Large-scale preparation of α-D-(1→4)-oligogalacturonic acids from pectic acid

Article abstract of DOI:10.1139/v02-055

An efficient and inexpensive method for large-scale preparation of α-D-(1→4)-oligogalacturonic acids (oligo-GalA), up to DP 5, from pectic acid is described. Pectic acid was digested with a commercially available pectinase to yield a mixture of oligo-GalA, which was effectively separated by a combination of low-pressure - size-exclusion chromatography based on ion-exchange chromatography to obtain pure oligo-GalA of DP 2-5.

Full text of DOI:10.1139/v02-055

900
Large-scale preparation of -D-(1J4)-
oligogalacturonic acids from pectic acid
Hong-Ni Fan, Mei-Zheng Liu, and Yuan C. Lee
Abstract: An efficient and inexpensive method for large-scale preparation of ꢀ-D-(1J4)-oligogalacturonic acids (oligo-
GalA), up to DP 5, from pectic acid is described. Pectic acid was digested with a commercially available pectinase to
yield a mixture of oligo-GalA, which was effectively separated by a combination of low-pressure – size-exclusion chro-
matography based on ion-exchange chromatography to obtain pure oligo-GalA of DP 2–5.
Key words: pectic acid, galacturonic acid, galabiose, galatriose, pectinase.
Résumé : On décrit une méthode efficace et peu coûteuse de préparation à grande échelle des acides ꢀ-D-(1J4)-
oligogalacturoniques (oligo-GalA), jusqu’à 5 DP, à partir de l’acide pectique. L’acide pectique est soumis à une diges-
tion avec une pectinase commercialement disponible pour conduire à un mélange d’oligo-GalA que l’on peut séparer
efficacement par une combinaison de chromatographie d’exclusion de taille à basse pression et de chromatographie
d’échange ionique pour obtenir les oligo-GalA de DP allant de 2 à 5.
Mots clés : acide pectique, acide galacturonique, galabiose, galatriose, pectinase.
[Traduit par la Rédaction] Fan et al. 903
Introduction
of polymerization (DP) (9–15). These methods, however, ei-
ther require non-commercial enzymes, an expensive special
ion-exchange resin, or preparative HPLC, and are not suit-
able for economical large-scale preparation of oligo-GalA.
We wanted to prepare large amounts of galabiose and oligo-
saccharides containing galabiose by an inexpensive proce-
dure, for the purpose of preparing high-affinity inhibitors for
some toxins (e.g., Shiga toxin, Shiga-like toxins) and for
certain pathogenic bacterial adhesion (16).
In this paper, we describe an efficient large-scale prepara-
tion of oligo-GalA (DP 2–5) from pectic acid by partial hy-
drolysis of pectic acid with a commercially available
pectinase and a combination of inexpensive low-pressure
column-chromatographic separation methods based on size
exclusion and ion exchange.
Pectic acid, derivable from an abundant plant polymer,
pectin, is essentially composed of a repeating unit of ꢀ-
(1J4)-linked D-galacturonic acid. ꢀ-D-(1J4)-Oligogalact-
uronic acids (oligo-GalA) have been shown to be physiologi-
cally active elicitors of plant-defense responses to pathogens
(1). On the other hand, galabiose (ꢀ-Gal-(1J4)-Gal) has the
same anomeric and positional linkages as the repeating unit
of pectic acid, differing only by the presence of hydroxy-
methyl group rather than the carboxyl group at C-6.
Galabiose is an important structural unit of some glyco-
lipids, such as Gb3 and Gb4, and is the recognition marker
for bacterial toxins (e.g., Shiga toxin and Shiga-like toxins)
as well as for certain microbial invasions of human tissues,
e.g., uropathogenic Escherichia coli, which causes hemor-
rhagic colitis and hemolytic uremic syndrome (2–6). Syn-
thetic glycoconjugates containing the carbohydrate moiety
of Gb3 (globotriose, ꢀ-Gal-(1J4)-ꢁ-Gal-(1J4)-Glc) proved
to be effective biomimetics for detecting and trapping of cer-
tain bacteria toxins and viruses (6–8). Galabiose has been
prepared from pectic acid by first obtaining (ꢀ-GalA-(1J4)-
ꢀ-GalA)n oligomers by partial digestion with pectinase
followed by chemical reduction of the carboxylic groups
(9–11). There are also some reports describing analysis, sep-
aration, and preparation of oligo-GalA with different degrees
Materials and methods
Materials
Apple pectic acid was obtained from Sigma-Aldrich-Fluka
(Milwaukee, WI). Pectinase from Aspergillus niger (EC
3.2.1.15), galacturonic acid (GalA), di- and tri-galacturonic
acid ((GalA)2 and (GalA)3, respectively), Dowex-50 W × 8
resin (H+ form, 200 mesh), Dowex-1 × 4 resin (400 mesh),
and Sephadex G-25 were from Sigma (St. Louis, MO).
Ultrafiltration membranes (YM3, NMWL 3 000) were from
Millipore (Bedford, MA).
Received 1 November 2001. Published on the NRC Research
Press Web site at http://canjchem.nrc.ca on 5 July 2002.
General methods
Analysis of GalA-oligomers was carried out with high-
performance anion-exchange chromatography, HPAEC (17)
using a Bio-LC (Dionex, Sunnyvale, CA) with a CarboPac
PA-1 column (4.6 × 250 mm) in combination with a pulsed
amperometric detector (PAD-2). The detector sensitivity was
set at 1 A. Potential and time settings of the detector were:
Dedicated to the memory of Professor Raymond U. Lemieux.
H.-N. Fan, M.-Z. Liu, and Y.C. Lee.1 Biology Department,
3400 N. Charles St., Johns Hopkins University, Baltimore,
MD 21218, U.S.A.
1Corresponding author (e-mail: yclee@jhu.edu).
Can. J. Chem. 80: 900–903 (2002)
DOI: 10.1139/V02-055
© 2002 NRC Canada

Fan et al.
901
Fig. 1. Time course of hydrolysis of pectic acid with pectinase
examined by HPAEC. The number labels mark the DP of the
oligogalacturonic acid for the indicated peaks. The time labels
on the right of each curve indicate the lengths of hydrolysis, and
GalA and (GalA)2 are standards. Equal amounts of the reaction
mixture were injected for each run.
Fig. 2. Elution profiles of pectic acid digested with different
amounts of pectinase by HPAEC. A: pectinase (20 units)/pectic
acid (1.0 g); B: pectinase (10 units)/pectic acid (1.0 g). The
numbers show the DP of the oligogalacturonic acid in the indi-
cated peaks.
1750
1
1
5000
1500
3
2
4
1250
1000
4000
24h
5
7
6
3000
2000
1000
0
3h
750
500
250
0
3
4
2
5
1h
B
A
0.5h
(Gal A)2
Gal A
0
10
20
30
40
50
0
10
20
30
40
50
60
Elution time (min)
Elution time (min)
pectinase digestion. During the digestion with 10 or 20 units
of pectinase–pectic acid (1.0 g) in 0.1 M acetate buffer,
pH 4.0 (see below) at room temperature for 24 h, portions of
the digest were removed after 0.5, 1, 3, and 24 h, immedi-
ately frozen in a dry-ice acetone bath, and stored frozen. Be-
fore analysis, the samples were thawed and heated at 100°C
for 10 min to inactivate the pectinase and the precipitate was
removed by microcentrifugation. Digestion with 20 units of
pectinase–pectic acid (1.0 g) yielded ꢀ-(1J4)-oligo-GalA
(DP 1–4) as the main products, in which GalA was predomi-
nant (Fig. 1). On the other hand, when 10 units of pectinase–
pectic acid (1.0 g) were used under the same conditions, a
much lower amount of GalA was produced, and the yields
of oligo-GalA (DP 2–5) were higher (Fig. 2).
Table 1. Elution gradient (%) of HPAEC for analysis of (GalA)n.
E2
E3
E4
Time
(min)
0.20 M NaOH/
1 M NaOAc
0.20 M NaOH
Water
0–2
2–9
9–51
51–52
52–62
50
50
50
50
50
48
2
48–30
30–0
0–48
48
2–20
20–50
50–2
2
E1 = +0.05 V (t1 = 0.42 s), E2 = +0.65 V (t2 = 0.18 s), and
E3 = ꢂ0.10 V (t3 = 0.36 s). Oligo-GalA samples were eluted
using the gradients shown in Table 1.
Preparation of oligo-GalA (DP 1–5) mixtures
Carbohydrates were determined with a version of the phe-
nol–sulfuric acid method (18). Thin layer chromatography
(TLC) was performed on a 0.25-mm layer of silica gel 60
coated on aluminum sheets (E. Merck, Darmstadt, Ger-
many).
Pectic acid powder (10.0 g) was suspended in HOAc–
NaOAc buffer (each 0.1 M, 500 mL, pH 4.0) and the mix-
ture was incubated at 55°C, sonicated, and stirred until the
pectic acid was totally dissolved. The homogeneous but vis-
cous solution was again adjusted to pH 4.0 with 0.1 M
NaOAc, and the pectinase (250 L, ca. 100 units) was
added. The solution was gently stirred for 24 h at room tem-
perature; the enzymatic hydrolysis was terminated by heat-
ing the mixture at 100°C for 10 min. After cooling, the
precipitate and the remaining polymer were removed by
ultrafiltration with a stirred cell (200-mL, Millipore, Bed-
ford, MA) using a YM3 membrane. To remove the cations,
the filtrate was applied to a column (2.5 × 17.6 cm) of
Dowex-50W × 8 resin (H+ form, 200 mesh) and washed
with distilled water (200 mL). The washing was concen-
trated by rotary evaporation, and the residue was lyophilized
to afford 7.68 g of oligo-GalA mixture (76.8% yield).
Results and discussion
Optimizing conditions for generation of oligo-GalA with
DP 1–5 by pectinase digestion
Mixtures of oligo-GalA with different DPs can be ob-
tained by hydrolysis of pectic acid with autoclaving or enzy-
matic digestion (9–12), but the enzymatic method was found
to be the more efficient to degrade pectic acid. For example,
depolymerization of pectic acid or pectin (partially esterified
pectic acid) with an endopolygalacturonase (19) produces
the desired saturated oligo-GalA, while with pectin lyases or
pectate lyases, both saturated and unsaturated oligo-GalAs
are produced. Unfortunately, such enzymes are not readily
available, and thus we chose to use a commercially available
pectinase to generate oligo-GalA from pectic acid.
Size-exclusion chromatography
The oligo-GalA mixture (4.0 g) in HOAc (0.1 M, 20 mL)
was applied to a Sephadex G-25 column (5.0 × 218 cm)
in 0.1 M HOAc and eluted at a flow rate of 4 mL min–1
in 0.1 M HOAc. Fractions (16 mL) were analyzed for
carbohydrates by the phenol–H2SO4 method (Fig. 3). The
The optimal conditions for generating oligo-GalA (DP 1–5)
mixtures were determined by TLC and HPAEC–PAD analy-
sis of the oligosaccharides released in the process of
© 2002 NRC Canada

902
Can. J. Chem. Vol. 80, 2002
Fig. 3. Elution of oligo-GalA on a Sephadex G-25 column (5.0 ×
218 cm). The y axis is A480 nm obtained by the phenol–sulfuric
acid method. Approximate positions of the DP of the oligoga-
lacturonic acid are indicated.
Fig. 5. Identification of each oligo-GalA peak isolated from the
Dowex-1 × 4 column (see Fig. 4) with HPAEC.
2000
3
2
4
5
2
1
4
1500
1000
500
0
(Gal A)5
(Gal A)4
(Gal A)3
3
1
(Gal A)2
Gal A
2
5
Oligo-Gal A
mixture
0
0
10
20
30
40
50
50
100
150
200
Elution time (min)
Fractions (16 mL)
Fig. 4. Separation of oligo-GalA on Dowex-1 × 4 column (2.5 ×
46 cm). The column was eluted with a linear gradient generated
with 1 L each of 0.4 M and 1.0 M NaOAc (pH 6.0). The num-
bers indicate the DP of oligo-GalA in the peaks.
content by the phenol–H2SO4 method; the results are shown
in Fig. 4. The carbohydrate-containing fractions were ana-
lyzed by HPAEC for identification of DP. The purity of
each of the isolated peaks is shown in Fig. 5. The individual
oligo-GalA peaks were pooled and passed through a column
(17.6 × 2.5 cm) of Dowex-50W × 8 resin (H+ form, 200
mesh) for decationization; the effluent was concentrated by
rotary evaporation, followed by lyophilization to give di-,
tri-, tetra-, and pentagalacturonic acid ((GalA)2, (GalA)3,
(GalA)4, (GalA)5, respectively), with the yields of 1.8, 18.2,
19.9, and 4.9%, respectively.
The alkaline treatment of the oligo-GalA mixture (pH 12
for 1 h) was to open any lactone rings that might have been
formed during the gel filtration procedure. Lactonization
under acidic condition is often a problem for separation of
anionic oligo/polysaccharides with anion-exchange chroma-
tography (20). The separation of oligo-GalA mixture on
Dowex 1, a common anion-exchange resin, was excellent
(Fig. 4), separating the oligomers better than those reported
previously with sophisticated instrumentation (14, 15). Al-
though the column described above has a loading capacity of
-1.0 g of the oligo-GalA mixture, it can be used repeatedly
or can be readily scaled up.
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
4
3
5
2
0
100
200
300
400
500
Fraction number
carbohydrate-containing fractions were examined by TLC
using silica gel 60 sheets with 1-butanol–formic acid–water
(v/v, 4:6:1) as the developing solvent. The Rf values of the
oligo-GalA with DPs 1, 2, 3, 4, and 5 were ca. 0.48, 0.35,
0.28, 0.22, and 0.17, respectively. The fractions containing
oligo-GalA of DP 2–5 were combined, concentrated by ro-
tary evaporation, and lyophilized to give 2.58 g of product
(64.5% yield). Although the Sephadex G-25 column did not
fully separate oligo-GalA of DP 2–5, most of the monomer
(GalA), which constituted a fairly large proportion of the to-
tal digest, could be removed.
Summary
We present here a simple and efficient preparative method
of oliogalacturonic acids, up to DP 5, from an inexpensive
source, pectic acid. The advantages of our method are the
use of an inexpensive commercial enzyme and low-cost
chromatographic procedures to yield pure oligo-GalA of DP
2–5 in good overall yield.
Anion-exchange chromatography of oligo-GalA
An example of the anion-exchange chromatography of
oligo-GalA is described below. The oligo-GalA mixture (DP
2–5, 1 g) was dissolved in water (10 mL), and the solution
was adjusted to pH 12 with 50% aqueous NaOH solution.
The mixture was left at room temperature for 1 h and the pH
was re-adjusted to 6.0 with 2 M HOAc. The resulting solu-
tion was applied to a column (46.5 × 2.5 cm) of Dowex-1 ×
4 resin (400 mesh) that had been equilibrated in 0.4 M
NaOAc buffer, pH 6.0. The column was washed with 25 mL
of the equilibration buffer, and eluted with a linear gradient
generated with 1 L each of 0.4 M and 1.0 M NaOAc
(pH 6.0). Finally, the column was washed with 1 L of 2.0 M
NaOAc (pH 6.0). Fractions were analyzed for carbohydrate
Acknowledgment
The authors are grateful to Dr. Reiko T. Lee for carefully
reading the manuscript and offering valuable advice.
References
1. C.A. Ryan and E.E. Farmer. Annu. Rev. Plant Physol. Mol.
Bio. 42, 651 (1991).
2. M.A. Karmali, B.T. Steele, M. Petric, and C. Lim. Lancet,
1, 619 (1983).
© 2002 NRC Canada

Fan et al.
903
3. N. Cimolai, J.E. Carter, B.J. Morrison, and J.D. Anderson. J.
Pediatr. 116, 589 (1990).
11. J. Dahmen, T. Freid, G. Magnusson, G. Noori, and A.-S. Carlstrom.
Carbohydr. Res. 127, 15 (1984).
4. G.D. Armstrong, E. Fodor, and R. Vanmaele. J. Infect. Dis.
164, 1160 (1991).
5. L.C.A. Boyd and B., Nephron, 51, 207 (1989).
6. P.I. Kitov, J.M. Sadowska, G. Mulvey, G.D. Armstrong,
H. Ling, N.S. Pannu, R.J. Read, and D.R. Bundle. Nature
(London), 403, 669 (2000).
12. M.D. Spiro, K.A. Kates, A.L. Koller, M.A. O’Neill, P. Albersheim,
and A.G. Darvill. Carbohydr. Res. 247, 9 (1993).
13. T.L. Nuederhauser, J. Halling, N.A. Polson, and J.D. Lamb, J.
Chromatgraphy A, 804, 69 (1998).
14. A.T. Hotchkiss, K.B. Hicks, L.W. Doner, and P.L. Irwin.
Carbohydr. Res. 215, 81 (1991).
7. M. Mammen, S.-K. Choi, and G.M. Whitesides. Angew.
Chem. Int. Ed. 37, 2754 (1998).
15. A.T. Hotchkiss, S.L. Lecrinier, and K.B. Hicks. Carbohydr.
Res. 334, 135 (2001).
8. G.D. Armstrong, P.C. Rowe, P. Goodyer, E. Orrbine, T.P. Klassen,
G. Ells, A. Mackenzie, H. Lior, C. Blanchard, F. Auclair,
B. Thompson, D.J. Rafter, and P.N. McLaine. J. Infect. Dis.
171, 1042 (1995).
9. J. Ohlsson and G. Magnusson. Carbohydr. Res. 329, 49 (2000).
10. J. Dahmen, T. Freid, G. Magnusson, G. Noori, and A.-S. Carlstrom.
Carbohydr. Res. 129, 63 (1984).
16. M.-Z. Liu, H.-N. Fan, and Y.C. Lee. Biochimie, 83, 693
(2001).
17. Y.C. Lee. J. Chromatog. A, 720, 137 (1996).
18. J.F. McKelvy and Y.C. Lee. Arch. Biochem. Biophys. 132, 99
(1969).
19. G. Dongowski. J. Chromatography A, 756, 211 (1996).
20. Y. Zhang and Y.C. Lee. J. Biol. Chem. 274, 6183 (1999).
© 2002 NRC Canada