Site-specific and asymmetric hydrolysis of prochiral 2-phenyl-1,3- propanediol diacetate by a bacterial esterase from an isolated strain

Article abstract of DOI:10.1271/bbb.70019

Bacillus cereus 809A and Burkholderia sp. 711C were isolated from soil. These strains demonstrate hydrolysis activity towards prochiral 2-phenyl-1,3-propanediol diacetate and accumulated the corresponding chiral monoacetates into the reaction mixture. When 2-phenyl 1,3-propanediol diacetate was used as a substrate, the produced monoacetates with Burkholderia sp. 711C were obtained in a racemic form but that produced by Bacillus cereus 809A showed an excess of the (S)-form. The resting cell reaction revealed that for Bacillus cereus 809A, there was an enrichment of one of the enantiomers of the monoacetate such that the enantiomeric excess (e.e.) of the (S)-form was over 95%. The purified enzyme from Bacillus cereus 809A hydrolyzed diacetate to monoacetate, and the e.e. value of the (S)-form increased by prolonged reaction in a way similar to the resting cell reaction. From N-terminal amino acids, this esterase is conserved in some strains of Bacillus for which the genomic sequences have been reported.

Full text of DOI:10.1271/bbb.70019

Biosci. Biotechnol. Biochem., 71 (8), 1858–1864, 2007
Site-Specific and Asymmetric Hydrolysis of Prochiral 2-Phenyl-1,3-propanediol
Diacetate by a Bacterial Esterase from an Isolated Strain
y
Ryoji MITSUI,1; Seiho SHINYA,1 Yuichi ICHIYAMA,1 Kenta KUDO,1
1
Takuo TSUNO,2 and Mitsuo TANAKA
1Department of Biochemistry, Faculty of Science, Okayama University of Science,
1-1 Ridai-cho, Okayama 700-0005, Japan
2Tsuno Food Industrial Co., Ltd., 94 Shinden, Katsuragi-cho, Ito-gun, Wakayama 649-7194, Japan
Received January 11, 2007; Accepted May 8, 2007; Online Publication, August 7, 2007
[doi:10.1271/bbb.70019]
Bacillus cereus 809A and Burkholderia sp. 711C were
isolated from soil. These strains demonstrate hydrolysis
activity towards prochiral 2-phenyl-1,3-propanediol
diacetate and accumulated the corresponding chiral
monoacetates into the reaction mixture. When 2-phenyl
1,3-propanediol diacetate was used as a substrate, the
produced monoacetates with Burkholderia sp. 711C
were obtained in a racemic form but that produced by
Bacillus cereus 809A showed an excess of the (S)-form.
The resting cell reaction revealed that for Bacillus
cereus 809A, there was an enrichment of one of the
enantiomers of the monoacetate such that the enantio-
meric excess (e.e.) of the (S)-form was over 95%. The
purified enzyme from Bacillus cereus 809A hydrolyzed
diacetate to monoacetate, and the e.e. value of the (S)-
form increased by prolonged reaction in a way similar
to the resting cell reaction. From N-terminal amino
acids, this esterase is conserved in some strains of
Bacillus for which the genomic sequences have been
reported.
there are two reported methods of lipase-based asym-
metric synthesis of 2-substituted-1,3-propanediol mono-
acetate that give high yields (more than 50%). One is
esterification of 2-substituted-1,3-propanediol with vinyl
acetate5,6) while the other is hydrolysis of prochiral 2-
substitued-1,3-propanediol diacetate.7)
This study was an attempt to produce optically pure 2-
substituted-1,3-propanediol monoacetate as a useful
chiral building block. We used the prochial propanediol
derivatives, 2-phenyl-1,3-propanediol diacetate (PPdAc)
and 2-methyl-1,3-propanediol diacetate to screen for
novel microbial esterases in cells. Some strains showed
activities for producing monoacetates from both diac-
etates, but 2-methyl-1,3-propanediol diacetate was not
suitable for producing optically pure substances, be-
cause all strains used produced monoacetate with low
optical purity (data not shown). However, when PPdAc
was used as the substrate, strain 809A, which was
screened in this study, produced monoacetate of the (S)-
form in excess in both resting and whole cell reactions
(Fig. 1). There are no reports of approaches that employ
resting or whole cells to prepare these compounds
cheaply. It would however be a useful method for the
production of optically pure compounds, because com-
mercial esterases and lipases are expensive. Further-
more, it is interesting to note that we found that the
optical purity of 2-phenyl-1,3-propanediol monoacetate
(PPmAc) increased with reaction time. In order to
understand the hydrolysis process in detail, we purified a
novel esterase from the crude extract of strain 809A and
characterized some of its enzymatic properties.
Key words: esterase; asymmetric hydrolysis; 2-phenyl-
1,3-propanediol diacetate; Bacillus cereus
The chiral pool synthetic method was developed to
produce useful chiral compounds efficiently.1) Optically
pure 2-substituted-1,3-propanediol derivatives are useful
chiral building blocks as one of the minimum pool
required for the synthesis of biologically active com-
pounds.2,3) Since the strict substrate specificities of
enzymes is a distinct advantage for chiral pool synthesis,
microbial enzymes are often used to obtain optically
pure substances. The stereospecific hydrolysis of race-
mic esters by esterase is an efficient strategy for optical
resolution,4) but the maximum yield of the targeted
optically pure compound is below 50%. In contrast,
Materials and Methods
Materials. PPdAc and PPmAc were synthesized by
and were gifts from Dr. Ohira and Dr. Kuboki (Depart-
y
To whom correspondence should be addressed. Fax: +81-86-256-9708; E-mail: rmitsui@dbc.ous.ac.jp
Abbreviations: PPdAc, 2-phenyl-1,3-propanediol diacetate; PPmAc, 2-phenyl-1,3-propanediol monoacetate; PPol, 2-phenyl-1,3-propanediol; e.e.,
enantiomeric excess; DEAE, diethyl aminoethyl

Enzymatic Hydrolysis of Prochiral Propanediol Derivatives
1859
Ph
Ph H
AcO
AcO
OH
OH
AcO
OAc
(S)-PPmAc
PPdAc
Ph
H
Ph
HO
OH
(R)-PPmAc
PPol
Fig. 1. Summary of This Study for Producing (S)-2-Phenyl-1,3-propanediol Monoacetate from Prochiral-2-phenyl-1,3-propanediol Diacetate Using
a Bacterial Esterase.
ment of Biochemistry, Faculty of Science, Okayama
University of Science). 2-Phenyl-1,3-propanediol (PPol)
was purchased from Tokyo Kasei Kogyo (Tokyo).
potassium phosphate (pH 7.0), and an appropriate
amount of enzyme. This reaction was performed at
30 ꢀC for 30 min. After the reaction, 1 ml of ethyl acetate
was added to the mixture to extract the remaining
substrate and products. One unit was defined as the
amount of enzyme needed for the formation of 1 mmol
PPmAc from PPdAc per min at 30 ꢀC. Protein was
measured by the Lowry method,9) with bovine serum
albumin as the standard. Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS–PAGE) was
performed using 10% polyacrylamide slab gels by the
method of Laemmli.10) The gels were stained for protein
with Coomassie brilliant blue R-250. The molecular
weight of the enzyme was estimated by elution time
from a G-3000SW HPLC column (Tosoh, Tokyo) with
detection at 280 nm. MW-Marker (Oriental Yeast,
Osaka, Japan) was used as the molecular weight
standard. Amino terminal sequence analysis of the
purified enzyme, which was blotted onto PVDF mem-
brane, was performed using the Edman methodology on
a 467A Protein Sequencer (Perkin Elmer Japan, Yoko-
hama, Japan).
Screening method. Soil samples (0.1 g each) were
suspended in 5 ml of physiological saline. The suspen-
sions were diluted adequately, spread on solid Luria-
Bertani medium,8) and cultivated for 2 to 5 d at 28 ꢀC.
The single colonies obtained were picked out and
inoculated into LB liquid medium for 2 d. The substrate
was dissolved in ethanol and then added to the medium
to a final concentration of 10 mM. After 24 h, the pro-
ducts in the supernatant were extracted with ethyl
acetate and analyzed by Gas-Liquid Chromatography
(GLC), as described below.
Resting cell reaction. Cells were harvested by
centrifugation at 10;000 ꢁ g for 10 min and washed
once with 50 mM potassium phosphate buffer (pH 7.0) at
4 ꢀC. The cells were then resuspended in the same buf-
fer. Resting cell reactions were performed at 28 ꢀC with
shaking for 8 h in the reaction mixture, which contained
10 mM PPmAc and 50 mM potassium phosphate buffer
(pH 7.0). The final cell density in the reaction mixture
was adjusted to an absorbance value at 610 nm of 5.0.
Preparation of cell extract. Isolated strain 809A was
grown at 28 ꢀC for 24 h on LB medium. After culti-
vation, the cells were harvested by centrifugation
(10;000 ꢁ g, 10 min, 4 ꢀC) and washed twice with 10
mM Tris–HCl buffer (pH 8.0). The pellet was resus-
pended in buffer and the suspension was treated with an
ultrasonicator (9 kHz) for 10 min, with care taken to
keep the temperature below 5 ꢀC. The lysate was then
centrifuged at 12;000 ꢁ g for 20 min. The resulting
supernatant was used as the cell extract.
Analytical measurements. The residual substrates and
hydrolyzed products extracted with ethyl acetate were
analyzed by GLC with a non-polarity capillary DB-1
column (0:25 mm ꢁ 30 m, J&W Scientific, Folsom, CA)
using a flame ionization detector. The injector temper-
ature was 250 ꢀC and the column temperature was raised
2 ꢀC/min from 80 to 110 ꢀC. The mobile phase was
nitrogen flowing at 1.2 ml/min. The optical purity of the
PPmAc was analyzed by HPLC with a Chiralcel OD
column (4:6 ꢁ 250 mm, Daicel Chemical Industries,
Tokyo) with detection at 254 nm. The mobile phase
was hexane and methanol (95:5 vol/vol) and the flow
rate was 0.5 ml/min. The value of enantiomeric excess
(e.e.) was determined by the following equation using
the results of HPLC analysis: e.e.% of (S) = ((S)-
isomer ꢂ (R)-isomer)/((S)-isomer + (R)-isomer) ꢁ 100.
The enzyme activity was estimated based on the
formation of PPmAc by hydrolysis of PPdAc in a 1-ml
reaction mixture containing 10 mM PPdAc, 50 mM
Protein purification. The enzyme, which catalyzed the
hydrolysis of PPdAc to PPmAc, was purified from the
cell extract as follows: The cell extract was applied to a
DEAE-Sepharose FF (GE Healthcare Bio-Science, Little
Chalfont, Buckinghamshire, UK) column (5:0 ꢁ 20 cm).
After the column was washed with 10 mM Tris–HCl
buffer (pH 8.0), the enzyme was eluted with a gradient
of increasing NaCl concentrations (0 to 0.4 M). The
active fractions were collected and dialyzed against
50 mM potassium phosphate buffer (pH 7.0). Solid NaCl
was added to dialyzed active solution to a concentration

1860
R. MITSUI et al.
of 2 M, and the solution applied to a Butyl-Sepharose FF
(GE Healthcare Bio-Science) column (2:6 ꢁ 20 cm)
equilibrated with the same buffer containing 2 M NaCl.
After the column was washed with the same buffer,
elution was performed with a gradient of decreasing
NaCl concentrations (2.0 to 0 M) and increasing ethylene
glycol concentrations (0 to 20%). The active fractions
were collected and dialyzed against 10 mM Tris–HCl
buffer (pH 8.0). The dialyzed solution was applied to a
Resource Q prepacked column (1 ml) (GE Healthcare
Bio-Science) equilibrated with 10 mM Tris–HCl buffer
(pH 8.0). After the column was washed with the same
buffer, the enzyme was eluted with a gradient of in-
creasing NaCl concentrations (0 to 0.6 M). The active
fractions were collected and dialyzed against 10 mM
Tris–HCl (pH 8.0) and stored at ꢂ20 or ꢂ80 ꢀC until
use.
100
75
50
25
0
10
8
6
Ph
H
AcO
OH
e.e.(%) of (S)-PPmAc
4
2
0
0
2
4
6
8
Reaction time (h)
Fig. 2. Hydrolysis of PPdAc in Resting-Cell Reactions with Strain
809A.
The final cell density in the reaction mixture was adjusted to an
Results and Discussion
absorbance value of 610 nm of 5.0.
e.e. (%).
PPdAc;
PPmAc; PPol;
Accumulation of PPmAc by hydrolysis of PPdAc by
the isolated strains
About 500 isolated strains were cultivated on LB
medium at 28 ꢀC for 24 h. After cultivation, PPdAc was
added to the culture and incubated with the cells for
24 h. The reaction mixture was extracted with ethyl
acetate in order to measure the accumulation of the
corresponding monoacetate, as described in ‘‘Materials
and Methods.’’ In most of the samples (about 70%) no
products were detected in the ethyl acetate extract, but
we found four strains that accumulated PPmAc in the
culture. These strains were examined further with regard
to hydrolysis of PPdAc with increasing reaction time
(data not shown). In the case of two of the strains,
further hydrolysis to 2-phenyl-1,3-propanediol (PPol)
occurred, but the other two strains, named 711C and
809A, accumulated PPmAc in the medium at high yield.
PPmAc at high yield, but measurement of the optical
purity of the PPmAc produced by HPLC showed that the
material produced by strain 711C was racemic at all
time points (data not shown), whereas the optical purity
of that produced by strain 809A increased with reaction
time (Fig. 2). For strain 809A, the value of e.e. after all
the PPdAc was hydrolyzed was more than 99% by
HPLC. The specific optical rotation of the PPmAc
25
produced was measured. It gave a value of ½ꢀꢃD
¼
ꢂ58:8ꢀ (c 1.2, CHCl3), demonstrating it to be the (S)
form. This value corresponds well with the previously
reported value of ꢂ59:6ꢀ.11) In order to understand the
hydrolysis mechanisms, the enzyme was purified from
strain 809A.
Identification of the isolated strains
711C was found to be a gram-negative aerobic strain
that was rod-shaped, formed light yellow colonies, and
was positive for oxidase and catalase. The 16S riboso-
mal DNA sequence, which was examined by NCIMB
Japan, showed 711C to be closely related to Burkhol-
deria cepacia. 809A is a gram-positive aerobic end-
spore forming rod-shaped strain that is negative for
oxidase but positive for catalase. The 16S ribosomal
DNA sequence identified this organism as a strain of
Bacillus cereus. From the above results, strains 711C
and 809A were identified as Burkholderia sp. 711C and
Bacillus cereus 809A respectively.
Purification and properties of the enzyme
The enzyme that hydrolyzed PPdAc to PPmAc was
purified from a cell-free extract of strain 809A, as
described in ‘‘Materials and Methods.’’ The crude
extract was purified by column chromatography. One
milligram of purified enzyme was obtained from 55 g of
cells (wet cell weight), which was from 5-liter culture.
The results of the purification are summarized in
Table 1. Purified enzyme migrated as a single band,
and the relative molecular weight of the purified enzyme
was estimated to be about 38,000 by SDS–PAGE
(Fig. 3A), and to be about 70,000 by gel-filtration
(Fig. 3B). These results suggest that this enzyme is a
dimer of two identical subunits. The pH- and temper-
ature-optimum for the activity of enzyme were estimat-
ed from the hydrolysis activities. The enzyme showed
highest activity towards PPdAc between pH 7 and 9
(Fig. 4A), and was most stable within a pH range of
6 to 10 in 30 min incubations at 30 ꢀC (Fig. 4B). The
Resting-cell reaction
Activity towards PPdAc was found in cells of strains
711C and 809A but not in the culture supernatants. In
order to improve the hydrolysis rate of the substrate and
the yield of product, resting-cell reactions were inves-
tigated. The cells of both strains hydrolyzed PPdAc to

Enzymatic Hydrolysis of Prochiral Propanediol Derivatives
1861
Table 1. Purification of PPdAc Esterase from B. cereus 809A
Total protein
(mg)
Total activity
(U)
Specific activity
(U mgꢂ1
Yield
(%)
Purification
)
Cell-free extract
DEAE-Sepharose
Butyl-Sepharose
Resource Q
12,000
700
320
130
80
0.1
0.7
100
46
1
7
470
4
32.5
80.0
19
11
325
800
1
A
B
1
2
10 6
97,400
66,200
10 5
7x104
45,000
31,000
10 4
3
3.5
4
4.5
5
5.5
Elution volume (ml)
Fig. 3. Molecular Weight Analysis.
SDS–PAGE (A) and gel-filtration (B) of purified esterase from strain 809A. A, Lane 1, molecular weight marker; lane 2, 3 mg of purified
enzyme. B, , Molecular weight standard, from large weight to small weight: glutamate dehydrogenase (2:90 ꢁ 105), lactate dehydrogenase
(1:42 ꢁ 105), enolase (6:70 ꢁ 104), adenylate kinase (3:20 ꢁ 104), and cytochorome C (1:24 ꢁ 104). , Purified esterase.
stability of this enzyme was clearly lower at pH levels
lower than 6 and higher than 10. The enzyme showed
highest activity at 60 ꢀC (Fig. 4C), but it was unstable
after prolonged incubation at this temperature (Fig. 4D).
pH-dependent non-enzymatic hydrolysis of PPdAc was
observed at pH 10 and higher, but was not observed at
any temperature under the experimental conditions we
examined.
Inhibition of the enzyme by certain metal ions and
other reagents was investigated. The activity was
inhibited strongly by Agþ (100%), and partially by
Cu2þ (50%) at a final concentration of 1 mM. Chelating
agents such as ethylenediaminetetraacetic acid did not
inhibit the activity. The enzyme activity was inhibited
by phenyl hydrazine (63%) and phenyl methyl sulfonyl
fluoride (80%). These results suggest that a serine
residue in the enzyme might play an important role in
activity.12)
esting that the value of e.e. (%) was almost constant
(about 60% e.e.) while sufficient PPdAc remained in the
mixture. After PPdAc was consumed, PPmAc was
further hydrolyzed to PPol and the excess of (S)-PPmAc
increased. Based on these results, we suggest the
following means of hydrolysis of PPdAc by this
enzyme: First, it catalyzes pro(R)-specific hydrolysis of
PPdAc, and the (S)-form accumulates preferentially in
the reaction mixture. The ratio of the (S) to (R)-form
products was approximately 4 to 1 (60% e.e.). This
value corresponds well to the simulation described by
Chen et al.13) That is, the enantiomeric ratio or E value is
read as around 4 as calculated from the production rate
of each enantiomer, and this value gives an e.e.(%) of
about 60 when the substrate conversion rate is 60%.
Secondly, the PPmAc produced is further hydrolyzed
slowly to PPol, and the amount of the (S)-form increases
even though the yield of PPmAc decreases. These
results suggest that the stereospecificity of the purified
enzyme is not high for PPmAc isomers. A reaction such
as that just described changes the ratio of the remaining
isomers. Under these conditions, the maximum yield of
optically pure (S)-PPmAc (about 55%) was obtained
after 2 h, after which the yield of (S)-PPmAc decreased
as it was further hydrolyzed to PPol.
Hydrolysis of PPdAc by the purified enzyme
A time course study of the hydrolysis of PPdAc and
the optical purity of the product was set up using
purified enzyme (Fig. 5). The hydrolysis reaction pro-
ceeded until PPdAc was completely consumed, and
PPmAc was found in the reaction mixture. The optical
purity of PPmAc increased with reaction time, as
observed in the reaction with resting cells. In order to
understand this observation, the stereoisomers of
PPmAc were measured quantitatively by HPLC in a re-
action with homogeneous, purified enzyme. It is inter-
N-Terminal amino acid sequence
The enzyme was blotted onto PVDF membrane and
the N-terminal amino acid sequence was determined
with a protein sequencer, as described in ‘‘Materials and

1862
R. MITSUI et al.
A
B
100
80
60
40
20
0
100
80
60
40
20
0
4
6
8
10
12
4
6
8
10
12
pH
pH
C
D
100
80
60
40
20
0
100
80
60
40
20
0
20
30
40
50
60
70
80
20
30
40
50
60
70
80
Temperature (°C)
Temperature (°C)
Fig. 4. pH Optimum (A) and Stability (B) of Purified Esterase from Strain 809A.
pH optimum was measured at 30 ꢀC using PPdAc as substrate. pH stability was measured by incubation at 30 ꢀC for 1 h and then by
measurement of residual activity at pH 7.0 using PPdAc as substrate. , Acetic acid buffer at pH 4.0–6.0; , potassium phosphate buffer at
pH 6.0–8.0; , Tris–HCl buffer at pH 8.0–10.0 , glycine–NaOH buffer at pH 10.0–12.0. Temperature optimum (C) and stability (D) of
purified esterase from strain 809A. Residual activity was measured for each temperature by incubation for 30 min in potassium phosphate buffer
at pH 7.0 using PPdAc as substrate.
Methods.’’ The N-terminal amino acid sequence ob-
tained was MKTRVNPELLQGLEMFPDLDLRPE. The
sequence was used to search genomes of Bacillus
species by BLAST. This search showed that the
sequence had similarities with some B. cereus esterases
(Fig. 6), but had highest similarity to esterases in
B. thuringiensis. These results suggest that this esterase
has been conserved widely among Bacillus species, but
it is not known whether these species have an esterase
with hydrolysis activity, site-specificity, and asymmetric
specificity towards PPdAc.
The resting cell reaction exhibited a high specific
hydrolysis rate for PPdAc. Furthermore, we have
established a method of producing PPmAc cheaply with
high optical purity. However, the maximum yield of
PPmAc with high optical purity (90% e.e.) was about
55% under the conditions imposed in this study, because
the optically pure material obtained was produced by
further hydrolysis of the PPmAcs produced earlier in the
reaction. In order to improve the yield of optically pure
PPmAc, further investigations are necessary to deter-
mine the reaction conditions. These include temperature,
pH, and addition of agents. These attempts are better
suited to the enzyme reaction rather than to the resting
cell reaction. In order to synthesize chiral reagents by
chemo-enzymatic methods, recombinant techniques
have been employed in order to express and purify
useful microbial enzymes.14) These recombinant pro-
teins have been purified on an affinity resin, Ni-NTA,
in a single step, through the addition of a hexa-histidine
tag to the N-terminus. Furthermore, recent genetic
approaches have progressed dramatically. Molecular
breeding has been applied to a variety of enzymes to
improve substrate specificity, catalytic efficiency, stabil-
ity, pH optima, and function by mutation of genes.15) In
order to improve the stereospecificity of enzymes,

Enzymatic Hydrolysis of Prochiral Propanediol Derivatives
1863
10
8
100
75
6
Ph
H
50
AcO
OH
(S)-PPmAc
4
Ph
H
25
0
AcO
OH
2
(R)-PPmAc
0
0
0.5
1.0
1.5
2.0
2.5
3.0
Reaction time (h)
Fig. 5. Hydrolysis of PPdAc by Purified Esterase from Strain 809A.
, PPdAc; , (S)-PPmAc; , (R)-PPmAc; , PPol; , e.e. (%). The reaction was performed with 0.2 U of purified enzyme per ml in 50 mM
potassium phosphate buffer (pH 7.0) at 30 ꢀC.
B. cereus 809A
B. thuringiensis ATCC 35646
B. anthracis str. Sterne
B. anthracis str. A2012
B. cereus subsp. cytotoxis NVH 391-98
B. cereus E33L
B. cereus ATCC 10987
Fig. 6. Comparison of N-Terminal Sequence of Esterase from Strain 809A with Esterases from Other Bacillus Strains.
Asterisks indicate identical amino acid residues. The gene bank accession numbers are as follows: B. thuringiensis ATCC 35646, ZP
00743698; B. anthracis str. Sterne, YP 028797; B. anthracis str. A2012, ZP 00392985; B. cereus subsp. cytotoxis NVH 39, ZP 01178861.
(2000).
molecular breeding has been applied to other lipases or
esterases.16) In these cases, the modified enzymes had
improved stereospecificity. Furthermore, some enzymes
had increased catalytic activity and stability in the
presence of an organic solvent.17) We are currently
trying to make recombinant esterase from strain 809A in
E. coli, which should allow easy purification of this
enzyme and efficient synthesis of optically active
propanediol derivatives.
2) Nalampang, K., and Johnson, A. F., Kinetics of
polyesterification: modeling and simulation of unsaturat-
ed polyester synthesis involving 2-methyl-1,3-propane-
diol. Polymer, 44, 6103–6109 (2003).
3) Shahid, K. A., Li, Y. N., Okazaki, M., Shuto, Y., Goto,
F., and Kiyooka, S., A short synthesis of the C15–C21
segment of (+)-discodermolide, based on an asymmetric
approach from achiral 2-methyl-1,3-propanediol versa-
tile enantiopure stereotriads. Tetrahedron Lett., 43,
6373–6376 (2002).
Acknowledgments
4) Yanase, H., Arishima, T., Kita, K., Nagai, J., Sumio, M.,
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efficient chemo-enzymatic approach to the enantioselec-
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This work was supported financially by the Program
for Social Collaborative Research of the Ministry of
Education, Culture, Sports, Science and Technology of
Japan. The authors thank Drs. Susumu Ohira and
Atsuhito Kuboki of Okayama University of Science
for substrate synthesis and technical assistance.
6) Tsuji, K., Terao, Y., and Achiwa, K., Lipase-catalyzed
asymmetric synthesis of chiral 1,3-propanediols and its
application to the preparation of optically pure building
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