Microbial production of optically active β-phenylalanine through stereoselective degradation of racemic β-phenylalanine

Article abstract of DOI:10.1271/bbb.60099

The ability to produce (R)- or (S)-β-phenylalanine from racemic β-phenylalanine through stereoselective degradation was screened for. Variovorax sp. JH2 and Arthrobacter sp. the faculty of Agriculture, Kyoto University (AKU) 638 were found to be potential catalysts for (R)- and (S)-β-phenylalanine production respectively. On 192 h cultivation of Variovorax sp. in medium containing 1.0% (w/v) racemic β-phenylalanine, 0.46% (w/v) (R)-β-phenylalanine with an enantiomeric purity of 99% e.e. was obtained. The initial step of the (S)-isomer degradation was stereoselective transamination. On 312 h cultivation of Arthrobacter sp. in medium containing 1.0% (w/v) racemic β-phenylalanine, 0.51% (w/v) (R)-β-phenylalanine with an enantiomeric purity of 90% e.e. was obtained. The initial step of the (R)-isomer degradation was supposed to be oxidative deamination. Resting cell reaction with vigorous shaking, with cells of Arthrobacter sp. as the catalyst, resulted in production of 0.49% (w/v) of (S)-β-phenylalanine with an enantiomeric purity of 99% e.e. from 1.0% (w/v) racemic β-phenylalanine in 45 h.

Full text of DOI:10.1271/bbb.60099

Biosci. Biotechnol. Biochem., 70 (8), 1941–1946, 2006
Microbial Production of Optically Active ꢀ-Phenylalanine
through Stereoselective Degradation of Racemic ꢀ-Phenylalanine
Junichi MANO, Jun OGAWA,y and Sakayu SHIMIZU
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
Received February 20, 2006; Accepted April 16, 2006; Online Publication, August 1, 2006
[doi:10.1271/bbb.60099]
The ability to produce (R)- or (S)-ꢀ-phenylalanine
from racemic ꢀ-phenylalanine through stereoselective
degradation was screened for. Variovorax sp. JH2 and
Arthrobacter sp. the faculty of Agriculture, Kyoto
University (AKU) 638 were found to be potential
catalysts for (R)- and (S)-ꢀ-phenylalanine production
respectively. On 192 h cultivation of Variovorax sp. in
medium containing 1.0% (w/v) racemic ꢀ-phenylala-
nine, 0.46% (w/v) (R)-ꢀ-phenylalanine with an enantio-
meric purity of 99% e.e. was obtained. The initial step
of the (S)-isomer degradation was stereoselective trans-
amination. On 312 h cultivation of Arthrobacter sp. in
medium containing 1.0% (w/v) racemic ꢀ-phenylala-
nine, 0.51% (w/v) (R)-ꢀ-phenylalanine with an enantio-
meric purity of 90% e.e. was obtained. The initial step
of the (R)-isomer degradation was supposed to be
oxidative deamination. Resting cell reaction with vig-
orous shaking, with cells of Arthrobacter sp. as the
catalyst, resulted in production of 0.49% (w/v) of (S)-ꢀ-
phenylalanine with an enantiomeric purity of 99% e.e.
from 1.0% (w/v) racemic ꢀ-phenylalanine in 45 h.
optically pure BPA is theoretically 50%, which limits
the efficiency of the process. We expected that there
must be a more efficient microbial process for optically
pure BPA-production that gives a higher production
yield, and hence investigated microbial reactions in-
volving BPA. We collected BPA-assimilating micro-
organisms and analyzed their BPA metabolism. Through
this investigation, we discovered microorganisms that
assimilate BPA with high enantioselectivity. Their BPA-
degradation did not permit optically active BPA pro-
duction with a yield of more than 50%, but, they were
useful for convenient production of optically active BPA
through stereoselective degradation of racemic BPA
without the tedious chemical esterification step required
for the enzymatic chemical resolution of BPA esters.6–8)
Furthermore, their BPA-degradation was expected to
involve unique enzymes that would give a higher
production yield.
In this report, we describe the results of screening of
microorganisms degrading BPA stereoselectively, ap-
plication of the potential strains for the microbial
production of optically active BPA, and biochemical
analysis of the reactions involved in their BPA-degra-
dation.
Key words: ꢀ-amino acid; ꢀ-phenylalanine; chiral reso-
lution; Variovorax; Arthrobacter
ꢀ-Phenylalanine (3-amino-3-phenylpropionic acid,
BPA) is a ꢀ-amino acid present in several bioactive
molecules, such as antibiotics and enzyme inhibitors.1,2)
Because of its bioactivity, optically active BPA and its
derivatives are becoming important chiral building
blocks for the synthesis of pharmaceutical agents. For
example, (S)-BPA is a chiral building block of fibri-
nogen receptor antagonists.3,4)
Materials and Methods
Chemicals. DL-3-Amino-3-phenylpropionic acid
(BPA) was purchased from Ardrich Chemical (Milwau-
kee, WI). 3-Oxo-3-phenylpropionic acid (benzoyl acetic
acid, BAA) was a kind gift from Kaneka (Osaka, Japan).
All other chemicals used were of analytical grade and
were commercially available.
We have reported screening for microorganisms that
hydrolyze BPA ethyl ester stereoselectively and have
developed a process for the production of optically pure
BPA ethyl ester with cells of the selected strains as
biocatalysts.5) By this method, the maximal yield of
Isolation of BPA-assimilating microorganisms. Small
amounts of soil samples were added to test tubes (16 ꢀ
165 mm) containing 5 ml of isolation liquid medium
comprising 0.1% (w/v) KH2PO4, 0.1% (w/v) K2HPO4,
y
To whom correspondence should be addressed. Fax: +81-75-753-6128; E-mail: ogawa@kais.kyoto-u.ac.jp
Abbreviations: AKU, the faculty of Agriculture, Kyoto University; BAA, benzoyl acetic acid; BPA, ꢀ-phenylalanine; e.e., enantiomer excess;
GITC, 2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranosyl isothiocyanate; HPLC, high-performance liquid chromatography; 2-OG, 2-oxoglutaric acid; PLP,
pyridoxal-50-phosphate; UV, ultraviolet

1942
J. MANO et al.
.
0.03% (w/v) MgSO4 7H2O, 0.01% (w/v) yeast extract,
0.2% (w/v) NH4Cl, and 0.15% (w/v) racemic BPA
(9.1 mM) (pH 7.0), followed by incubation at 28 ꢁC with
shaking (300 strokes/min) for 1–2 d. Fifty ml of me-
dium, in which the growth of microorganisms was
observed, was transferred to another test tube containing
the same medium, followed by further cultivation at
28 ꢁC with shaking (300 strokes/min) for 1–2 d. The
culture media were streaked onto agar plates with the
same composition as the isolation medium and culti-
vated at 28 ꢁC for 2–3 d. After cultivation, the colonies
that appeared were isolated as BPA-assimilating micro-
organisms. Microorganisms obtained from the culture
collection of the Faculty of Agriculture of Kyoto
University (AKU) were also inoculated onto the iso-
lation media, and strains with the ability to assimilate
BPA were selected by the same methods. These BPA-
assimilating strains from soil samples and the culture
collection were used for the subsequent experiments.
20 mM BPA was incubated at 28 ꢁC with or without
shaking (300 strokes/min). After 10 h reaction, the
decomposition of BPA was assayed as described
below.
Analytical methods. Qualitative analysis of BPA,
BAA, and L-glutamic acid was performed by thin-layer
chromatography (TLC) with Kieselgel 60 F254 (Merck,
Rahway, NJ). The developing system consisted of n-
butanol, acetic acid, and H2O in a ratio of 4:1:1 (v/v/v).
Detection was carried out with 0.2% (v/v) ninhydrin in
acetone and ultra-violet (254 nm). Quantitative analysis
of BPA was performed by reverse-phase high-perform-
ance liquid chromatography (HPLC) with a Shimadzu
LC-VP system (Shimadzu, Kyoto, Japan) equipped
with a Cosmosil 5C8-MS column (0:46 ꢀ 250 mm;
Nacalai Tesque, Kyoto, Japan). The mobile phase
was acetonitrile–water (9:1 by vol., pH 2.0, adjusted
with phosphoric acid) at a flow rate of 1.0 ml/min, and
the effluent was monitored by means of 210 nm ultra-
violet (UV) detection. For detection of BAA, the same
HPLC method was used. Liquid chromatography–mass
spectrometry (LC–MS) analysis was carried out under
the condition described above with LC–MS 2010A
(Shimadzu).
Determination of the enantiomeric purity of BPA was
carried out by reverse-phase HPLC with a Shimadzu
LC-VP system equipped with a Cosmosil 5C18-AR-II
column (0:46 ꢀ 250 mm; Nacalai Tesque) after deriva-
tization with 2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranosyl
isothiocyanate (GITC).9–11) The mobile phase was
methanol–water (55:45 by vol., pH 2.5, adjusted with
phosphoric acid) at a flow rate of 1.0 ml/min, and
the effluent was monitored by means of 250 nm UV
detection. For detection of ammonia, the same HPLC
method was used after derivatization with GITC. For
detection of L-glutamic acid, the same HPLC method
was used after derivatization with GITC except that
methanol–water (40:60 by vol., pH 2.5, adjusted with
phosphoric acid) was used as the mobile phase.
Screening method. The medium used for screening
was the same as the isolation liquid medium except that
the concentration of racemic BPA was 1.0% (w/v)
(61 mM). BPA-assimilating microorganisms were inocu-
lated into 5 ml of the medium and then cultivated at
28 ꢁC with shaking (300 strokes/min) for 1–10 d. Dur-
ing cultivation, aliquots of the media were withdrawn
and the supernatants obtained on cetrifugation (15,000
rpm, 3 min, MX-150; Tomy Seiko, Tokyo) were
analyzed as described below.
Preparation of cell-free extracts. Cells of the selected
strains, Variovorax sp. JH2 and Arthrobacter sp. AKU
638 (about 1 g of wet cells obtained by cultivation in
1-liter of the isolation medium), were suspended in 3.3
ml of buffer and then disrupted for 10 min with an
ultrasonic oscillator (Kubota, Osaka, Japan). Samples
were ultracentrifuged at 40,000 rpm for 1 h (L-80
Ultracentrifuge; Beckman Coulter, Fullerton, CA). The
supernatants were used as soluble cell-free extracts. The
pellets obtained on ultracentrifugation were suspended
in 3.3 ml of 50 mM potassium phosphate buffer (pH 7.0)
and used as the insoluble fraction.
Results
Screening for microorganisms that stereoselectively
degrade BPA
Effects of pyridoxal-50-phosphate (PLP) and 2-oxo-
glutaric acid (2-OG) on (S)-BPA degradation. A
reaction mixture comprising, in 100 ml, 50 ml of the
soluble cell-free extract of Variovorax sp. JH2, 10 mM
BPA, and 5 mM each chemical mentioned below was
incubated at 28 ꢁC with shaking (300 strokes/min). The
chemicals examined were as follows: NADþ, NADPþ,
2-OG, pyruvate, and PLP. After 10 h reaction, the
decomposition of BPA was assayed as described
below.
Three hundred-thirty BPA-assimilating microorgan-
isms were obtained from soil samples and the AKU
culture collection. Three hundred of them were bacteria
and the others were molds. BPA was degraded in
various portions of enantiomers by them (data not
shown), and some strains exhibited stereoselectivity as
to BPA degradation. (R)-BPA production through (S)-
BPA selective degradation was observed for six strains.
Among them, the highest optical purity and yield of (R)-
BPA were observed with strain JH2 (Table 1A). (S)-
BPA production through (R)-BPA selective degradation
was observed for three strains. Among them, the highest
optical purity and yield of (S)-BPA were observed for
Effect of agitation on (R)-BPA degradation. A
reaction mixture comprising, in 100 ml, 50 ml of the
insoluble fraction of Arthrobacter sp. AKU 638 and

Microbial Production of Optically Pure ꢀ-Phenylalanine
1943
Table 1. Microorganisms Producing (R)-BPA through (S)-BPA Selective Degradation (A), and Microorganisms Producing (S)-BPA through (R)-
BPA Selective Degradation (B)
Remaining BPA
Cultivation time
(h)
A
Strain
Type of strain
% (w/v)
e.e. (%) for (R)-BPA
JH1
JH2
JR1
JT1
JT2
JT3
Bacterium
Bacterium
Bacterium
Bacterium
Bacterium
Bacterium
192
192
192
192
192
192
0.41
0.46
0.43
0.40
0.43
0.38
97
98
97
96
96
96
Remaining BPA
Cultivation time
(h)
B
Strain
Type of strain
% (w/v)
e.e. (%) for (S)-BPA
JW1
JW2
Mold
Mold
120
120
0.52
0.40
33
63
Arthrobacter sp.
AKU 638
Bacterium
240
0.55
76
e.e., enantiomer excess
1.0
0.9
0.8
0.7
0.6
0.5
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
A
B
90% e.e.
99% e.e.
0.4
0.3
0.2
0.1
0
0
50
100
150
200
0
100
200
300
Cultivation time (h)
Cultivation time (h)
Fig. 1. Preparative Production of (R)-BPA during Cultivation of Variovorax sp. JH2 (A), and of (S)-BPA during Cultivation of Arthrobacter sp.
AKU 638 (B).
The cultivations were carried out under the same conditions as those for screening, as described in ‘‘Materials and Methods.’’ The % e.e.
values for obtained BPA are presented with arrows. The averages of three separate experiments, the results of which were reproducible within
ꢂ10%, are presented in the figure. , (R)-BPA; , (S)-BPA; , total BPA.
Arthrobacter sp. AKU 638 (Table 1B). Strain JH2 and
Arthrobacter sp. AKU 638 were thus selected as potent
strains with the ability to produce (R)- and (S)-BPA
respectively.
biological traits of this strain were determined to be
as follows: methophilic (grew at 30 ꢁC); colony shape,
circular, smooth, and yellow in color; cell shape, rod-
shaped (0:6{0:7 ꢀ 1:0{1:2 mm); motile; Gram-negative;
and nonspore-forming. These biological traits supported
our assumption that strain JH2 belonged to the genus
Variovorax, so the strain was named Variovorax sp.
JH2.
Identification of strain JH2
Strain JH2 was identified by phylogenetic analysis
and from its biochemical characteristics. The partial 16S
rDNA sequence of this strain showed high similarity to
that of a strain of Variovorax paradoxus (100%), and
formed a sister group with that of V. paradoxus in the
phylogenetic tree (data not shown). Thus the isolated
strain might belong to the genus Variovorax, probably to
a group closely related to Variovorax paradoxus. The
Preparative production of (R)- and (S)-BPA
Preparative production of (R)-BPA through (S)-iso-
mer selective degradation during the cultivation of
Variovorax sp. JH2 was carried out (Fig. 1A). After
192 h cultivation in medium containing 1.0% (w/v)

1944
J. MANO et al.
None
2-OG
A
BPA
PLP
Degradation product
2-OG+PLP
B
C
4
0
1
2
3
Degradation of BPA (mM)
BAA
Fig. 2. Effects of PLP and 2-OG on BPA-Degradation by the Soluble
Cell-Free Extract of Variovorax sp. JH2.
The amount of BPA (mM) degraded on 10 h reaction with 5 mM
2-OG, 5 mM PLP, or both is shown.
30
0
10
20
racemic BPA (61 mM) as the sole carbon source, 0.46%
(w/v) BPA (28 mM) was obtained with an optical purity
of 99% e.e. for the (R)-isomer.
Preparative production of (S)-BPA through (R)-iso-
mer selective degradation during the cultivation of
Arthrobacter sp. AKU 638 was also carried out
(Fig. 1B). After 312 h cultivation in medium containing
1.0% (w/v) racemic BPA (61 mM) as the sole carbon
source, 0.51% (w/v) BPA (31 mM) was obtained with an
optical purity of 90% e.e. for the (S)-isomer.
Retention time (min)
Fig. 3. HPLC Chromatograms of the Product of (S)-BPA Degrada-
tion by the Soluble Cell-Free Extract of Variovorax sp. JH2 and
Authentic BAA.
HPLC analysis was performed under the conditions given in
‘‘Materials and Methods.’’ A, HPLC chromatogram of the reaction
mixture before incubation; B, HPLC chromatogram of the reaction
mixture after incubation for 30 min; C, HPLC chromatogram of
authentic BAA (2.5 mM).
Reaction analysis of the (S)-BPA degradation by
Variovorax sp. JH2
L-Glutamic acid
D-Glutamic acid
The (S)-BPA degrading activity of Variovorax sp.
JH2 was found in the soluble cell-free extract but not in
the insoluble fraction (data not shown). The effects of
various cofactors on (S)-BPA degradation by soluble
cell-free extracts of Variovorax sp. JH2 were inves-
tigated. Among the five chemicals examined, PLP and
2-OG accelerated the degradation (Fig. 2). The combi-
nation of PLP and 2-OG further enhanced the degrada-
tion. The degradation products were identified by TLC,
HPLC, and LC–MS. One ultra-violet positive product
was observed on TLC analysis, and it showed the same
Rf value as that of authentic BAA (data not shown).
BAA was further identified on HPLC analysis (Fig. 3),
but BAA was not detected on LC–MS, because it is not
stable under ionization conditions. One ninhydrin posi-
tive product was also detected on TLC analysis, and it
showed the same Rf value as that of L-glutamic acid.
(data not shown). L-Glutamic acid was further identified
on HPLC analysis (Fig. 4). These results suggest that the
degradation products were BAA and L-glutamic acid,
but the stoichiometry of the reaction was not confirmed
because these products were further degraded by the
cell-free extracts of Variovorax sp. JH2. Based on these
results, we tried to produce optically active (R)-BPA
using resting cells of Variovorax sp. JH2 as the catalyst
in the presence of cofactors. On 36 h reaction with 1.0%
(w/v) racemic BPA (61 mM), 0.44% (w/v) 2-OG
(30 mM), 0.12% (w/v) PLP (5 mM), and 10% (wet cells,
w/v) washed cells of Variovorax sp. JH2 obtained on
24 h-cultivation in the isolation liquid medium, it was
A
B
C
Degradation product
15
0
5
10
Retention time (min)
Fig. 4. HPLC Chromatograms of the GITC-Derivatized Product of
(S)-BPA Degradation by the Soluble Cell-Free Extract of Variovor-
ax sp. JH2 and a Standard Mixture of GITC-Derivatized Racemic
Glutamic Acid.
HPLC analysis was performed under the conditions given in
‘‘Materials and Methods.’’ A, HPLC chromatogram of a standard
mixture containing racemic glutamic acid (10 mM); B, HPLC
chromatogram of the reaction mixture before incubation; C, HPLC
chromatogram of the reaction mixture after incubation for 30 min.
almost the case that only (S)-BPA was degraded, 0.48%
(w/v) of (R)-BPA being obtained in a high yield and
with a high enantiomeric purity (molar yield as to
racemic BPA, 48%; optical purity, 99% e.e. for (R)-
BPA).

Microbial Production of Optically Pure ꢀ-Phenylalanine
1945
cells of Arthrobacter sp. AKU 638 as the catalyst with
vigorous shaking. On 45 h reaction with 1.0% (w/v)
racemic BPA (61 mM) and 10% (wet cell, w/v) of
washed cells of Arthrobacter sp. AKU 638 obtained on
24 h of cultivation in the isolation liquid medium, it
was almost the case that only (R)-BPA was degraded,
0.49% (w/v) of (S)-BPA being obtained in a high yield
and with a high enantiomeric purity (molar yield as to
racemic BPA, 49%; optical purity, 99% e.e. for (S)-
BPA).
(R)-BPA
(S)-BPA
A
B
Degradation
product
C
NH3
Discussion
Variovorax sp. JH2 and Arthrobacter sp. AKU 638
were found to be (S)- and (R)-BPA degrading micro-
organisms, and selected as potential catalysts for the
production of optically pure (R)- and (S)-BPA respec-
tively.
0
10
Retention time (min)
20
Fig. 5. HPLC Chromatograms of GITC-Derivatized Samples Con-
taining the Product of (R)-BPA Degradation by the Insoluble
Fraction of Arthrobacter sp. AKU 638 and a Standard Mixture of
GITC-Derivatized Ammonia.
The (S)-BPA degrading activity of Variovorax sp.
JH2 was found in the soluble cell-free extract. The
enzyme catalyzing the initial step of the (S)-BPA
metabolism appeared to be a soluble protein. On
analysis of the biochemical properties of the reaction,
PLP and 2-OG were found to accelerate it. In addition,
the products of the reaction were identified as BAA and
L-glutamic acid. These facts suggest that the reaction
comprises stereoselective transamination and that the
enzyme degrading (S)-BPA can be classified as (S)-3-
amino-3-phenylpropionic acid: 2-oxoglutaric acid ami-
notransferase. Concerning the microbial metabolism of
ꢀ-amino acids, such as ꢀ-alanine, ꢀ-aminoisobutyrate,
ꢀ-aminobutyrate and taurine, metabolism through trans-
amination catalyzed by !-amino acid transaminases has
been reported.12–14) Until now, there has been no report
on BPA metabolism. In this study, it was found that one
type of BPA metabolism involves transamination that is
the same as some other ꢀ-amino acid metabolisms.
(R)-BPA degrading activity was found in the insolu-
ble fraction of Arthrobacter sp. AKU 638, suggesting
that the enzyme catalyzing the initial step of (R)-BPA
metabolism is a membrane protein. The reaction with
the insoluble fraction as the catalyst proceeded slowly
under the static condition but faster with shaking,
suggesting that the concentration of soluble oxygen in
the reaction mixture affects the enzyme activity in-
volved in the (R)-BPA degradation. Ammonia was
identified as the reaction product under the shaking
condition and hence it was speculated that (R)-BPA is
degraded through an oxidative deamination process.
In summary, we report here two novel types of
microbial BPA metabolism with stereoselectivity, viz.,
transamination and oxidative deamination. This micro-
bial degradation should allow efficient production of
both isomers of BPA.
HPLC analysis was performed under the conditions given in
‘‘Materials and Methods.’’ A, HPLC chromatogram of the reaction
mixture before incubation; B, HPLC chromatogram of the reaction
mixture after incubation for 10 h; C, HPLC chromatogram of the
standard mixture containing NH3 (10 mM).
Reaction analysis of the (R)-BPA degradation by
Arthrobacter sp. AKU638
The (R)-BPA degrading activity of Arthrobacter sp.
AKU 638 was found in the insoluble fraction but not in
the soluble cell-free extract (data not shown). By
shaking the reaction mixture, the (R)-BPA degradation
by the insoluble fraction of Arthrobacter sp. AKU 638
was greatly enhanced. Under the static condition, only
0.8 mM BPA in the reaction mixture was degraded in
10 h while 6.7 mM of BPA was degraded under the
shaking condition. One of the products of the reaction
under the shaking condition was identified as ammonia
on HPLC chromatography (Fig. 5), and further by LC–
MS analysis showing the same molecular weight
(m=z ¼ 407; ESI positive) of that of GITC-derivatized
ammonia. However, a possible deamination product,
BAA, was not found in the reaction mixture. The
stoichiometry of the reaction was not confirmed because
these products were further degraded by the insoluble
cell-free fractions of Arthrobacter sp. AKU 638. These
results suggest that (R)-BPA is degraded through an
oxidative deamination process, but the reaction mech-
anism could not be elucidated because general cofactors
for enzymatic oxidation or dehydrogenation, such as
NADþ, NADPþ, NADH, NADPH, FAD, FMN, phena-
zine methosulfate, 2,6-dichlorophenolindophenol, meth-
ylene blue, potassium ferricyanide, 1,4-benzoquinone,
1,4-naphthoquinone, pyrroloquinoline quinone, cyto-
chrome c, thioredoxin, and rubredxin, did not accelerate
the reaction (data not shown). Based on these results, we
tried to produce optically active (S)-BPA using resting
References
1) Achilles, K., Schirmeister, T., and Otto, H. H., ꢀ-lactam

1946
J. MANO et al.
derivatives as enzyme inhibitors: 1-peptidyl derivatives
8) Faulconbridge, S. J., Holt, K. E., Sevillano, L. G., Lock,
C. J., Tiffin, P. D., Tremayne, N., and Winter, S.,
Preparation of enantiomerically enriched aromatic ꢀ-
amino acids via enzymatic resolution. Tetrahedron Lett.,
41, 2679–2681 (2000).
of 4-phenylazetidin-2-one as inhibitors of elastase and
papain. Arch. Pharm. Pharm. Med. Chem., 333, 243–253
(2000).
2) Nagano, Y., Ikedo, K., Fujishima, A., Izawa, M.,
Tsubotani, S., Nishimura, O., and Fujino, M., Pylorici-
dins, novel anti-Helicobacter pylori antibiotics produced
by Bacillus sp. J. Antibiotics, 54, 934–947 (2001).
3) Bovy, P. R., Tjoeng, F. S., Rico, J. G., Rogers, T. E.,
Lindmark, R. J., Zablocki, J. A., Garland, R. B.,
McMackins, D. E., Dayringer, H., Toth, M. V., Zupec,
M. E., Rao, S., Panzer-Knodle, S. G., Nicholson, N. S.,
Salyers, S., Taite, B. B., Herin, M., Miyano, M., Feigen,
L. P., and Adams, S. P., Design of orally active, non-
peptide fibrinogen antagonists: an evolutionary process
from the RGD sequence to novel anti-platelet aggrega-
tion agents. Bioorg. Med. Chem., 2, 881–895 (1994).
4) Stilz, H. U., Guba, W., Jablonka, B., Just, M., Klingler,
O., Konig, W., Wehner, V., and Zoller, G., Discovery of
orally active non-peptide fibrinogen receptor antagonist
based on the hydantoin scaffold. J. Med. Chem., 44,
1158–1176 (2001).
5) Ogawa, J., Mano, J., and Shimizu, S., Microbial
production of optically active ꢀ-phenylalanine ethyl
ester through stereoselective hydrolysis of racemic ꢀ-
phenylalanine ethyl ester. Appl. Microbiol. Biotechnol.,
70, 663–669 (2006).
6) Roche, D., Prasad, K., and Repic, O., Enantioselective
acylation of ꢀ-aminoesters using penicillin G acylase in
organic solvents. Tetrahedron Lett., 40, 3665–3668
(1999).
9) Kinoshita, T., Kasahara, Y., and Nimura, N., Reversed-
phase high-performance liquid chromatographic resolu-
tion of non-esterified enantiomeric amino acids by
derivatization with 2,3,4,6-tetra-o-acetyl-ꢀ-D-glucopyra-
nosyl isothiocyanate and 2,3,4-tri-o-acetyl-ꢁ-D-arabino-
pyranosyl isothiocyanate. J. Chromatogr., 210, 77–81
(1981).
10) Nimura, N., Ogura, H., and Kinoshita, T., Reversed-
phase liquid chromatographic resolution of amino acid
enantiomers by derivatization with 2,3,4,6-tetra-o-ace-
tyl-ꢀ-D-glucopyranosyl isothiocyanate. J. Chromatogr.,
202, 375–379 (1980).
11) Nimura, N., Toyama, A., and Kinoshita, T., Optical
resolution of amino acid enantiomers by high-perform-
ance liquid chromatography. J. Chromatogr., 316, 547–
552 (1984).
12) Yonaha, K., Suzuki, K., and Toyama, S., Streptomyces
ꢀ-alanine:ꢁ-ketoglutarate aminotransferase, a novel !-
amino acid transferase. J. Biol. Chem., 260, 3265–3268
(1985).
13) Toyama, S., Misono, H., and Soda, K., Properties of
taurine:alpha-ketoglutarate aminotransferase of Achro-
mobacter superficialis: inactivation and reactivation of
the enzyme. Biochim. Biophys. Acta, 523, 75–81 (1978).
14) Yun, H., Lim, S., Cho, B. K., and Kim, B. G., !-Amino
acid:pyruvate transaminase from Alcaligenes definitrifi-
cans Y2k-2: a new catalyst for kinetic resolution of ꢀ-
amino acids and amines. Appl. Environ. Microbiol., 70,
2529–2534 (2004).
7) Cohen, S. G., and Weinstein, S. Y., Hydrolysis of D(ꢃ)-
ethyl ꢀ-phenyl-ꢀ-hydroxypropionate and D(ꢃ)-ethyl ꢀ-
phenyl-ꢀ-acetamidepropionate by ꢁ-chymotrypsin. J.
Am. Chem. Soc., 86, 725–728 (1964).