Liao et al.: The cell wall galactan of Catenella nipae
189
Botanica Marina
Vol. 36, pp. 189-193, 1993
The Cell Wall Galactan of Catenella nipae Zanardini
from Southern Australia
M.-L. Liao, S. L. A. Munro*, D. J. Craik*, G. T. Kraft and A. Bacic
School of Botany, University Of Melbourne, Parkville, Victoria 3052, Australia
* Victorian College of Pharmacy, Monash University, Parkville, Victoria 3052, Australia
(Accepted 4 April 1993)
Abstract
The cell wall polysaccharide of Catenella nipae Zanardini collected from southern Australia was extracted
and analysed by chemical and physical methods. Galactose and 3,6-anhydrogalactose were the major monosaccharide constituents of this preparation in a molar proportion of 6 : 4, with minor amounts of xylose also
present. No mono-O-methylgalactosyl residues were detected. The high sulphate ester content (ca. 30% as
SO3Na) and gelling in the presence of Ca2+ are indicative of a carrageenan type of polysaccharide. Infrared
and 13C-NMR spectroscopy established that the dominant component of the extracted polysaccharide preparation is iota (i)-carrageenan.
Introduction
Carrageenans are a spectrum of sulphated galactans
extracted with hot water from cell walls of some red
algae, especially of the order Gigartinales. They have
an essentially linear backbone of alternating β-(1—>4)and a-(l—»3)-linked D-galactopyranosyl residues,
which can carry various substituents in different quantities. Although some carrageenans have been found
to exhibit pyruvation (Hirase and Watanabe 1972) or
methylation (Chopin et al. 1990), usually to a low
extent, the overriding structural features are the position and degree of sulphation (for a review, see
Craigie 1990). In addition, the existence of some 1,4linked galactosyl residues in the form of 3,6-anhydrogalactose (AnGal) has a major influence on the solution properties of carrageenans.
Zablackis and Santos (1986) reported the presence of
a novel type of carrageenan in Catenella nipae Zanardini obtained from Burma. The structure of the
polymer, based mainly on infrared (IR) spectroscopy,
was proposed as having a repeating disaccharide unit
of -(l—>4)-D-galactose and oe-(l—»3)-3,6-anhydrogalactose-2-sulphate, and the carrageenan was designated as alpha (oc)-carrageenan. Interestingly, the carrageenan was shown to exhibit strong milk reactivity
at low concentration (260 ppm), making it commercially attractive for its great ability to suspend cocoa
powder in milk.
In our surveys of potentially commercial Australian
carrageenophytes, we have discovered species from
several genera that produce varying amounts of octype carrageenan. These results will be reported separately, but a particular anomaly that we describe
now concerns our reinvestigation of Australian representatives of the species from which Zablackis and
Santos (1986) initially reported α-carrageenan. Iota
(i)-carrageenan, rather than α-carrageenan, has been
identified in Catenella nipae from Victorian habitats
near the southern-most range limits of Avicennia marina (Forsk.) Vierh., the world's highest latitude mangrove species.
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Liao et al.: The cell wall galactan of Catenella nipae
Material and Methods
Monosaccharide analysis '
Plant Material
The reductive hydrolysis procedure recently developed by Stevenson and Furneaux (1991) was used to
prepare alditol acetate derivatives, which were then
identified by their gas-liquid chromatography (GLC)
retention times relative to /wy -inositol hexaacetate
and by their mass spectra. For GLC, the alditol
acetates were separated and quantified on a BPX70
(SGE, Melbourne, Australia) capillary column (25 m
χ 0.22 mm i.d.) in a Hewlett-Packard (Avondale,
PA, U.S.A.) 5890A Gas Chromatograph equipped
with a flame-ionisation detector. The initial oven temperature, 190 °C, was maintained for l min following
split injection, then raised to 260 °C at 3 °C/min, and
kept at 260 °C for 3.5 min. The detector temperature
was held at 240 °C. For combined GLC-mass spectrometry (MS), the oven temperature program was
the same as described for GLC, except that the final
temperature was kept at 260 °C for 10 min. Compounds that eluted from the GLC column were detected by electron impact (El) ionisation MS in a
Finnigan (Sunnyvale, CA, U.S.A.) 1020B GC/MS,
using the total ion current (reconstructed ion chromatogram, RIC) by scanning from m/z 100 to m/z
350 in 0.5 s.
The alga was collected from mangrove pneumatophores at Tooradin, Victoria, Australia. Infrared spectroscopy indicated that there was no change of polysaccharide type with different reproductive phases
(data not shown), so that the algal material was
analysed without any sexual differentiation.
Plants were hand cleaned of epiphytes and then rinsed
in fresh water to remove dirt and surface salts. Material was dried in an oven at 50 °C for 24 h, and then
ground to a powder using a Wiley Mill with a 40mesh screen.
Extraction of polysaccharide
The pigments of dry algal meal were extracted with
acetone, ethanol and ether according to the procedure
of Craigie and Leigh (1978). The resulting meal (5 g)
was extracted by continuous stirring in water (750
mL) at 95 °C for 1.5 h. The pH of the mixture was
monitored before and after cooking, and was found
to be in the range of 6.0 — 6.5. Diatomaceous earth
(10 g) was then added to the extract, which was subsequently filtered through Whatman paper No. 541.
The residue was extracted (500 mL, 1 h) and filtered,
two additional times. The three extracts were pooled
and refined, first through Whatman GF/A glass-fibre
filter, and then through a 0.45 μιη Millipore membrane filter. After concentrating the refined extract in
vacuo to about 800 mL, NaCl was added to a concentration of 0.1 M, and the polysaccharide was precipitated by adding 2.5 volumes of hot (60 °C) isopropanol. The precipitate was washed with 80% (v/v)
aqueous isopropanol, followed with 99.5% isopropanol, and then dried overnight in vacuo at 60 °C over
P205.
Starch digestion of extracted polysaccharide
To remove co-precipitated floridean starch, the extracted polysaccharide (1 g) was treated with amyloglucosidase (1 mg, Sigma, from Rhizopus) in aqueous
solution (200 mL), dialysed and the product recovered
by lyophilisation as described by Adams et al. (1988).
Alkali modification of amyloglucosidase-treated polysaccharide preparation
Alkali modification of the amyloglucosidase-treated
polysaccharide preparation was carried out as described by Craigie and Leigh (1978). The yield of
modified polysaccharide was about 80% of the amyloglucosidase-treated polysaccharide preparation.
Sulphate determination
Sulphate ester content of polysaccharide was quantified by the modified turbidimetric method of Craigie
et al. (1984).
Infrared (IR) spectroscopy
Films for IR analysis were prepared by evaporating
aqueous solutions of polysaccharide (10 mg in 2.5 mL
H2O) in 3.2 cm plastic dishes at 60 °C. The IR spectra
were recorded with a Βίο-Rad FTS-60A Fourier
Transform infrared (FTIR) spectrophotometer. To
compare the spectra of native and alkali-modified
polysaccharide preparations, the band at 2920 cm"1,
which is attributed to C-H and thus a good index for
total sugar content (Rochas et al. 1986), was used as
an internal reference. Only the region of the spectrum
between 1500—400 cm"1 for each preparation is
shown, which contains the structurally relevant region
for these classes of polysaccharides.
J3
C-nuclear magnetic resonance (13C-NMR) spectroscopy
The samples were dissolved in D2O (40 mg/mL). Proton decoupled 13C-NMR spectra were recorded on
Bruker AM300 spectrometer (75.5 MHz) at 80 °C
using a 5 mm inverse probe. A spectral width of 15.2
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Liao et al: The cell wall galactan of Catenella nipae
kHz was used with a 45° pulse, an acquisition time
of 0.54 s, a relaxation delay of 0.2 s, and approximately 58000 scans. Free Induction Decays (FIDs)
of 16K data points were acquired. Prior to Fourier
transformation the FIDs were multiplied by an exponential function to improve the signal to noise ratio
that resulted in 10 — 14 Hz line-broadening in the
transformed spectra. Chemical shifts (ppm) were
measured relative to internal dimethyl sulfoxide
(DMSO, 39.6 ppm) and converted to values relative
to external tetramethylsilane (TMS).
Table I. Sulphate ester content and monosaccharide composition of the amyloglucosidase-treated polysaccharide extracted
from Catenella nipae.
Sample
Native
Alkali-treated
Sulphate
ester3
(% dry wt.)
31.3
29.4
Constituent sugars
(mole %)b
Gal
AnGal
57.0
57.3
41.2
42.7
Xyl
1.8
* trace
a
Expressed as SO3Na.
Gal, galactose; AnGal, 3,6-anhydrogalactose; Xyl, xylose;
trace, < 0.8%.
b
Optical rotation determination
Optical rotation measurement was done at room temperature in a Perkin-Elmer D-polarimeter using 0.3%
(w/v) aqueous solutions of polysaccharide.
Rheological properties
The gelling behavior was determined on a 2% solution
of the polysaccharide preparation containing 0.2%
CaCU and visually monitored.
Results and Discussion
Extraction of de-pigmented algal meal produced 60%
crude polysaccharide, which was treated with amyloglucosidase to remove small amounts of co-precipitated floridean starch. The monosaccharide composition and sulphate ester content of the amyloglucosidase-treated polysaccharide preparation are listed in
Table I. Galactose and 3,6-anhydrogalactose (AnGal)
were the major constituent sugars in a molar proportion of 6 : 4. A small amount (1.8 mole %) of xylose
was present in the native sample, but was destroyed
or removed after alkali treatment. Only trace amounts
of glucosyl residues and no mono-0-methylgalactosyl
residues were detected. Hot alkali treatment has been
used on phycocolloids for the conversion of 1,4-linked
galactosyl residues bearing sulphate ester group at
C(O)6 position to the corresponding AnGal residues
(Rees 1961). After this treatment, the sulphate ester
content of the polysaccharide extract decreased from
31.3 to 29.4%, whereas the AnGal content increased
slightly, indicating the presence of only low quantities
of precursor residues in the native polysaccharide.
In the presence of Ca2+, 2% solutions of both native
and alkali-treated polysaccharides gave resilient gels,
which showed no syneresis during a 1-month storage
at 4 °C. The monosaccharide composition, high sulphate ester content, gelling capacity in the presence
of Ca2+, as well as a positive optical rotation ([oc]D
+ 59°) suggested a carrageenan type of polysaccharide from C. nipae.
The FTIR spectra (Fig. 1) of the polysaccharide from
C. nipae exhibited the typical triplet pattern characteristic of an iota (i)-carrageenan (Stancioff and Stanley 1969): a peak at 805 cm"1 (axial 2-sulphate of the
AnGal), a peak at 845 cm""1 (axial 4-sulphate of the
1,3-linked galactose), and a peak at 930 cm"1
(AnGal). In addition, a broad band at 1240 cm"1
(total ester sulphate) indicated the relatively high sulphate ester content of the polysaccharide. The decrease of absorbance around 820 — 830 cm"1 region
and a concomitant increase of the absorbance at 930
cm"1 following alkali-treatment reflected the conversion of some 1,4-linked galactose-2,6-bisulphate precursor to the corresponding AnGal-2-sulphate.
The 13C-NMR spectrum (Fig. 2) had the 12 peaks
expected for t-carrageenan. The assignments are consistent with data reported by Greer et al. (1985).
Nu(v)-carrageenan, the precursor of t-carrageenan,
was reported to give two anomeric carbon signals at
98.4 and 104.8 ppm (Greer and Yaphe 1984). No such
signals were evident in the anomeric region for the
native polysaccharide of C. nipae (spectrum not
shown), indicating, if present, the precursor was at
too low a concentration to be detected by 13C-NMR.
This result is consistent with the monosaccharide composition data shown in Table I. However, the spectra
of both native and alkali-treated polysaccharides contained several minor resonances, which could indicate
the presence of low levels of other substitution patterns. This is not unusual, since most of the carrageenophytes yield two or more recognisable carrageenan types (Craigie 1990). Minor resonances Xi
and X2 were observed at 67.4 (6.0 ppm downfield
from G6) and 66.5 ppm (5.7 ppm upfield from G4),
respectively. These two resonances, which were also
present in the native polysaccharide and survived the
alkali treatment, are tentatively assigned to C6 and
C4 of the 1,3-linked, 6-sulphated galactopyranosyl
residues by comparison with data reported by Usov
and Shashkov (1985) and Mollion et al. (1986). Further work is required to isolate or enrich this minor
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Liao et al.: The cell wall galactan of Catenella nipae
1500
1400
1300
1200
1100
1000
900
800
Wavenumbers (cm-i)
700
600
500
400
Fig. 1. FTIR spectra of the amyloglucosidase-treated polysaccharide extracted from Catenella nipae. A, native; B, alkali-treated.
100
I
90
1
ι
60
PPM
ι '
70
[
60
ι ·
50
13
Fig. 2. C-NMR spectrum of the amyloglucosidase- and alkali-treated polysaccharide from Catenella nipae. G and A denote
carbon atoms of -(l—»4)-linked D-galactose-4-sulphate and oc-(l—>3)-linked 3,6-anhydro-D-galactose-2-sulphate, respectively. X{
and X2 indicate minor signals that are tentatively assigned and have not been confirmed.
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Liao et αϊ.: The cell wall galactan of Catenella nipae
carrageenan component by fractionation and confirm
its fine structure by chemical and 13C-NMR procedures.
The results obtained from chemical analyses and
physical characterisation clearly indicated that i-carrageenan was the dominant cell wall polysaccharide
of C. nipa from southern Australia. This would suggest the need to reexamine, if available, the voucher
specimen of C. nipae obtained from Burma by Zablackis and Santos (1986), in order to verify its taxonomy and perform more.detailed analysis of the
193
carrageenan. It is interesting to note that the carrageenan extracted from the Burmese alga does not gel
in the presence of Ca2+, indicating different rheological properties between the carrageenans isolated from
the Burmese specimen and the Australian specimen.
Acknowledgements
We wish to thank E. Lau for her technical support.
This work was supported by a grant from the Australian Research Council.
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