Microstructure of poly(γ-glutamic acid) produced by Bacillus subtilis consisting of clusters of D- and L-glutamic acid repeating units

Article abstract of DOI:10.1021/jf8001262

Poly(γ-glutamic acid) (PGA) produced by a strain of Bacillus subtilis was partially hydrolyzed into various oligopeptides so that the dipeptide fraction was isolated by the preparative thin-layer chromatography. HPLC analysis was applied to the detection of each of the four stereoisomers in this fraction using chemically synthesized authentic samples. The fraction consisted of N-γ-D-glutamyl-D-glutamic acid, N-γ-L-glutamyl-L-glutamic acid, N-γ-D-glutamyl-L-glutamic acid, and N-γ-L-glutamyl-D-glutamic acid at a ratio of 5.9:6.0:1.0:1.0. On the basis of this result, a model was proposed for the microstructure of the bacterial PGA, in which D- and L-glutamic acid repeating units are alternately linked in a single chain of the molecule.

Full text of DOI:10.1021/jf8001262

J. Agric. Food Chem. 2008, 56, 4225–4228 4225
Microstructure of Poly(γ-glutamic acid) Produced by
Bacillus subtilis Consisting of Clusters of D- and
L-Glutamic Acid Repeating Units
FEI WANG,† MASAJI ISHIGURO,† MAI MUTSUKADO,‡ KEN-ICHI FUJITA,‡ AND
TOSHIO TANAKA*,‡
Suntory Institute for Bioorganic Research, 1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka
618-0024, Japan, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto,
Sumiyoshi-ku, Osaka 558-8585, Japan
Poly(γ-glutamic acid) (PGA) produced by a strain of Bacillus subtilis was partially hydrolyzed into
various oligopeptides so that the dipeptide fraction was isolated by the preparative thin-layer
chromatography. HPLC analysis was applied to the detection of each of the four stereoisomers in
this fraction using chemically synthesized authentic samples. The fraction consisted of N-γ-D-glutamyl-
D-glutamic acid, N-γ-L-glutamyl-L-glutamic acid, N-γ-D-glutamyl-L-glutamic acid, and N-γ-L-glutamyl-
D-glutamic acid at a ratio of 5.9:6.0:1.0:1.0. On the basis of this result, a model was proposed for the
microstructure of the bacterial PGA, in which D- and L-glutamic acid repeating units are alternately
linked in a single chain of the molecule.
KEYWORDS: Poly(γ-glutamic acid); Bacillus subtilis; microstructure
INTRODUCTION
of both D- and L-glutamic acids (10, 11). The existence of such
a stereochemically heterogeneous peptide unit in PGA should
be more directly demonstrated by the detection of a covalently
bound dipeptide unit of D- and L-glutamic acids in a linear chain
of PGA.
Poly(γ-glutamic acid) (PGA) is an extremely viscous polypep-
tide of glutamic acid, which elongates via the amide linkage
between the γ-carboxyl and amino groups (1). Both D- and
L-glutamic acid stereoisomers are commonly detected in the acid
hydrolysate of PGA produced by various strains of Bacillus
subtilis (2, 3). On the other hand, D-glutamic acid is predomi-
nantly detected in the hydrolysate of PGA produced by Bacillus
licheniformis and Bacillus anthracis (4, 5), whereas L-glutamic
acid is a major component of PGA produced by the alkalophilic
strain of Bacillus sp (6). More recent studies revealed the
polymerization of L-glutamic acid alone in PGAs from a
eukaryotic organism such as Hydra and a strain of an extremely
halophilic Arcahaeon (7, 8). These findings suggest the pref-
erential polymerization of either D- or L-glutamic acid residues
by PGA synthesizing enzyme with a strict specificity toward
either of these stereoisomers. We analyzed the microstructure
of PGA produced by B. subtilis F-2-01 with the aid of PGA
hydrolase that specifically hydrolyzes the linkage between
L-glutamic acid residues. A model was thus proposed for the
microstructure of the bacterial PGA, in which D- and L-glutamic
acids are copolymerized in a single chain of the molecule (9).
This model was later supported by the 13C NMR analysis of
poly(R-ethlyl γ-D,L-glutamate), which was prepared from PGA,
as well as the stereospecificity of the PGA synthetic system-
associated cell membrane of B. subtilis in the polymerization
Recent progress in peptide synthetic chemistry has enabled
the syntheses of N-γ-D-glutamyl-L-glutamic acid (D-L) and N-γ-
L-glutamyl-D-glutamic acid (L-D). We therefore attempt to
detect such stereochemically heterogeneous dipeptides in the
partial acid hydrolysate of PGA by HPLC using chemically
synthesized dipeptides as their authentic markers. A model is
proposed for the microstructure of the bacterial PGA on the
basis of the ratio of four stereoisomers constituting the dipeptide
fraction.
MATERIALS AND METHODS
Materials. Diethyl phosphorocyanidate (DEPC), 1-hydroxy-7-aza-
benzotriazole hydrochloride (HOAt), and the derivatives of D- and
L-glutamic acid (1, 2) were purchased from Watanabe Chemical
Industries, Ltd. (Hiroshima, Japan). Pd/C (20 wt %), citric acid, and
sodium bicarbonate were purchased from Nacalai Tesque Inc. (Kyoto,
Japan). PGA of B. subtilis F-2-01 is a product of Yakult Yakuhin Co.
(Tokyo, Japan), formerly manufactured by Meiji Seika Kaisha Co.
(Tokyo, Japan). Unless otherwise indicated, all commercially available
reagents were used without further purification.
NMR and MS. 1H and 13C NMR spectra were recorded respectively
at 400 and 100 MHz in CDCl3 and D2O on a JEOL JNM-EX400
spectrometer. Chemical shifts are recorded as δ values in parts per
million (ppm). Coupling constants (J) are reported in hertz (Hz). The
abbreviations of multiplicity are as follows: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; and br, broad. Infrared spectra were
* To whom correspondence should be addressed. Tel: 81-6-6605-
3163. Fax: 81-6-6605-3164. E-mail:tanakato@sci.osaka-cu.ac.jp
† Suntory Institute for Bioorganic Research.
‡ Osaka City University.
10.1021/jf8001262 CCC: $40.75  2008 American Chemical Society
Published on Web 05/20/2008

4226 J. Agric. Food Chem., Vol. 56, No. 11, 2008
Wang et al.
Scheme 1. Syntheses of Four Stereochemical Isomers of Glutamic Acid Dimer
Scheme 2. Syntheses of F-moc Derivatives of D-D, L-L, D-L, and L-D
recorded on a Shimadzu IRPrestige-21 spectrometer. The absorption
bands of IR spectra are in cm-1. High-resolution mass spectra (HRMS)
were acquired using a Shimadzu LCMS-IT-TOF mass spectrometer.
Syntheses of F-moc Derivatives of N-γ-D-Glutamyl-D-glutamic
acid (D-D), N-γ-L-Glutamyl-L-glutamic acid (L-L), D-L, and L-D.
Syntheses of Four Dimers of Glutamic Acid (Scheme 1). To a solution
of 1 (111.4 mg, 0.3 mmol) and 2 (98.2 mg, 0.3 mmol) in anhydrous
dichloromethane were added diethyl phosphorocyanidate (DEPC) (58.7
mg, 0.36 mmol) and 1-hydroxy-7-azabenzotriazole hydrochloride
(HOAt) (44.9 mg, 0.33 mmol) at 0 °C. The resulting mixture was stirred
at room temperature overnight. The reaction mixture was diluted with
ethyl acetate, and the organic layer was washed with 10% citric acid
(50 mL × 2), saturated sodium bicarbonate (50 mL × 2), and brine
(50 mL); dried over anhydrous magnesium sulfate; and concentrated
under vacuum. Flash chromatography afforded 3 in 93-96% yield.
N-(O-Benzyl-N-benzyloxycarbonyl-D-γ-glutamyl)-D-glutamic acid diben-
zyl ester: Yield ) 192 mg (94%). 1H NMR (400 MHz, δ ppm, CDCl3):
1.92-2.03 (2H, m, CH2CH), 2.19 (4H, br, CH2COO), 2.29-2.44 (2H,
m, CH2CH), 4.38 (1H, br, CH), 4.60 (1H, br, CH), 5.03-5.17 (8H, m,
CH2Ph), 5.62 (1H, br d, J ) 7.2 Hz, NH), 6.31 (1H, br d, J ) 6.4 Hz,
NH), 7.12-7.35 (20H, m, Ar-H). 13C NMR (100 MHz, δ ppm, CDCl3):
27.19, 28.20, 30.23, 31.99, 51.82, 53.51, 66.52, 67.06, 67.30, 67.32,
127.99 (d), 128.06 (d), 128.15 (d), 128.20 (d), 128.21 (d), 128.27 (d),
128.41 (t), 128.48 (d),128.55 (t), 135.06, 135.12, 135.62, 136.10,
156.04, 171.42, 171.46, 171.56, 172.43. IR (cm-1): 3315 (NH), 2960
(CH), 1732 (ester CdO), 1690 (amide CdO), 1647 (amide CdO), 1541
(NH), 1449, 1389, 1275, 1171, 1059, 960. HRMS (m/z): calcd. for
C39H40N2NaO9 (M + Na+) 703.2626, found 703.2640. The NMR and
HRMS spectral data were identical or fully comparable to those detected
with each of N-(O-benzyl-N-benzyloxycarbonyl-L-γ-glutamyl)-L-
glutamic acid dibenzyl ester, N-(O-benzyl-N-benzyloxycarbonyl-D-γ-
glutamyl)-L-glutamic acid dibenzyl ester, and N-(O-benzyl-N-benzy-
loxycarbonyl-L-γ-glutamyl)-D-glutamic acid dibenzyl ester.
A mixture of 3 (100 mg, 0.147 mmol) and Pd/C (20 wt %, 50 mg)
in 95% ethanol was stirred at room temperature for 24 h under hydrogen
atmosphere. The reaction mixture was filtered, and the filtrate was
concentrated under vacuum. The residue was triturated with methanol/
CHCl3 to precipitate white powder, then the white powder was dissolved
in water and freeze-dried after filtration to give 4 in 64-71% yield.
D-D: Yield ) 26 mg (64%). 1H NMR (400 MHz, δ ppm, D2O):
1.83-1.93 (1H, m, CHCH2), 1.97-2.13 (3H, m, CHCH2), 2.35 - 2.48
(4H, m, CH2COO), 3.70 (1H, t, J ) 6.8 Hz, NH2CHCH2), 4.22 (1H,
dd, J ) 4.8 Hz, 8.8 Hz, NHCHCH2). 13C NMR (100 MHz, δ ppm,
D2O): 26.98, 27.07, 31.28, 32.24, 54.03, 54.84, 174.38, 175.13, 177.14,
178.17; IR (cm-1): 3325 (NH, COOH), 2947 (CH), 1717 (CdO), 1636
(CdO), 1558 (NH), 1406, 1250, 1080. HRMS (m/z): calcd for
C10H17N2O7 (M + H+) 277.1030, found 277.1033. The NMR and
HRMS spectral data were identical or fully comparable to those detected
with each of L-L, D-L, and L-D.
9-fluorenylmethylcarbonyl chloride (F-moc Cl) (3.5 mg, 0.0135 mmol)
and NaHCO3 (22.8 mg, 0.271 mmol) were added. The reaction mixture
was stirred overnight. After removing the solvent under vacuum, the
residue was dissolved in 3 mL of hexane:2-propanol (75:25) and the
solution was filtered using a 0.45 µm membrane for HPLC.
HPLC Separation of F-moc Derivatives of D-D, L-L, D-L, and
L-D. HPLC was performed using a Waters 2690 HPLC system
equipped with a photodiode array detector and a scanning fluorescence
detector, in which F-moc derivatives were separated by a Chiralcel IA
column (4.6 × 250 mm) at 20 °C and were detected with excitation at
250 nm and emission at 335 nm. Isocratic elution was performed using
a mixture of hexane and 2-propanol (75:25) as the mobile phase at a
flow rate of 0.7 mL/min. The standard F-moc derivatives were fully
separated under the above analytical conditions, in which the derivatives
of D-D, D-L, L-D, and L-L were eluted at the retention times of 25.1,
28.9, 31.2, and 35.0 min, respectively. Their peak areas were
proportional to the quantities injected into the column in the range of
10-200 pmol with the correlation coefficients of more than 0.999. The
relative standard deviation was kept less than 3.1% of the average
quantity in three different experiments for the measurement of each
derivative.
Preparation of Dipeptide Fraction from PGA. PGA of B. subtilis
F-2-01 (50 mg) was dissolved in 5 mL of 1.0 N HCl and partially
hydrolyzed to yield various oligopeptides at 100 °C for 2.5 h. The
hydrolyzed sample was then evaporated to dryness and the resulting
pellet was dissolved in distilled water. This procedure was repeated
several times for the complete removal of HCl. Oligopeptides were
separated by preparative thin-layer chromatography using a silica gel
plate (Kieselgel 60, Merck) and a mixture of acetic acid, butanol, and
water (1:1:1) as the solvent. The silica gel layers containing each of
the glutamic acid, dipeptide, and tripeptide fractions were removed from
the plate, and these components were extracted with distilled water.
The extracts were appropriately concentrated and their purity was
analyzed by thin-layer chromatography. As shown in Figure 1, the
spots of glutamic acid, dipeptides, and tripeptides were fully separated
Figure 1. Thin-layer chromatography of glutamic acid (lane 3), dipeptide
(lane 4), and tripeptide (lane 5) from acid hydrolysate of PGA (lane 2).
Each sample was spotted onto a silica gel plate (Kieselgel 60, Merck)
and developed together with L-glutamic acid (G1) and L-L (G2) (lane 1
and 6) as markers. The plate was sprayed with a ninhydrin reagent and
heated at 100 °C to visualize each spot.
Syntheses of F-moc DeriVatiVes (Scheme 2). To the sample in
anhydrous methanol was added 0.5 mL of trimethylchlorosilane
(TMSCl), and the resulting mixture was stirred for 3 h. After
evaporating the solvent of the reaction mixture under vacuum, the
residue was dissolved in 5 mL of dioxane:water (1:1), and then

Microstructure of Poly(γ-glutamic acid) Produced by B. subtilis
J. Agric. Food Chem., Vol. 56, No. 11, 2008 4227
number of glutamic acid residues in each of the repeating units
can be larger than seven in few regions of PGA. Mart´ınez de
Ilarduya et al. analyzed the microstructure of poly(R-ethyl
γ-glutamate) obtained from PGA with the aid of 13C NMR and
for the first time revealed its existence as the racemic stereo-
copolymers together with a minor amount of a mixture of the
two enantiomerically pure homopolymers (10). In agreement
with our results, stereoblocks with a number-average sequence
length of 7-11 enantiomeric units were found to be the most
probable sequences existing in biosynthetic PGA.
In our previous study, the bacterial PGA was converted into
a peptide (20K-40K) in which D- and L-glutamnic acid residues
are commonly detected, with the accompanying production of
dipeptides, tripeptides, and tetrapeptides of L-glutamic acid alone
by the action of PGA hydrolase with a strict stereospecificity
toward L-glutamic acid (9). This peptide was found to contain
two or three L-glutamic acid residues at both N- and C-terminals
of the molecule. This result suggests that the enzyme can split
the bacterial PGA in the region where L-glutamic acid residues
are sequentially linked to a heptapeptide unit. As shown in
Figure 2C, however, the heptapeptide unit seems to be too short
to be the site of the enzymatic hydrolysis, since this unit most
frequently appears in the structure of PGA (Figure 2B) and its
enzymatic hydrolysis should degrade PGA to peptides much
smaller than the experimentally obtained peptide (20K-40K).
The region susceptible to the enzyme action should have a
cluster of L-glutamic acid repeating units with a more increased
size that are rarely distributed in the linear chain of PGA.
However, it remains to be solved how D- and L-glutamic acid
residues sequentially elongate to form clusters and how these
clusters are alternately linked in a linear chain of the bacterial
PGA. There seems to be a clue to the mode of polymerization
of glutamic acid residues by PGA synthesizing enzyme in the
relationship between the spatial structure of the growing PGA
molecule and the stereochemical conformation of glutamic acid
isomers incorporated into this growing chain of PGA.
Figure 2. Proposed microstructures of PGA deduced from the ratio of
D-D, L-L, D-L, and L-D. (A) D- (O) And L- (b) glutamic acid residues are
randomly linked at a ratio of 1.0:1.0 in a single chain of PGA, in which
D-D, L-L, D-L, and L-D are arranged at an approximate ratio of 1.0:1.0:
1.0:1.0. (B) D- (O) And L- (b) glutamic acids are linked in a linear chain
of PGA at a ratio of 1.0:1.0, in which D-D, L-L, D-L, and L-D are arranged
at an approximate ratio of 6.0:6.0:1.0:1.0. The arrows indicate every
combination of the sites for the complete hydrolysis of PGA into dipeptides.
(C) The mode of production of a 20K-40K peptide by the action of PGA
hydrolase is expected on the assumption that the enzyme cannot split
the regions where L-glutamic acid residues are sequentially linked to a
unit with the size of a heptapeptide. The arrows indicate possible hydrolysis
sites of PGA hydrolase.
from other components so that these extracted samples could be used
in the analysis of their compositions in terms of D- and L-glutamic
acid.
The mixture of synthetic dipeptide stereoisomers was heated in 1.0
N HCl at 100 °C for 2.5 h and analyzed for their contents by HPLC.
These stereoiomers were detected at the ratios with no significant
differences before and after hydrolysis. This indicates that the ratio of
stereoisomers in the dipeptide fraction from PGA primarily depends
on their mode of arrangements in the molecule.
ABBREVIATIONS USED
RESULTS AND DISCUSSION
PGA, poly(γ-glutamic acid); D-D, N-D-γ-glutamyl-D-glutamic
acid; L-L, N-L-γ-glutamyl-L-glutamic acid; D-L, N-D-γ-glutamyl-
L-glutamic acid; L-D, N-L-γ-glutamyl-D-glutamic acid; HRMS,
high-resolution mass spectra; DEPC, diethyl phosphorocyani-
date; HOAt, 1-hydroxy-7-azabenzotriazole hydrochloride; TM-
SCl, trimethylchlorosilane.
Glutamic acid isolated from the partial acid hydrolysate of
PGA was analyzed for its D- and L-glutamic acid contents by
HPLC according to our previously described method (9). D-
And L-glutamic acids were detected at a ratio of 1.0:1.0. The
dipeptide fraction was found to consist of D-D, L-L, D-L, and
L-D at a ratio of 5.9:6.0:1.0:1.0, and the overall ratio of D- and
L-glutamic acids therein agreed with a ratio of D- and L-glutamic
acids (1.0:1.0) obtained above. This study demonstrates the
copolymerization of D- and L-glutamic acid residues in a single
chain of PGA on the basis of the detection of D-L and L-D.
The ratio of D- to L-glutamic acid in PGA may represent equal
stereospecificities of glutamic acid ligase of the PGA synthesiz-
ing enzyme toward D- and L-glutamic acids as substrates for
polymerization.
Figure 2A shows a structure of PGA in which D- and
L-glutamic acid residues are randomly linked at a ratio of 1.0:
1.0 so that D-D, L-L, D-L, and L-D are available at an
approximate ratio of 1.0:1.0:1.0:1.0. Differently from this
structure, however, the experimental data on the ratio of these
stereoisomers indicate the existence of D- and L-glutamic acid
repeating units, which are alternately linked in a linear chain
of PGA. We thus propose a model of the structure of the
bacterial PGA (Figure 2B) in which the average numbers of
D- and L-glutamic acid repeating units are deduced to be seven
from experimental data (5.9:6.0:1.0:1.0). This means that the
LITERATURE CITED
(1) Candela, F.; Fouet, A. Poly-γ-glutamate in bacteria. Mol. Micro-
biol. 2006, 60, 1091–1098.
(2) Kubota, H.; Matsunobu, T.; Uotani, K.; Takebe, H.; Satoh, A.;
Tanaka, T.; Taniguchi, M. Production of poly(γ-glutamic acid)
by Bacillus subtilis F-2-01. Biosci. Biotechnol. Biochem. 1993,
57, 1212–1213.
(3) Ashiuchi, M.; Kamei, T.; Baek, D.-H.; Shin, S.-Y.; Sung, M.-H.;
Soda, K.; Yagi, T.; Misono, H. Isolation of Bacillus subtilis
(chungkookjang), a poly-γ-glutamate producer with high genetic
competence. Appl. Microbiol. Biotechnol. 2001, 57, 764–769.
(4) Troy, F. A. Chemistry and biosynthesis of the poly(γ-D-glutamyl)
capsule in Bacillus licheniformis. I. Properties of the membrane-
mediated biosynthesis reaction. J. Biol. Chem. 1973, 248, 305–
316.
(5) Hanby, W. E.; Rydon, H. N. The capsular substance of Bacillus
anthracis. Biochem. J. 1946, 40, 297–309.
(6) Aono, R. Characterization of structural component of cell walls
of alkalophilic strain of Bacillus sp. C-125. Biochem. J. 1987,
245, 467–472.

4228 J. Agric. Food Chem., Vol. 56, No. 11, 2008
Wang et al.
(7) Weber, J. Poly(γ-glutamic acid)s are the major constituents of
nematocysts in Hydra (Hydrozoa, Cnidaria). 1990, 265, 9664–
9669.
(8) Hezayen, F. F.; Rehm, B. H. A.; Tindall, B. J.; Steinbu¨chel, A.
Transfer of Natrialba asiatica B1T to Natrialba taiwanensis sp.
nov., a novel extremely halophilic, aerobic, non-pigmented
member of the Archaea from Egypt that produces extracellular
poly(glutamic acid). Int. J. Syst. EVol. Microbiol. 2001, 51, 1133–
1142.
(10) Mart´ınez de Ilarduya, A.; Ittobane, N.; Bermu´dez, M.; Alla, A.;
El Idrissi, M.; Mun˜oz-Guerra, S. Poly(R-alkyl γ-glutamate)s of
microbial origin. 2. On the microstructure and crystal structure
of poly(R-ethlyl γ-glutamate)s. Biomacromolecules 2002, 3, 1078–
1086.
(11) Ashiuchi, M.; Shimanouchi, K.; Nakamura, H.; Kamei, T.; Soda,
K.; Park, C.; Sung, M.-H.; Misono, H. Enzymatic synthesis of
high-molecular-mass poly-γ-glutamate and regulation of its ster-
eochemistry. Appl. EnViron. Microbiol. 2004, 70, 4249–4255.
(9) Tanaka, T.; Fujita, K.; Tekenishi, S.; Taniguchi, M. Existence of
optically heterogenous peptide unit in poly(γ-glutamic acid)
produced by Bacillus subtilis. J. Ferment. Bioeng. 1997, 84, 361–
364.
Received for review January 15, 2008. Revised manuscript received
March 25, 2008. Accepted March 28, 2008.
JF8001262