Selective phospholipid methanolysis catalyzed by a weakly acidic microbial polysaccharide, zooglan

Article abstract of DOI:10.1016/j.carres.2008.04.008

Zooglan, a weakly acidic exopolysaccharide produced by Zoogloea ramigera 115, catalyzed the preferential methanolysis of phospatidylcholine compared to other phospholipids when the reaction was carried out in pure methanol at 30 °C. The reaction was monitored by thin-layer chromatography (TLC) as well as ¹H and ³¹P nuclear magnetic resonance (NMR) spectrometry. Zooglan enhanced the rate of methanolysis of dipalmitoylphosphatidylcholine (DPPC) up to about 170-fold compared to controls such as DPPC alone, pyruvic acid, succinic acid and acetic acid. Furthermore, the methanolysis was different depending on the head groups of the phospholipids. Through this study, we have shown that zooglan can act as an environmentally benign catalytic polysaccharide for methanolysis in pure methanol solution.

Full text of DOI:10.1016/j.carres.2008.04.008

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1378–1382
Note
Selective phospholipid methanolysis catalyzed by a weakly
acidic microbial polysaccharide, zooglan
Sanghoo Lee and Seunho Jung*
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University,
1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
Received 21 December 2007; received in revised form 31 March 2008; accepted 5 April 2008
Available online 10 April 2008
Abstract—Zooglan, a weakly acidic exopolysaccharide produced by Zoogloea ramigera 115, catalyzed the preferential methanolysis
of phospatidylcholine compared to other phospholipids when the reaction was carried out in pure methanol at 30 °C. The reaction
was monitored by thin-layer chromatography (TLC) as well as 1H and 31P nuclear magnetic resonance (NMR) spectrometry. Zoo-
glan enhanced the rate of methanolysis of dipalmitoylphosphatidylcholine (DPPC) up to about 170-fold compared to controls such
as DPPC alone, pyruvic acid, succinic acid and acetic acid. Furthermore, the methanolysis was different depending on the head
groups of the phospholipids. Through this study, we have shown that zooglan can act as an environmentally benign catalytic poly-
saccharide for methanolysis in pure methanol solution.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Zooglan; Zoogloea ramigera; Catalytic polysaccharide; Catalyst; Phosphatidylcholine; Methanolysis
Zoogloea ramigera is an aerobic gram-negative bacte-
rium that is found primarily in organically enriched
aqueous environments; these organisms have the ability
to form flocks. Z. ramigera I-16-M produces no gelati-
nous matrix,1 while Z. ramigera 115 produces an extra-
cellular zoogloeal matrix in which the cells are
embedded.2 The structure of the biopolymer produced
from Z. ramigera 115 has been reported to have a pyr-
uvyl group as a substituent and is composed of glucose
and galactose as shown in Figure 1A. The polysaccha-
ride has a molecular weight of about 105.3–7 On the
other hand, the polysaccharide produced by the Z. ram-
igera 115SLR mutant has been reported to have more
varied substituents than does the wild type organism,
for example, inclusion of acetyl, succinyl and pyruvyl
groups.8
specific organic reactions has been identified. Recently,
we reported that zooglan isolated from Z. ramigera
115 catalyzes the methanolysis of the lactone ring in
4-benzylidene-2-phenyloxazolone in pure methanol.3
We also reported that some microbial carbohydrates
have catalytic potential in specific methanolysis
reactions.14–16
Methanolysis has also been performed during biodie-
sel production, using catalysts such as acids, alkalis, and
lipases.17 Biodiesel fatty acid methyl esters (FAMEs),
which are produced by the methanolysis of triglycerides
such as animal fats and vegetable oils, have been of
increasing interest as promising renewable alternative
energy sources to replace conventional fossil fuels. Thus,
environmentally benign catalysts and processes for mak-
ing the FAMEs are of importance in terms of environ-
mental protection. Thus, zooglan may have potential
as an environmentally friendly catalyst for producing
biodiesel.
With regard to physiochemical properties, zooglan
produced from Z. ramigera 115 has the ability to absorb
amino acids,9 metal ions,10–12 and cholesterol.13 In addi-
tion to these properties, a catalytic ability of zooglan for
As part of our early work in this area, we recently
reported that cyclic b-(1?2)-glucans (cyclosophoraose)
isolated from Rhizobium species catalyze the methanoly-
sis of biological phospholipids.14 However, no work on
*
Corresponding author. Tel.: +82 2 450 3520; fax: +82 2 452 3611;
e-mail: shjung@konkuk.ac.kr
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.04.008

S. Lee, S. Jung / Carbohydrate Research 343 (2008) 1378–1382
1379
A
Glc
β(1,3)
Glc
β(1,6)
β(1,3)
Glcβ(1,4) Glcβ(1,4) Gal β(1,4)Glcβ(1,4)Gal β(1,4)Glc
Gal β(1,4)Glc
β(1,3)
β(1,3)
Glc
Glc
β(1,3)
6
Glc
Pyr
Pyr
4
6
β(1,3)
Glc
30-40
4
O
B
P
O
O
R1O
R1O
P
O
OR2
O
R2O
O
Zooglan
CH3OH, 30ºC
+
O
O
CH3OR3
O
(CH2)14
O
(CH2)14
R1 = -H, -(CH2)2-N(CH3)3
R2 = H or -CO(CH2)14CH3
R3 = -CO(CH2)14CH3
Figure 1. Possible structure6 of zooglan, which is produced by Zoogloea ramigera 115 (A). Glc, Gal and Pyr represent glucose, galactose and pyruvic
acid, respectively. Schematic representation of the methanolysis reaction of DPPC catalyzed by zooglan (B).
the catalytic methanolysis of phospholipids induced by
microbial linear polysaccharides was described. In this
study, we report for the first time that zooglan, which
contains a pyruvyl group as a substituent, can function
as a catalytic polysaccharide in the production of
FAMEs when phospholipids are used as substrates.
Furthermore, the methanolysis catalyzed by zooglan
showed selectivity for the phospholipids (Fig. 1B). This
selective methanolysis process may be useful in the stud-
ies on the specific interactions between cellular carbo-
hydrates and lipids depending on head groups, and on
quantitative or qualitative analysis of a specific lipid like
phospatidylcholines among a mixture of phospholipids.
Prior to the reaction, we characterized both the iso-
lated and the commercial zooglan by FTIR spectro-
scopy. Based on the previous reports,3,18 we identified
the functional groups of the zooglan. From the spectra,
we confirmed no differences between the isolated and the
commercial zooglan. The functional groups of zooglan
were assigned as following: OH, 3445 cmÀ1; C–H,
2935–2920 cmÀ1; C@O of carboxyl groups, 1653 cmÀ1
and 1458 cmÀ1; tertiary CH–OH, 1165 cmÀ1; C–O
saccharide in pure water. A 3% aqueous solution of zoo-
glan has a pH of 6.75. This weakly acidic polysaccharide
functioned as a catalyst for the selective methanolysis of
phospholipids. Among the phospholipids tested, dipal-
mitoylphosphatidylcholine (DPPC) was preferentially
methanolyzed, while dipalmitoylphosphatidic acid
(DPPA) was a weaker substrate for the reaction.
We first identified the reactant and the products on
TLC using a colorimetric detection system composed
of an ammonium molybdate–copper solution in the
absence or the presence of zooglan (Fig. 2). After 48 h,
DPPC and DPPA were clearly converted into two spots
in the presence of zooglan, while DPPC and DPPA
alone were not (Fig. 2A). From the TLC image, we con-
firmed that dipalmitoylphosphatidylserine (DPPS) and
dipalmitoylphosphatidylethanolamine (DPPE) were
not methanolyzed by zooglan. The TLC image with
DPPA and DPPC was re-run with cospot lanes
with the known products for clarity. In Figure 2B, the
TLC image of DPPA and DPPC with cospot lanes of
the known products in the absence or the presence of
zooglan is shown. Lyso-palmitoylphosphatidic acid
and the lyso-palmitoylphosphatidylcholine were newly
observed in lanes 3 and 6, respectively. The TLC image
suggests that the intact DPPC and DPPA are converted
stretching of zooglan bonds, 950–1150 cmÀ1 3,18
.
Because the zooglan used in this study has a pyruvyl
group as a substituent, we measured the pH of the poly-

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S. Lee, S. Jung / Carbohydrate Research 343 (2008) 1378–1382
1H NMR spectrum of DPPC when zooglan was added
to a solution of the substrate dissolved in pure metha-
nol. After just 5 min, a signal corresponding to the
methoxy protons of the FAME product was clearly
observed at 3.66 ppm; the integration of this peak
increased with reaction time.
We further confirmed the reaction catalyzed by zoo-
glan through 31P NMR spectral analysis on the methan-
olysis of DPPC (Fig. 3C). In the presence of zooglan, the
reactant (DPPC) and the product (lyso-palmitoylphos-
phatidylcholine) were observed at 0.456 and 1.042 ppm
after just 5 min, respectively. After 48 h, the peaks
corresponding to the product increased further.
Because the zooglan itself is a heterogeneous biopoly-
mer, it is difficult to determine the origin of the selectiv-
ity of the methanolysis reaction. However, it is clear that
the zooglan more favorably catalyzes the methanolysis
of DPPC compared to other phospholipids. Although
a detailed mechanistic study is complicated by the high
molecular weight of zooglan and its insolubility in any
solvent, a possible reaction mechanism might be as-
sumed as follows based on the our previous study.3,14
One possibility is that either the hydroxyl groups or pyr-
uvyl groups of the zooglan, acting as a nucleophile,
might attack the ester carbonyl carbons of the DPPC,
which is bound to the polysaccharide. This process
would result in the formation of an acyl intermediate,
which would be subject to attack by the methanol as sec-
ond nucleophile thus generating the FAME product. A
second possibility is that zooglan might activate the
methanol via hydrogen bonding, resulting in the attack
of the hydroxyl group on the ester group of the DPPC.
In conclusion, we show that the natural polysaccha-
ride zooglan acts as a catalytic carbohydrate for the
selective methanolysis of the biological phospholipids.
The preferential catalytic methanolysis of zooglan
toward phosphatidylcholine was confirmed through
TLC, 1H and 31P NMR spectral analysis. Although
the exact mechanism remains unclear, zooglan definitely
has a potential to act as a catalyst for methanolysis,
which is environmentally benign compared to other
typical acid or base catalysts. Studies on other functions
of zooglan are in progress.
Figure 2. TLC image of the dipalmitoylphospholipids containing
DPPC in the absence or the presence of zooglan (3 mg) after 48 h at
30 °C (A). Lane 1, DPPS alone; lane 2, DPPS with zooglan; lane 3,
DPPA; lane 4, DPPA with zooglan; lane 5, DPPC; lane 6, DPPC with
zooglan; lane 7, DPPE; lane 8, DPPE with zooglan. The TLC image
rerun with cospot lanes with the known products for the DDPA and
DPPC in the absence or the presence of zooglan (3 mg) (B). Lane 1 is
L-a-lyso-oleoylphosphatidic acid alone as the known product, lane 2,
DPPA alone, lane 3, DPPA with zooglan, lane 4, L-a-lyso-palmitoyl-
phosphatidylcholine alone as the known product, lane 5, DPPC alone,
lane 6, DPPC with zooglan, respectively. Arrows indicate the products
newly made from the reaction mixtures in the presence of zooglan.
After the reaction mixtures were loaded and developed on TLC, the
solution of ammonium molybdate as a detecting agent was sprayed.
into lyso-palmitoylphosphatidylcholine or lyso-palmi-
toylphosphatidic acid as one of the products by zooglan
during the reaction, and then one of the fatty acyl
groups (at the position sn-1 or sn-2) is methanolyzed
to the FAME as shown in Figure 1B. To investigate
the effect of simple acids on methanolysis, we used suc-
cinic acid, acetic acid and pyruvic acid, and then moni-
tored the reaction with TLC. The acid did not have any
effect on the methanolysis of DPPC at 30 °C when an ex-
cess of the simple acid (100–170 mM) was mixed with
DPPC in anhydrous methanol (data not shown).
The catalytic reaction was also confirmed by 1H
NMR spectroscopic analysis (Fig. 3A and B). Fig. 3A
shows the conversion of DPPC and DPPA catalyzed
by zooglan with increasing reaction time, based on the
integration analysis of the reactant and the product
measured from 1H NMR spectra. In the presence of
zooglan, the methanolysis of DPPC was enhanced about
170-fold compared with DPPC alone at 30 °C. With the
results obtained from the 1H NMR spectral analysis, the
data were fitted to a single exponential to obtain a kcat
1. Experimental
1.1. Chemicals
(3.15 0.03) Â 10À2 hÀ1 and
a
kuncat of (1.85
Dipalmitoylphosphatidylcholine (DPPC), dipalmitoyl-
phosphatidylserine (DPPS), dipalmitoylphosphatidy-
lethanolamine (DPPE), dipalmitoylphosphatidic acid
(DPPA) and dipalmitoylphosphatidylglycerol (DPPG).
L-a-Lysophosphatidic acid, oleoyl and L-a-lysophos-
phatidylcholine, palmitoyl, anhydrous MeOH (99.8%),
0.05) Â 10À4 hÀ1. Also, the conversion rate of DPPA
was determined to have
a
kcat of (2.94
0.07) Â 10À3 hÀ1. DPPA alone in methanol was not con-
verted into products after 48h and so it was not possible
to calculate a rate constant. Figure 3B shows the partial

S. Lee, S. Jung / Carbohydrate Research 343 (2008) 1378–1382
1381
A
60
40
20
0
0
10
20
30
40
50
Time (h)
B
f
f
(H3C)3N
(H3C)3N
e
O
e
c
d
d
P
O
a
b
O
O
O
O
P
OR
Zooglan
RO
O
O
c
+
CH3OH, 30 °C
b
a
CH3OR
(R = H or -CO(CH2)14CH3)
O
O
O
(CH2)14
O
(CH2)14
C
Figure 3. Conversion of DPPC (d) and DPPA (j) into their product with increasing reaction time in the absence (s for DPPC alone) or the
presence of zooglan (A). No product peaks for DPPA alone in anhydrous methanol were observed. Three separate experiments were performed, and
then average values at each time were plotted. The data were fitted to a single exponential to obtain a kcat of (3.15 0.03) Â 10À2 hÀ1 and a kuncat of
(1.85 0.05) Â 10À4 hÀ1. Also, the conversion rate of DPPA was determined at a kcat of (2.94 0.07) Â 10À3 hÀ1. Partial 1H NMR spectra of DPPC
with increasing reaction time in the absence or the presence of zooglan (B). The asterisks indicate the resonances of the methoxy protons of the
methyl palmitate made from the methanolysis of DPPC catalyzed by zooglan. 31P NMR spectra of the reaction mixtures at 5 min (low view) and 48 h
(top view) when zooglan is added at 30 °C (C). All thereactions in this study were carried out in MeOH (1 mL) at 30 °C in the absence or the presence
of zooglan (3 mg). Then, the concentration of the phospholipids used was 20 mM.

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S. Lee, S. Jung / Carbohydrate Research 343 (2008) 1378–1382
pyruvic acid, succinic acid and acetic acid were pur-
chased from Sigma–Aldrich Inc. (St. Louis. MO).
under reduced pressure. 1H chemical shifts are expressed
in d (ppm) downfield from tetramethylsilane (Me4Si,
TMS), which was used as an internal standard. The time
course of the reaction was fitted to a single exponential
to obtain kcat or kuncat using the GRAPHPAD PRISM ver-
sion 4.0 (GRAPHPAD Software, Inc., San Diego, CA).
1.2. Isolation and characterization of zooglan
For the preparation of zooglan, Z. ramigera 115 (ATCC
25935) was grown in 5 L jar fermentor containing 3 L of
defined medium for 6 days at 30 °C, isolated and puri-
fied, as in our previous report.3 For this study, commer-
cial zooglan isolated from Z. ramigera 115 was also
purchased from Sigma–Aldrich Inc.
Acknowledgements
This research was supported by the National R&D pro-
ject for Biodiscovery in MOST 2004. Professor S. Jung
expresses his thank to Konkuk University for the finan-
cial support during his sabbatical year.
To characterize both the isolated and the commercial
zooglan, Fourier transform infrared (FTIR) spectro-
scopic analysis was performed. A pellet for FTIR anal-
ysis was obtained by grinding 2 mg of the zooglan with a
KBr and pressing in a mold. The FT-IR spectrum of
zooglan was obtained using a JASCO FT-IR-300E
spectrometer (USA) over the wavenumber range of
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