Technol. 1979, 12, 247-251. (26) Tollier, M.-T.; Robin, J.-P. Ann. Technol. Agric. 1979, 28, 1-15. (27) Englyst H. N.; Cummings, J. H. J. Assoc. Off. Anal. Chem. 1988, 71, 808-814. (28) Manning, G. S. J. Chem. Phys. 1969, 51, 924-933. (29) Manning, G. S. In Polyelectrolytes; Selegny, E., Ed.; Reidel Publishing Co.: Dordrecht, 1974; pp 9-37. (30) Manning, G. S. J. Phys. Chem. 1975, 79, 262-265. (31) Rinaudo, M.; Milas, M. J. Polym. Sci. 1974, 12, 2073-2081. (32) Brigand, G.; Denis, A.; Grall, M.; Lecacheux, D. Carbohydr. Polym. 1990, 12, 61-77. (33) de Vries, J. A.; Rombouts, F. M.; Voragen, A. G. J.; Pilnik, W. Carbohydr. Polym. 1984, 4, 89-101. (34) Ralet, M.-C.; Bonnin, E.; Thibault, J.-F. J. Chromatogr. B 2001, 753, 157-166. (35) Schols, H. A.; Reitsma, J. C. E.; Voragen, A. G. J.; Pilnik, W. Food Hydrocolloids 1989, 3, 115-121. (36) Kravtchenko, T. P.; Voragen, A. G. J.; Pilnik, W. Carbohydr. Polym. 1992, 19, 115-124. (37) de Vries, J. A.; Rombouts, F. M.; Voragen, A. G. J.; Pilnik, W. Carbohydr. Polymers 1983, 3 245-258. (38) Ravanat, G.; Rinaudo, M. Biopolymers 1980, 19, 2209-2222.

BM020055O

Interchain Heterogeneity of Enzymatically Deesterified Lime Pectins

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