Purification and structural data of a highly substituted exopolysaccharide from Pseudomonas stutzeri AS22

Article abstract of DOI:10.1016/j.carbpol.2014.06.003

Pseudomonas stutzeri AS22, when grown on media containing starch and yeast extract and incubated at 30 °C and 200 rpm for 24 h, was found to produce an acidic and high-molecular mass exopolysaccharide (EPS22). The EPS22 was purified and a yield of 1.3 g/l was achieved. The average molecular mass of the EPS22 was determined by high-performance size-exclusion chromatography (HPSEC) and showed an average molecular mass of 9.9 × 10⁵ Da and a polydispersity index M_w/M_n (M_w, weight-average and M_n, number-average) of 1.197 ± 0.015. Structural data of this EPS22 were determined using a combination approach including monosaccharide composition (HPAEC-PAD and GLC), methylation analysis (GC-MS) and NMR spectroscopy analysis. EPS22 was found to be a complex heteropolysaccharide with a repeating unit mainly composed of glucose, mannose and lactyl rhamnose in a molar ratio of 1:1.1:0.7. The acidic nature of the polysaccharide is due to the presence of three non-osidic substituents consisting of a lactyl, acetyl, and pyruvyl groups.

Full text of DOI:10.1016/j.carbpol.2014.06.003

Carbohydrate Polymers 112 (2014) 404–411
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Purification and structural data of a highly substituted
exopolysaccharide from Pseudomonas stutzeri AS22
Hana Maaleja, Claire Boissetb, Noomen Hmideta,∗, Laurine Buonb,
Alain Heyraudb, Moncef Nasria
a Laboratory of Enzyme Engineering and Microbiology, Sfax-University, National School of Engineering of Sfax (ENIS), BP 1173, Sfax 3038, Tunisia
b Centre de Recherches sur les Macromolécules Végétales, C.N.R.S., Université Joseph Fourier, BP 53, Grenoble Cedex 9 38041, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Pseudomonas stutzeri AS22, when grown on media containing starch and yeast extract and incubated at
30 ◦C and 200 rpm for 24 h, was found to produce an acidic and high-molecular mass exopolysaccharide
(EPS22). The EPS22 was purified and a yield of 1.3 g/l was achieved.
Received 18 April 2014
Received in revised form 23 May 2014
Accepted 2 June 2014
The average molecular mass of the EPS22 was determined by high-performance size-exclusion chro-
matography (HPSEC) and showed an average molecular mass of 9.9 × 105 Da and a polydispersity index
Mw/Mn (Mw, weight-average and Mn, number-average) of 1.197 0.015. Structural data of this EPS22 were
determined using a combination approach including monosaccharide composition (HPAEC–PAD and
GLC), methylation analysis (GC–MS) and NMR spectroscopy analysis. EPS22 was found to be a complex
heteropolysaccharide with a repeating unit mainly composed of glucose, mannose and lactyl rhamnose
in a molar ratio of 1:1.1:0.7. The acidic nature of the polysaccharide is due to the presence of three
non-osidic substituents consisting of a lactyl, acetyl, and pyruvyl groups.
Available online 10 June 2014
Keywords:
Pseudomonas stutzeri AS22
Purification
Exopolysaccharide EPS22
High molecular mass
Structural data
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
and animals (Donot et al., 2012). The genus Pseudomonas can be
included among the potential sources of promising biopolymers.
The ability to produce exopolysaccharides (EPSs) with greatly
varying composition and properties is a well-known biological
phenomenon among bacteria (Singha, 2012). On the basis of
monosaccharide composition in their backbone, bacterial EPSs have
been classified into homopolysaccharides and heteropolysaccha-
rides (Donot, Fontana, Baccou, & Schorr-Galindo, 2012). While
homopolysaccharides are made up of a single type of monosac-
charide, heteropolysaccharides, which constitute the majority of
bacterial EPSs, are composed of several types of monosaccharides as
well as non-osidic components (Donot et al., 2012; Notararigo et al.,
2013) leading therefore to a great structural diversity. Although the
acidic nature of some of these polymers is most often due to the
presence of uronic acids, other acidic substituents such as lactate,
pyruvate, succinate and acetate may also be present.
For example, alginate produced by P. aeruginosa and P. men-
docina, levan from P. fluorescens, succinoglycan synthesized by P.
marginalis, hyaluronan from P. aeruginosa and recently GalactoPol
synthesized by P. oleovorans, have gained considerable commercial
interest (Freitas, Alves, & Reis, 2011).
Increasing attention is being paid to such molecules because
of their physical properties such as gelling, stabilizing, thickening
and emulsifying, making them suitable for numerous commer-
cial applications in food industries (Freitas et al., 2011). Thanks to
their bioactive role such as anti-tumor (Okutani, 1984), immuno-
stimulating (Okutani, 1992), anticoagulant (Colliec et al., 2001),
hypoglycemic (Dahech et al., 2011) and antioxidant activities, these
biopolymers exhibit therefore large applications in biomedical, bio-
pharmaceutical and cosmetic industries.
The interest in the exploitation of microorganisms for the
production of valuable polysaccharides has greatly increased in
recent years, since these biopolymers often show advantages over
other natural polysaccharides derived from plants, marine algae
Because technological applications and biological properties of
exopolysaccharides are quite dependent on their structural proper-
ties, the determination of the chemical composition and structure
of an EPS is relevant for predicting its potential applications. There-
fore, in order to investigate the biological nature of the EPS22
secreted by Pseudomonas stutzeri AS22 for future applications, we
present in this manuscript the purification and structural data of
this polysaccharide.
∗
Corresponding author. Tel.: +216 22 248933; fax: +216 74 275 595.
E-mail address: hmidet noomen@yahoo.fr (N. Hmidet).
http://dx.doi.org/10.1016/j.carbpol.2014.06.003
0144-8617/© 2014 Elsevier Ltd. All rights reserved.

H. Maalej et al. / Carbohydrate Polymers 112 (2014) 404–411
405
2. Materials and methods
(TMS). Briefly, these derivatives were prepared by methanoly-
sis of 400 ␮g of EPS22 (3 N MeOH/HCl; 4 h at 110 ◦C), followed
by TMS-derivatisation performed with bis(trimethylsilyl)tri-
fluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS)
reagents in dry pyridine, at 80 ◦C for 20 min. After evaporation
to dryness under a stream of nitrogen gas, per-O-TMS-glycoside
samples were recovered from the dichloromethane organic phase
before injection. GLC analysis of the TMS methyl glycosides was
performed on an Agilent 6850 Series GC System, equipped with
a HP-5MS column (30 m × 0.25 mm) (Agilent Technologies, Palo
Alto, CA, USA) and using He as carrier gas and a Flam Ionization
Detector.
2.1. Production, isolation and purification of EPS22
The production of EPS22 by P. stutzeri AS22 was performed
on liquid medium composed of (g/l): starch 10, yeast extract 5
(ultrafiltered to remove molecular species larger than 3 × 104 Da
especially mannan), MgSO4 (7 H2O) 0.1, K2HPO4 1.4, KH2PO4 0.7
and NaCl 0.5. The medium was adjusted to pH 8.0. The strain was
cultured in 25 ml medium in 250 ml conical flasks maintained at
30 ◦C and incubated with agitation at 200 rpm for 24 h. Bacterial
cells were separated from the EPS preparation by dilution and cen-
trifugation (13,000 rpm for 15 min at 4 ◦C) of the culture broth. The
supernatant, containing the EPS fraction, was filtered under vac-
uum successively through Sartorius cellulose nitrate filters of 8.0,
5.0, 3.0, 1.2, 0.8 and 0.45 ␮m pore size (in diameter) to eliminate
cells and large cellular fragments and to give therefore the crude
exopolysaccharide. Then, to remove simple carbohydrates from
culture broth prior to the quantification of exopolysaccharides,
ultrafiltration (UF) using a 300 kDa cut-off cellulose membrane
was used. Finally, the exopolysaccharide fraction, collected from
the retentate, was dialyzed against distilled water, evaporated and
lyophilized and used for further study as the purified EPS22. The
purified EPS22 dry weight measurement was used as an indication
of the EPS yield, expressed as grams of polymer dry mass per liter
of fermented medium.
2.2.4. Methylation analysis
A sample of EPS22 (10 mg) was methylated by the method
of Hakomori (1964). After methylation, the reaction mixture was
diluted with water, dialyzed and freeze-dried. The methylation
procedure was repeated twice following the method of Purdie
and Irvine (1903). Methylated polysaccharide was hydrolyzed
with 90% formic acid at 100 ◦C for 2 h and then heated with 2 M
trifluoroacetic acid for 3 h at 100 ◦C. Methylated sugars were con-
verted into their alditol acetates (Albersheim, Nevins, English,
& Karr, 1967) and analyzed by GC-MS. Peaks identification was
based on their typical electron-impact break down profiles and
retention times compared to partially methylated alditol acetates
standards.
GC-MS analyses were performed using an Agilent 6850 Series
GC System coupled with an Agilent 5975 C MSD mass spectrometer
(France) connected to a capillary SP2380 column (30 m × 0.25 mm)
(Supelco), using He as carrier gas. The GC oven temperature was set
to 150 ◦C and held for 2 min, then increased to 240 ◦C at 3 ◦C/min
and held for 5 min. For MS detection, the ion source and transfer
line temperatures were set at 250 ◦C, with electron ionization (EI)
mode at 70 eV.
2.2. Structural characterization of EPS22
2.2.1. Molecular mass determination
The average molecular mass of the EPS22 was determined by
high-performance size-exclusion chromatography (HPSEC) using
Shodex OHpak SB-804 HQ and SB-805 HQ columns in series,
coupled to a differential refractometer, a multi-angle laser light-
scattering detector (MiniDAWN, Wyatt Technology, Dawn) and a
viscometer (Waters). EPS was separated with 0.1 M NaNO3 as elu-
ent.
2.2.5. Partial acid hydrolysis
Exopolysaccharide (20 mg/ml) was hydrolyzed with TFA 1 N at
100 ◦C for 0, 5, 15, 30, 60 and 240 min. After analysis of the different
hydrolysates, oligosaccharides obtained from 5 min EPS22 hydrol-
ysis were then fractionated by gel-filtration chromatography on a
Bio-Gel P2 column (1.5 cm × 200 cm, Biorad, Richmond, California),
eluted with water at a rate of 0.5 ml/min. The different collected
fractions were freeze dried before analysis by mass spectrometry
and NMR spectroscopy.
2.2.2. General procedures
Total carbohydrate content of the EPS22 from P. stutzeri AS22
was estimated by the phenol-sulfuric acid calorimetric method
(Dubois, Gilles, Hamilton, Rebers, & Smith, 1956).
Protein content was calculated according to the Bradford’s
method (1976).
Elemental analysis on the carbon (C), nitrogen (N), phosphorus
(P), sodium (Na), chloride (Cl) and sulfur (S) weight percentages
of the purified EPS22 was realized using an elemental analyzer
(SCA Instrumentation, CNRS Solaize, France) at “Service Central
d’Analyses” (SCA) (CNRS, Solaize-France).
2.2.6. Electrospray ionization mass spectrometry (ESI-MS)
ESI mass spectra, in the positive-ion mode, were recorded on a
ZQ Waters micromass spectrometer (capillary 3.5 kV, cone voltage
80 V).
2.2.3. Glycosyl composition analysis using HPLC and GLC
The monomer composition of the P. stutzeri exopolysaccharide
was determined after complete acid hydrolysis in acidic conditions.
The purified EPS22 (4 mg/ml) was treated with 2 N trifluoroacetic
acid (TFA) containing myo-inositol (1.05 mg/ml) at 100 ◦C for 4 h.
The identity of the released monomers was determined using
high performance anion exchange chromatography (HPAEC) with
pulsed amperometric detection (PAD) on a Dionex CarboPac PA1
column (4 mm × 250 mm). Neutral monosaccharides were eluted
isocratically using 18 mM of sodium hydroxide at a flow rate of
0.7 ml/min. The ratio of monomers was determined by comparison
of the detector response to calibration standards of the individual
monomers.
2.2.7. Nuclear magnetic resonance spectroscopy (NMR)
NMR spectra were recorded for EPS samples that were dissolved
(2–30 mg/ml) directly in D2O in 5 mm tubes. Chemical shifts are
given relative to external tetramethylsilane (TMS) (0 ppm) and cal-
ibration was performed using the signal of the residual protons of
the solvent (HOD) as a secondary reference.
1H NMR and 13C NMR experiments were recorded on a Bruker
Avance 400 spectrometer. Samples were examined as solution in
D2O at 300 K in 5 mm o.d. tube (internal acetone 1H (CH3) at 2.1 ppm
relative to Me4Si).
Two-dimensional spectra COSY (Correlation Spectroscopy) and
HMQC (HeteroMultiQuantum Coherence) were recorded using the
standard Bruker procedures.
The results were confirmed by gas–liquid chromatography
(GLC) analysis of the per-O-trimethylsilylated methyl glycosides

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H. Maalej et al. / Carbohydrate Polymers 112 (2014) 404–411
Fig. 1. Composition of EPS22 by (a) HPAEC–PAD and (b) GLC analysis after hydrolysis with TFA 2 N at 100 ◦C for 4 h.
3. Results and discussion
In the present study, the PI of EPS22 was close to 1, confirming
the homogeneity of the polymer. EPS from P. oleovorans, with a
weight-average molar mass of 1.0–5.0 × 106, have shown PI that
ranged between 1.43 and 2.15 (Freitas et al., 2009). Moreover,
Fishman et al. (1997), when studying the physical properties of
various Pseudomonas EPSs, reported a weight average molar mass
ranging from about 0.71 to 2.85 × 106 with a PI range of 1.0l–1.37.
3.1. Molecular mass estimation
The exopolysaccharide 22 (EPS22) was isolated by high-speed
centrifugation and ultrafiltration of the culture supernatant of P.
stutzeri AS22. Under these conditions, a 1.3 g/l yield of purified EPS,
estimated gravimetrically, was achieved.
The HPSEC elution profile (data not shown) showed that EPS22
from P. stutzeri AS22 was a homogeneous and high molecular mass
polymer with an average mass of 9.9 × 105 Da and a polydisper-
sity index Mw/Mn (Mw, weight-average and Mn, number-average)
of 1.197 0.015.
The EPS22 Mw value was found to be in the same range
as that of alginate (0.3–1.3 × 106), gellan (5.0 × 105), galactoPol
(1 × 106), cellulose (1.0 × 106), levan (1.0–3.0 × 106) and hyaluro-
nan (1.0–2.0 × 106) (Freitas et al., 2011; Freitas, Alves, Reis, Crespo,
& Coelhoso, 2014). Besides the molecular mass, the polydisper-
sity index (PI), which reflects the degree of heterogeneity of the
polymer’s chain lengths, is an important parameter influencing the
functional properties of polysaccharides and thereafter will deter-
mine their suitability for specific applications.
3.2. Composition analysis
Colorimetric assays of the purified EPS22 showed a total sugars
and protein amounts about 70% and 1% (w/w), respectively. Ele-
mental analysis indicated that the C and N content were 40.14%
and 1.22%, respectively. However, the S, P, and Na content were
less than 1%. In addition, the EPS22 was shown to be acidic since
it forms a precipitate in presence of cethyl trimethyl ammonium
bromide (CTAB).
The glycosyl residue composition was determined by high
performance anion exchange chromatography coupled with a
pulsed amperometric detector (HPAEC-PAD) after the total acid
hydrolysis of EPS22 and by gaz-liquid chromatography (GLC)
analysis of trimethylsilyl derivatives after methanolysis of the

H. Maalej et al. / Carbohydrate Polymers 112 (2014) 404–411
407
Fig. 2. HPAEC–PAD analysis of TFA hydrolysates obtained after different hydrolysis
conditions of EPS22.Glc: glucose; Man: mannose; X: unknown sugar.
same EPS (Fig. 1). EPS22 was found to be composed of man-
nose/glucose/rhamnose/ribose at a molar ratio of 1.1:1:0.062:0.02,
respectively. The later one, detected as trace, could probably be
originating from residual biomass. Another unknown sugar labeled
X has been detected but not identified. The absolute configura-
tion of the monosaccharides in the polysaccharide was determined
from the formation of trimethylsilylated (S)-2-butyl glycosides
and from GLC analysis. All the monosaccharides were in the
D configuration.
In the same context, it should be pointed out that degradation
of some sugars that are known to be sensitive to acidic conditions,
may occurs during the acidic treatment. To prove this, many hydrol-
ysis conditions such as 1 N TFA at 100 ◦C for 5–240 min have been
tested. As shown in Fig. 2, the unidentified sugar (X) is released after
5 min of mild hydrolysis. It was also remarkable that while the glu-
cose and mannose concentrations increased gradually from 15 min
to 4 h of hydrolysis reaction, the amount of the unknown sugar X
was decreasing. Because sensitivity to acid hydrolysis is shown for
deoxy-sugars, this result led us to suggest that the uncommon sugar
X, the first monosaccharide being released within the first 5 min, is
probably a branched deoxy sugar.
Then, the exopolysaccharide EPS22 was subjected to partial
hydrolysis with 1 N TFA for 5 min to avoid the contamination of
the unknown sugar X by glucose and mannose residues. Then, the
hydrolysate was separate by Size Exclusion Chromatography (SEC)
to isolate the monosaccharide fraction (P3) and oligosaccharides
(P1 and P2) from polysaccharide (Fig. 3a). After that, the purity
of the released monosaccharide (P3) was verified by HPAEC–PAD
analysis (Fig. 3b) and finally it was structurally characterized using
NMR and mass spectrometry experiments.
Fig. 3. (a) Elution profile on a Bio-Gel P2 column of the TFA hydrolysate obtained
after 5 min of hydrolysis of EPS22 (column 1.5 cm × 200 cm; eluent: H2O; flow rate:
30 ml/h; temperature: 55 ◦C). The P1, P2 and P3 peaks correspond to the void volume
V0, to oligosaccharides and to monosaccharides, respectively. Fractions correspond-
ing to the peak P3 are collected and pooled before being subjected to a HPAEC–PAD
analysis. (b) HPAEC–PAD analysis of the pool P3. X: the unknown sugar.
In order to confirm this hypothesis and to establish the iden-
tity of the deoxy sugar, this compound (X) was subjected to ESI-MS
analysis. The mass spectrum, reported in Fig. 4b, together with liter-
ature data, suggest the unknown sugar X to be a rhamnose residue
substituted with both acetyl and carboxyethyl substituents, in that
ions at m/z = 290 (corresponding to the sodiated parent ion) and 186
(corresponding to the sodiated rhamnose residue) were observed
(loss of 104 which was attributed to the carboxyethyl and acetyl
substituents).
These data are in good agreement with previous articles on the
structural studies of the Pseudomonas species exopolysaccharides,
where it has been reported the presence of a 1-carboxyethyl (lactic
acid) substituent. Osman and Fett (1993) reported that the car-
boxyethyl substituent was found to be linked to a glucose residue
in the case of P. marginalis strain ATCC 10844 exopolysaccharide. In
P. stutzeri strain 17588 (Osman, Fett, & Dudley, 1994) and P. stutzeri
ATCC-31258 (Hisatsuka, Ishiyama, Inoue, Tsumura, & Sato, 1984)
exopolysaccharides, the carboxyethyl substituent was found to be
linked to rhamnose residues.
The 1H NMR spectrum of the purified compound X (Fig. 4a)
showed a signal (ı 5.26 ppm) in the anomeric region suggesting
the saccharidic nature of this compound. Moreover, the high-field
region of the 1H NMR spectrum contained a doublet signal at 1.36
and 1.37 ppm, typical for CH3-groups of 6-deoxy sugars. We also
observe the presence of a signal at ı 1.25 which may be attributed
to the CH3-group of a non-osidic substituent.
3.3. Glycosyl linkage analysis
According to previous studies on Pseudomonas species and espe-
cially P. stutzeri exopolysaccharides, the acidic nature of these EPSs
is most often due to a carboxyethyl (lactic acid) substituent, a
common feature in Pseudomonas exopolysaccharides (Fett, Osman,
Wijey, & Singh, 1993). Consequently this finding together with the
presence of a signal at 4.09 ppm which is known to be characteris-
tic of a carboxyethyl (lactyl) substituent (Pillon et al., 2010; Severn
& Richards, 1993), let us suggest the presence of such substituent
linked to a deoxy sugar.
The EPS22 was permethylated, then hydrolyzed with acid and
converted into alditol acetates, and analyzed by GC–MS (Table 1).
Consistent with the glycosyl composition analysis, glycosyl link-
age analysis showed that the EPS22 is mainly composed of
4-linked glucopyranose, 3,4-linked mannopyranose and 3-linked
rhamnopyranose in a molar ratio of 1:0.92:0.79, with minor peaks
for other sugars also being found including 3-linked mannopyra-
nose and terminally-linked rhamnopyranose in a molar ratio of
0.08:0.085.

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H. Maalej et al. / Carbohydrate Polymers 112 (2014) 404–411
Fig. 4. 1-D 1H NMR spectrum (400 MHz, 300 K) (a) and mass spectrum (b) of the purified unknown sugar X. CH3: protons from methyl group in X residue.
The presence of a 3,4-disubstituted mannopyranose and both 3-
3.4. NMR spectroscopy analysis
monosubstitued and terminal rhamnopyranosyl residues revealed
branch points and substitutions. Moreover, we can deduce that
every 10 and 8 repeating units, one mannose residue was not linked
(in C-4) to rhamnose and one rhamnose residue was not substituted
(in C-3) with lactate residue, respectively.
The 1H NMR spectrum of the native EPS22 (Fig. 5a) exhibited
in the low-field region (ı 5.5–4.4) the presence of six signals at ı
5.32, 5.2 (A and Aꢀ), 5.0 (B), 4.8 (C), 4.65 and 4.47 (D) ppm. How-
ever, the HMQC spectrum (Fig. 6a) shows that the low-intensity
Table 1
GC–MS analysis of the methylated EPS22.
Type of sugar
PMAAa
Mode of linkage
Molar ratiob
Rhamnose
Mannose
Rhamnose
Glucose
1,3,5-Tri-O-acetyl-1-deuterio-6 deoxy 2,4-di-O-methyl d-rhamnitol
1,3,5-Tri-O-acetyl-1-deuterio-2,4,6-tri-O-methyl d-mannitol
1,5-Di-O-acetyl-1-deuterio-6 deoxy 2,3,4-di-O-methyl d-rhamnitol
1,4,5-Tri-O-acetyl-1-deuterio-2,3,6-tri-O-methyl d-glucitol
1,3,5-Tri-O-acetyl-1-deuterio-2,4,6-tri-O-methyl d-mannitol
→3)-Rha-(1→
→3)-Man-(1→
Rha-(1→
0.79
0.08
0.085
1
→4)-Glc-(1→
→3,4)-Man-(1→
Mannose
0.93
a
Partially methylated alditol acetate.
Results are expressed in molar ratios relative to the 1,4-linked-glucose residue.
b

H. Maalej et al. / Carbohydrate Polymers 112 (2014) 404–411
409
Fig. 5. 1-D 1H NMR (400 MHz, 300 K) (a) and 13C NMR (100 MHz, 300 K) (b) spectra of EPS22 (5 and 30 g/l in D2O). A/H1, Aꢀ/H1, B/H1, C/H1 and D/H1: H1 in A, Aꢀ, B, C and D
residues; A/C1, Aꢀ/C1, B/C1, C/C1 and D/C1: C1 in A, Aꢀ, B, C and D residues.
signal at ı 5. 2 ppm correspond to two different protons A and Aꢀ,
with 1H/13C signals at ı 5.2/98.8 and 5.2/104 ppm, respectively,
corresponding therefore to two anomeric protons with ˛ config-
uration. In addition, we note the presence of three other major
and well defined anomeric protons, two with ␣ configuration at
ı 5.0/93.13 ppm (B) and 4.8/98.8 ppm (C) and another one with
␤ configuration at 4.47/100.08 ppm (D). In contrast, the lowest
field signal at ı 5.32 ppm and the signal at ı 4.65 ppm corre-
lated with non-anomeric carbons resonating at ı 69 and 61.8 ppm,
respectively and thus couldn’t be attributed to anomeric protons.
Integration data showed that the ratios between the peak areas of
A/Aꢀ, B and C were equal to 0.3:0.79:1.08, respectively, which was
consistent with glycosyl composition and linkage analysis. Based
on these results, major components of the EPS22 repeating unit,
designated B, C and D were assigned to ␣-rhamnose, ␣-mannose
and ␤-glucose residues, suggesting that the exopolysaccharide
under study consists mainly of a trisaccharide repeating unit, in
which minor amount of two other residues (A and Aꢀ) could be
present.
In the high-field region of the 1H NMR spectrum, the signal at
ı 2.18 was attributed to the CH3 of an acetyl group, whereas the
methyl signals in the 1.1–1.4 ppm region were assigned to a deoxy
sugar (1.22–1.24 ppm) and non-osidic substituents (1.28 ppm),
which were identified, based on the results described above and
1D/2D NMR analysis, as a rhamnose residue and both carboxyethyl
(lactyl) and pyruvyl substituents, respectively.
The 13C NMR spectrum (Fig. 5b) showed a signal at 15.15 ppm
which was correlated with methyl proton signal at 1.28 ppm in
HMQC spectrum (Fig. 6a), and another one at 179.22 ppm which
was assigned to a carboxyl group. The COSY spectrum (Fig. 6b)
showed correlation between the methyl protons CH3 at 1.28 ppm
and CH at 4.17 ppm. Taken together, these results confirmed the
presence of a lactyl substituent (Parolis, Parolis, Niemann, & Stirm,
1988). However, the integral of the methyl signal at 1.28 ppm
was estimated to be relatively high, suggesting that such sig-
nal resulted of the overlapping signals of the lactate and another
substituent. The carbon resonances at 17.55 ppm which corre-
lated with the methyl protons CH3 at 1.28 ppm, and at 172.4 ppm

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H. Maalej et al. / Carbohydrate Polymers 112 (2014) 404–411
by the signal at 171.60 ppm, characteristic of the CO group of
such substituent. These results are consistent with the presence
of an O-acetyl group in the approximate amount of 1.5 O-acetyl
groups per sugar, estimated, thanks to the integration of protons
of acetates reported (1.9–2.2 ppm) to the integration of all H-1 sig-
nal integrations (4.6–5.2 ppm) (Tavernier et al., 2008), suggesting
therefore a highly acetylated EPS22. Consequently, the signals at
5.32 and 4.65 ppm, which were not observed in the deacetylated
EPS (data not shown), were assigned to ring protons attached to O-
acetyl groups. As a result of the O-acetylation chemical shift effect,
these signals shifted into the anomeric region. Since the signal at
5.32 ppm was attributed to H-2 of rhamnose as shown is the COSY
spectrum, this indicated the presence of an O-acetyl substituent
linked to the C-2 of rhamnose, as previously suggested after NMR
and MS analysis of the purified sugar X. Concerning the signal at
4.65 ppm, it correlated according to the HMQC spectrum with a
carbon signal at 61.6 ppm, suggesting the glucose and/or mannose
residues to be acetylated at position 6.
Signals characteristic of 6-deoxy sugar methyl groups were
observed at 1.23 and 1.24 ppm and were assigned to the H-6 of
a rhamnose residue. This finding is in agreement with the meth-
ylation analysis results that indicated the existence of rhamnose
residues as a branch and terminal points and with previously sug-
gested structure of the unknown sugar X purified from the EPS22
acid hydrolysate. Although the complexity of the EPS22 structure
due to its high substitution degree, COSY experiments of EPS22
afforded the assignment of most of the rhamnose proton reso-
nances (Fig. 6b) and indicated a correlation between the proton of
the lactate CH group and the C-3 of the rhamnose residue B, but also
H-3 B and the carbon of the lactate CH group, thus demonstrating
the location of the lactyl group with an ether linkage at position
3 of rhamnose. Methylation analysis of the EPS22 revealed a 1,3
linked rhamnopyranose, thus supporting this mode of attachment.
In addition, the presence in few amount of a terminal rhamnopy-
ranose also confirms this statement, since a loss of the lactyl
substituent may occur during methylation and EPS hydrolysis.
Moreover, the detection of few amount of 1,3-linked mannose may
be explained by the dissociation of the terminal rhamnose from C-4
of the mannose residue and thus suggesting a Manp-(4→1)-Rhap
linkage.
Fig. 6. 2D 1H/13C HMQC (a) and 1H/1H COSY (b) spectra of EPS22.
are consistent with the presence of a pyruvate methyl and car-
boxyl groups, respectively. The overlapping signals at 1.28 ppm
prevented the estimation of the lactyl and pyruvyl substituent pro-
portions.
Unfortunately, the complexity of the EPS22 structure, due to
its high molecular mass and to the presence of three different
non-osidic substituents (lactyl, acetyl and pyruvyl) prevented the
complete assignment of the chemical shifts. The partial chemical
shift assignment of the NMR is shown in Table 2.
Besides, the 13C NMR spectrum of the native EPS showed a sig-
nal at 18.86 ppm, which correlated in the HMQC spectrum with
the methyl protons CH3 at 2.18 ppm, and thus may be attributed
to an O-acetyl substituent. This latter assignment is confirmed
Table 2
1H and 13C NMR data on the residues that occur in EPS22 polysaccharide.
Compound
Residue
Isotope
Chemical shift (ppm) at indicated position
1
2
3
4
5
6
B
C
D
1H
5.14
93.19
4.8
98.80
4.52
100.08
5.32
69.85
4.3
77
3.3
3.6
73
4
75
3.7
NI
3.4
75
3.7
NI
3.7
NI
4.4
80
3.6
NI
3.6
NI
1.2
11.4
3.8
61.6
3.9
61.6
→3)-␣-Rha-(1→
→3,4)-␣-Man-(1→
→4)-␤-Glc-(1→
13C
1H
13C
1H
13C
75
Compound
Lac
CH
CH3
CO
4.17
1.28
–
78.92
15.15
179.22
O-Ace
–
–
2.18
18.86
–
171.6
Pyr
1.28
–
17.55
172.4
Lac, lactate; Ace, acetate; Pyr, pyruvate; Rha, rhamnose; Man, mannose; Glc, glucose; NI, non-identified.

H. Maalej et al. / Carbohydrate Polymers 112 (2014) 404–411
411
On the basis of all these results, a major structural motif of the
heteropolysaccharide was assigned as follows:
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It is composed of a backbone of alternating →4)-␤-Glcp-(1→
and →3,4)-␣-Manp-(1→ residues, in which a considerable part of
the →3,4)-␣-Manp-(1→ residues is branched at the C-4 position
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substituents, lactate (carboxyethyl), acetate and pyruvate, respon-
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located due to problems of solubility related to the high molecular
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vaccines, since O-acetyl groups are important immunogenic deter-
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The research on microbial exopolysaccharides is attracting
increased attention. In this study, the EPS22 produced by P. stutzeri
AS22, was purified and its composition and structure were partially
elucidated. The EPS was an anionic, high molecular mass and highly
substituted heteropolysaccharide, mainly composed of mannose,
glucose and a carboxyethyl-substituted rhamnose in an approxi-
mate ratio of 1.1:1:0.7. Although the main sugar components of the
P. stutzeri AS22 exopolysaccharide were similar to those reported
by other researchers, the primary structure of this polymer is rather
unusual in three ways; the O-2 acetylation of the carboxyethyl
rhamnose residue, the presence of pyruvic groups and the high
acetylation degree. All these structural features of the P. stutzeri
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Acknowledgements
This work was funded by the Ministry of Higher Education
and Scientific Research of Tunisia. The authors thank Redouane
Borsali, Philippe Colin-Morel and Marie-France Marais (Centre de
Recherches sur les Macromolécules Végétales, France) for help dur-
ing the structural analysis and for valuable discussions.
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