Highly enantioselective hydrolysis of phenyl-1,2-ethanediol cyclic carbonates by newly isolated Bacillus sp. ECU0015

Article abstract of DOI:10.1016/j.molcatb.2010.03.011

A bacterial strain (No. ECU0015), which catalyzes the hydrolysis of phenyl-1,2-ethanediol cyclic carbonates (4-phenyl-1,3-dioxolan-2-one, 1) to (S)-1-phenyl-1,2-ethanediol (2) with high enantioselectivity, was newly isolated from soil samples utilizing the cyclic carbonate as sole carbon and energy source. The bacterium was later identified as Bacillus species by its 16S rDNA sequence and phylogenetic analysis. The optimal reaction temperature and pH for the asymmetric hydrolysis of 1 using whole cells were 35 °C and pH 7.3, respectively. Partial bio-oxidation of the produced (R)-diol was observed, resulting in an increase in the ee_p (enantiomeric excess of product) of the main product (S)-diol. Under the improved reaction condition, the target product (S)-diol was prepared in gram scale, affording an excellent ee _p (>99%) with a moderate yield (27.8%) as compared to the maximum theoretical yield of 50% for kinetic resolution. This strain of Bacillus sp. also displayed fairly good activity and enantioselectivity towards some other compounds tested, such as 2-acetoxy-2-phenylacetic acid (3) and its derivatives.

Full text of DOI:10.1016/j.molcatb.2010.03.011

Journal of Molecular Catalysis B: Enzymatic 66 (2010) 95–100
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Highly enantioselective hydrolysis of phenyl-1,2-ethanediol cyclic carbonates by
newly isolated Bacillus sp. ECU0015
Lei Chang, Li-Ming Ouyang∗, Yi Xu, Jiang Pan, Jian-He Xu∗
Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, Shanghai 200237,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A bacterial strain (No. ECU0015), which catalyzes the hydrolysis of phenyl-1,2-ethanediol cyclic carbon-
ates (4-phenyl-1,3-dioxolan-2-one, 1) to (S)-1-phenyl-1,2-ethanediol (2) with high enantioselectivity,
was newly isolated from soil samples utilizing the cyclic carbonate as sole carbon and energy source. The
bacterium was later identified as Bacillus species by its 16S rDNA sequence and phylogenetic analysis. The
optimal reaction temperature and pH for the asymmetric hydrolysis of 1 using whole cells were 35 ◦C and
pH 7.3, respectively. Partial bio-oxidation of the produced (R)-diol was observed, resulting in an increase
in the eep (enantiomeric excess of product) of the main product (S)-diol. Under the improved reaction
condition, the target product (S)-diol was prepared in gram scale, affording an excellent eep (>99%) with
a moderate yield (27.8%) as compared to the maximum theoretical yield of 50% for kinetic resolution.
This strain of Bacillus sp. also displayed fairly good activity and enantioselectivity towards some other
compounds tested, such as 2-acetoxy-2-phenylacetic acid (3) and its derivatives.
Received 21 October 2009
Received in revised form 24 March 2010
Accepted 27 March 2010
Available online 7 April 2010
Keywords:
Cyclic carbonate
1-Phenyl-1,2-ethanediol
Enantioselective hydrolysis
Hydrolase
Bacillus sp.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
tioselective hydrolysis of racemic cyclic carbonate catalyzed by the
microorganism whole-cell systems would be much cheaper and
Because optically active diols and their cyclic carbonates are
important intermediates for the synthesis of pharmaceutical and
natural products, many efforts had already been devoted to effec-
tive preparation of such compounds with different methods [1–4].
However, the chemical synthesis of optically pure diols or cyclic
carbonates is still rather difficult and thus needs to be further
improved.
Enzymatic hydrolysis of cyclic carbonates is an attractive
method for the preparation of optically active diols because of the
unique advantages of bioreaction, such as no need of cofactors for
the hydrolytic reaction [5,6] and the high selectivity of hydrolase.
The carbonic acid produced in the reaction can be decomposed to
carbon dioxide and quickly released from the system, which will
facilitate the bioconversion to diol and the product purification as
well. Over the past several years, the application of porcine pan-
creas lipase (PPL) or pig liver esterase (PLE) in the enantioselective
hydrolysis of mono-substituted cyclic carbonates to obtain chiral
1,2-diols had received considerable attentions [7–11]. However,
PPL or PLE is relative expensive and unstable as a free enzyme for
the transformations of cyclic carbonates. On the contrary, enan-
more convenient. To the best of our knowledge, only one microbial
strain, Pseudomonas diminuta, has so far been reported and used
for such a biocatalytic hydrolysis. However, it could only hydrolyze
C2-symmetrical cyclic carbonates [12,13]. Therefore, the screening
for new microorganisms with high activity and stereoselectivity for
the hydrolysis of racemic cyclic carbonates become imperative.
The objective of the present study was to screen for a microbial
strain that harbors a stereoselective cyclic carbonate hydrolase for
the biocatalytic resolution of racemic cyclic carbonate (1) to (S)-diol
(2) with high conversion and good selectivity (Scheme 1).
2. Experimental
2.1. Soil sample, reagents and medium
Soil samples used for the isolation of bacteria with hydrolytic
activity were collected from different places of Shanghai, Jiangsu
and Shandong Provinces, China. Styrene oxide was purchased
from Sigma Chemical Co., USA. Cyclic carbonate 1, racemic diol
2, 2-acetoxy-2-phenylacetic acid 3 and its derivative (2-acetoxy-
2-(3ꢀ-chlorophenyl) acetic acid) 4 were prepared by our lab with
details described in Sections 2.2 and 2.3. All other chemicals were
obtained from commercial suppliers as reagent grade.
∗
Corresponding authors. Fax: +86 21 6425 0840.
E-mail addresses: ouyanglm@ecust.edu.cn (L.-M. Ouyang),
The mineral salts medium (MSM) for enrichment culture con-
sisted of the following components (per liter): (NH4)2SO4 1.0 g,
jianhexu@ecust.edu.cn (J.-H. Xu).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.03.011

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L. Chang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 95–100
Scheme 1. Enantioselective hydrolysis of phenyl-1,2-ethanediol cyclic carbonate 1 to (S)-diol 2 by the resting cells of Bacillus sp. ECU0015.
K2HPO4·3H2O 6.0 g, KH2PO4 3.0 g, NaCl 0.5 g, MgSO4·7H2O 0.5 g,
CaCl2 0.05 g, pH 7.0. The rich medium consisted of the following
components (per liter): glycerol 15 g, yeast extract 5.0 g, pep-
tone 5.0 g, Na2HPO4·12H2O 2.7 g, KH2PO4 1.0 g, MgSO4 0.5 g. The
medium was adjusted to pH 7.0 with NaOH before autoclaved at
121 ◦C for 20 min.
were identified by the TLC, were inoculated onto the agar plate of
the rich medium. The grown colonies were inoculated individually
to the rich liquid medium (2 ml) in test tubes. After 1–2 days cul-
ture, the hydrolytic activity was measured by TLC again. Positive
strains were plated onto the rich medium agar and preserved for
future use.
2.4.2. Secondary screening for microbial strains with high
enantioselectivity
2.2. Preparation of 1-phenyl-1,2-ethanediol (PED) and the
corresponding cyclic carbonate (PDC)
In the second round of screening, about 0.5–1.0 g (wet weight) of
washed cells were suspended in 9.5 ml of 50 mM potassium phos-
phate buffer (KPB, pH 7.0) and 0.5 ml of 200 mM cyclic carbonate
1 in methanol was added to give a final concentration of 10 mM.
The mixture was incubated at 30 ◦C and 160 rpm for 36 h. After
extraction with ethyl acetate and dehydration overnight with anhy-
drous Na2SO4, samples (0.5 ml each) of the reaction mixture were
withdrawn for measuring the substrate conversion and product
enantiomeric excess (eep) by HPLC.
The racemic diol 2 was prepared from styrene oxide. Styrene
oxide (1.5 g) was dissolved in 100 ml THF containing 10 ml water.
Then 0.2 ml H2SO4 was added and the reaction mixture was stirred
at 50 ◦C for 24 h. When the conversion reached 100%, saturated
NaHCO3 aqueous solution was added to neutralize the H2SO4 and
then the product (diol 2) was extracted with ethyl acetate. The
organic phase was dried over anhydrous Na2SO4 and then most of
the solvent was removed by vacuum distillation. The product was
crystallized with ethyl acetate and petroleum ether. The purity of
the product was tested by HPLC and the structure of product and
substrate was confirmed by NMR.
2.5. Identification of the bacterial strain ECU0015
The cyclic carbonate 1 was prepared from racemic diol 2 by
transesterification with dimethyl carbonate catalyzed by heat and
strong alkaline. The product, cyclic carbonate 1, was separated by
silica gel column chromatography with mobile phase of petroleum
ether and ethyl acetate (3/1, v/v). The purity of the product was
tested by HPLC and the structure of product was confirmed by NMR
too.
Cell morphology of the bacterial strain ECU0015 was observed
via an optical microscope. The bacterial genome DNA was extracted
according to the procedure of Wilson [14]. The 16S rDNA was
amplified by the polymerase chain reaction (PCR) with the uni-
versal primers (forward primer, 5ꢀ-AGAGTTTGATCCTGGCTCAG-3ꢀ;
reverse primer, 5ꢀ-AAGGAGGTGATCCAGCCGCA-3ꢀ). The PCR reac-
tions were performed with initial denaturation at 94 ◦C for 5 min,
30 cycles of denaturation at 94 ◦C for 1 min, annealing at 52 ◦C
for 1 min, extension at 72 ◦C for 1.5 min, and final extension at
72 ◦C for 5 min. PCR product was ligated into pMD18-T vector
(Takara, Dalian, China) and sequenced. DNA analysis was com-
pleted using BLAST network services provided by the National
Center for Biotechnology Information (NCBI), USA.
2.3. Preparation of 2-acetoxy-2-phenylacetic acid 3 and its
derivative 4
The organic acid 3 and its derivative 4 were prepared from the
corresponding mandelic acid. The mandelic acid and equal equiv-
alent of acetyl chloride were dissolved in 1,4-dioxane and refluxed
for 12 h. When the conversion reached 100%, the reaction was
stopped and the product mixture was vacuum distilled and the
resultant white crystals were dried for NMR verification.
2.6. Monitoring of cell growth and enzyme production of Bacillus
sp. ECU0015
The isolated strain, Bacillus sp. ECU0015, was grown aerobically
in 250-ml Erlenmeyer flasks containing 50 ml of the rich medium at
30 ◦C and 160 rpm for 60 h. The sterilized rich medium was inocu-
lated with 5% (v/v) of a 12 h pre-culture. At different time intervals,
two flasks were withdrawn for the determination of enzyme activ-
ity, dry cell weight (DCW) and medium pH. The cells were harvested
by centrifuge and used for hydrolytic activity assay. The pH value
was determined by a pH meter and the dry cell weight was mea-
sured after drying the wet cells harvested from 10 ml of culture
broth at 50 ◦C till a constant weight.
2.4. Isolation of hydrolase-producing microorganisms with high
activity on cyclic carbonate 1
2.4.1. Primary screening for strains with high conversion rate
A tiny of each soil sample was suspended in test tubes containing
2 ml of mineral salts medium. Then the suspension was supple-
mented with 40 ␮l of 0.5 M cyclic carbonate (1) in methanol as
the carbon source, giving a final substrate concentration of 10 mM.
The enrichment culture was shaken aerobically at 160 rpm and
30 ◦C for 1–2 days. Then the reaction was stopped and the mixture
was extracted by addition of 0.3 ml ethyl acetate. After centrifuge,
the ethyl acetate phase was dried over anhydrous Na2SO4. Thin-
layer chromatography (TLC) assay was performed using petroleum
ether/ethyl acetate (3:1, v/v) as developing agent. And then, the soil
culture samples with hydrolase-producing microorganisms, which
2.7. Enzyme assay
The hydrolytic activity of rest cells was measured by HPLC
as follows. The cells harvested from 2 ml of the culture broth
were washed with physiological saline solution (0.85% NaCl), then

L. Chang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 95–100
97
resuspended in 0.48 ml of KPB (50 mM, pH 7.3). The mixture was
2.11. Bioconversion of other ester substrates with resting cells of
Bacillus sp. ECU0015
preincubated on a mini-shaker (Thermomixer Compact, Eppendorf,
Germany) at 30 ◦C and vortexed at 1100 rpm for 10 min. Then the
reaction was started with the addition of 20 ␮l methanol solution
containing 250 mM cyclic carbonate 1. After 10 min of incubation,
the reaction was stopped by extracted with 0.5 ml ethyl acetate.
After centrifuge, the organic phase was withdrawn and dried over
anhydrous Na2SO4, and then subjected to HPLC analysis to deter-
mine the quantity of (S)-diol (2) formed. One unit of hydrolytic
activity was defined as the amount of enzyme catalyzing the for-
mation of 1.0 ␮mol (S)-diol per minute under the above conditions.
We also examined the hydrolytic reactions of other ester sub-
strates, viz. 3, 4 and dimethyl carbonate (DMC) 5 with resting
cells of Bacillus sp. ECU0015. The reaction condition was the
same as cyclic carbonate 1. As for substrates of 3 and 4, after
36 h of bioconversion, the cells were removed by centrifugation
and the supernatant was acidified with concentrated H2SO4 to
pH 1.0 and extracted with ethyl acetate. The ethyl acetate layer
was collected, dried over anhydrous Na2SO4 and concentrated
under reduced pressure to obtain a crude crystal of (R)-mandelic
acid or (R)-3-chloro-mandelic acid. The acids were purified by
silica gel column chromatography. The eluent components are
toluene–ethyl acetate–formic acid (5:1:0.2, v/v/v) for mandelic acid
and 3-chloromandelic acid.
2.8. Catalytic characteristics of Bacillus sp. ECU0015 resting cells
For the determination of optimal pH for the transformation by
whole cells, 0.2 g wet resting cells and 10 mM cyclic carbonate 1
was added into 2 ml of buffer with pH varying from 5.0 to 9.0. Two
kinds of buffers were used, viz. KPB for pH 5.0, 6.0, 7.0 and 8.0, and
Na2CO3–NaHCO3 buffer for pH 9.0. The reactions were carried out
at 30 ◦C and 1000 rpm for 30 min.
To investigate the optimal reaction temperature, the reactions
were set at different temperatures from 20 to 50 ◦C in buffer of pH
7.3 and the other reaction conditions were same as the above.
To investigate the thermal stability of the microbial enzyme,
the cells were suspended (10%, w/v) in 20 ml KPB (100 mM, pH 7.3)
and preserved at different temperature (4, 30 and 60 ◦C) for 5 days.
Enzyme activity was determined each day by the standard method
as described in Section 2.7.
As for the substrate of DMC, the residual substrate was detected
by gas chromatography (SHIMADZU GC-14C, ␤-cyclodextrin
capillary column, length = 30 m, diameter = 0.5 mm, column tem-
perature 150 ◦C, injector temperature 280 ◦C, detector temperature
320 ◦C) after 36 h of transformation.
2.12. HPLC method
The concentrations and enantiomeric excess (ee) of the
diol 2 and its cyclic carbonate 1 were determined by HPLC
(LC-10AT, Shimadzu, Japan) using a chiral column (Chiralcel
OD, Ø4.6 mm × 250 mm, Daicel, Japan). The mobile phase was
hexane/2-propanol (95:5, v/v) and the flow rate was 1.0 ml/min.
Detection was made at 254 nm. The retention times for (R)-, (S)-2
and (R)-, (S)-1 were 20.8, 22.1, 38.9 and 44.9 min, respectively. The
ee of (S)-2 was calculated as follows, where [S] and [R] denote the
concentrations of (S)-2 and (R)-2, respectively:
To investigate the pH stability of the whole-cell biocatalyst, the
cells were suspended (10%, w/v) in 20 ml 100 mM buffer with dif-
ferent pH (KPB for pH 5.0 and 7.3, Na2CO3–NaHCO3 for pH 9.0) and
preserved at 30 ◦C for 5 days. Enzyme activity was determined each
day with the standard method as described in Section 2.7.
S − R
[ ] [ ]
S + R
[ ] [ ]
ee =
× 100%
2.9. Bioconversion process of PDC with resting cells of Bacillus sp.
ECU0015
2.13. Compound characterization
The bioconversion of cyclic carbonate 1 with resting cells was
performed in three-necked flask. Resting cells (1.0 g wet cell) was
suspended in 9.6 ml of KPB (50 mM, pH 7.3), and 400 ␮l methanol
solution of 250 mM cyclic carbonate 1 was added to give a final
concentration of 10 mM. The reaction was carried out at 30 ◦C and
160 rpm. The bioconversion process was monitored with HPLC by
withdrawing 500 ␮l each of samples at fixed time intervals. The
reaction was stopped by adding 500 ␮l of ethyl acetate to 500 ␮l
of the sample. The cells were removed by centrifugation and the
organic phase was used for the determination of the product con-
centration and eep with HPLC.
1: 1H NMR (500 MHz, MeOH), ı/ppm: 4.37 (t, J = 8.7 Hz, 1H), 4.82
(t, J = 8.2 Hz, 1H), 5.77 (t, J = 7.9 Hz, 1H), 7.41 (m, 5H).
(S)-2: [˛]2D5 + 56.5 (c 0.5, MeOH). 1H NMR (500 MHz, MeOH),
ı/ppm: 3.60 (t, J = 5.48 Hz, 2H), 4.67 (t, J = 6.03 Hz, 1H), 7.20–7.40
(m, 5H) [11].
(R)-Mandelic acid: [˛]2D5 − 147.8 (c 0.5, MeOH). 1H NMR
(500 MHz, MeOH), ı/ppm: 5.19 (s, J = 5.17 Hz, 1H), 7.33 (m, 5H).
(R)-3-Chloro-mandelic acid: [˛]2D5 − 124.1 (c 0.5, MeOH). 1H
NMR (500 MHz, MeOH), ı/ppm: 5.18 (s, J = 8.86 Hz, 1H), 7.29 (m,
3H), 7.44 (m, 1H) [15].
2.10. Preparation of enantiopure (S)-diol 2 by resting cells of
Bacillus sp. ECU0015 in gram scale
3. Results and discussion
3.1. Isolation and identification of microorganisms for
biohydrolysis of PDC
Fifteen grams of resting wet cells of Bacillus sp. ECU0015 were
suspended in 96 ml of KPB (50 mM, pH 7.3), and 4.0 ml cyclic car-
bonate 1 solution (250 mM, dissolved in methanol) was added
to give a final substrate concentration of 10 mM. The mixture
was incubated at 30 ◦C and 160 rpm with rotated shaker. After
incubation for 36 h, the mixture was saturated with NaCl and
then extracted for four times with 50 ml ethyl acetate each time.
After dried over anhydrous Na2SO4, the ethyl acetate was evap-
orated under reduced pressure. The crude product of (S)-diol 2
was purified by silica gel column chromatography with petroleum
ether/ethyl acetate (4:1, v/v) as eluent. The product structure was
confirmed by 1H NMR. The absolute configuration was determined
by comparing the optical rotation measured with that of literature.
In the first round of screening, many kinds of microorganisms
were found to have hydrolytic activity on cyclic carbonate (1).
Using different rich media, 500 strains were isolated from over
200 soil samples by enrichment culture and used for the analysis
of hydrolytic activity on cyclic carbonate 1. The amount of prod-
uct formed was determined by TLC. Then 66 strains with obvious
product (diol, 2) spots on TLC plates were subjected to next round
of screening.
During the secondary round of screening, the activities and
enantioselectivities of the 66 strains in the bioconversion of cyclic
carbonate (1) were investigated with HPLC analysis, and the

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L. Chang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 95–100
Fig. 2. Profiles of cell growth and enzyme production of Bacillus sp. ECU0015. Sym-
bols: (ꢀ) cyclic carbonate hydrolase activity; (♦) biomass; (ꢁ) pH.
Fig. 1. The results of secondary round screening. Sixty-six strains of microorganisms
with obvious carbonate hydrolase activity were found, which could enantioselec-
tively hydrolyze substrate 1 into product 2. Symbols: (᭹) the strain of No. ECU0015;
(ꢁ) the other strains.
anism is proposed for the biosynthesis of (S)-2 from the racemic
cyclic carbonate (1) by the bacterial strain ECU0015, as shown in
Scheme 2.
This newly isolated microbial strain (ECU0015) was a rod-shape,
gram positive bacterium by observation via an optical microscope.
The 16S rDNA sequence of our strain was deposited in the Gen-
Bank with an accession number of GQ304942. The sequence was
100% identical with the 16S rDNA sequence of Bacillus sp. N6 in the
Genbank, so the strain ECU0015 was identified as Bacillus species
and subsequently marked as Bacillus sp. ECU0015. This strain has
been deposited in China General Microbiological Cultures Center
(CGMCC), with an accession number of CGMCC 2874.
results were shown in Fig. 1. Among the strains preferentially
producing (S)-isomer of the diol (2), 7 strains showed relatively
high enantioselectivities (eep > 70%). This result indicates that the
microorganisms preferentially producing (S)-2 were widespread in
nature, while those preferentially producing (R)-2 were relatively
rare.
Among the active strains, three bacterial strains showing good
enantioselectivity, with eep higher than 90%, were selected for
further study. In the subsequent experiments, it was found surpris-
ingly that with one of them the ee of (S)-2 could be improved up to
higher than 99% when the reaction time was lengthened. This result
indicates that the enzymatic hydrolysis may afford the optically
pure (S)-diol. On the other hand, the increase of ee of (S)-2 during
the reaction may be due to the further oxidation of the opposite iso-
mer (R)-2. It was confirmed that the bacterium could preferentially
oxidize the (R)-2 and the formation of ␣-hydroxyacetophenone
was also demonstrated in an independent biodegradation exper-
iment of rac-2. And then the hydroxy ketone could be transformed
to some other substances (data not shown) and it was difficult to
quantify the amount of hydroxy ketone. Therefore, a reaction mech-
3.2. Cell growth and enzyme production of Bacillus sp. ECU0015
To further characterize the new bacterial isolate, time course of
the enzyme production was monitored by cultivating Bacillus sp.
ECU0015 in 250-ml flasks containing 50 ml fermentation medium.
As shown in Fig. 2, the maximum cyclic carbonate hydrolase activ-
ity (5.08 U/l) was observed at about 36 h and the hydrolase activity
increased in parallel with the cell growth during the first 36 h. Then
the enzyme activity began to decline whereas the cell growth con-
tinued until 48 h. The change of pH during the cultivation was little,
with a trend of slight decline.
Scheme 2. The proposed reaction mechanism for Bacillus sp. ECU0015 whole-cells catalyzed bioconversion of phenyl-1,2-ethanediol cyclic carbonate to ␣-
hydroxyacetophenone.

L. Chang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 95–100
99
Fig. 4. Effects of preservation temperature (A) and pH (B) on the stability of the
enzyme. Symbols: A: thermal stability. The cells were suspended (10%, w/v) in 20 ml
KPB (100 mM, pH 7.3) and preserved at different temperature for 5 days. (ꢂ) 4 ◦C;
(ꢃ) 30 ◦C; (ꢀ) 60 ◦C. B: pH stability. The cells were suspended (10%, w/v) in 20 ml
100 mM buffer with different pH and preserved at 30 ◦C for 5 days. The initial enzyme
activity was regarded as 100%. Under certain conditions, the ratio of enzyme activity
measured after preservation and the initial value was defined as the residual activity.
(ꢂ) pH 5.0; (ꢃ) pH 7.3; (ꢀ) pH 9.0.
The cells kept similar enzyme activity when preserved at differ-
ent pHs for 5 days, varying from 40 to 50%, while the neutral pH of
7.3 is the best, followed by pH 9.0 and 5.0 (Fig. 4B).
Based on the results above, the hydrolysis of cyclic carbonate
1 by resting cells of Bacillus sp. ECU0015 was performed under
the optimal reaction conditions, 30 ◦C and pH 7.3, with an initial
substrate concentration of 10 mM.
Fig. 3. Effects of reaction temperature (A) and pH (B) on the cyclic carbonate
hydrolase activity of Bacillus sp. ECU0015 resting cells. Experimental methods were
described in Section 2.8.
3.3. Catalytic performance of Bacillus sp. ECU0015 resting cells
and the preparation of enantiopure (S)-2 in gram scale
As shown in Fig. 5, an ee of 97.3% for the product was achieved
after 40 h in reaction volume of 10 ml, at 36.5% conversion. How-
The strain of Bacillus sp. ECU0015 could catalyze the asymmetric
hydrolysis of substrate 1 with high enantioselectivity. To further
optimize the catalytic conditions for the biohydrolysis of ester 1,
the initial rate of enzymatic hydrolysis (10 mM) catalyzed by the
resting cells was measured at different pHs and temperatures by the
method described in Section 2.8. As shown in Fig. 3A, the enzyme
showed the maximum activity at 35 ◦C. However, considering the
thermo stability of enzymes, we chose 30 ◦C as the optimal reaction
temperature. As shown in Fig. 3B, the optimum pH was about 7.0.
When the pH was over 7.0, the whole-cell activity decreased and
the spontaneous hydrolysis of substrate increased, and when the
pH was below pH 6.0 the activity became very low. Finally we chose
7.3 as the pH optimum to reduce the inhibition caused by the by-
product CO2.
As for the stability of microbial cells under different stor-
age conditions, low temperature and mild pH were helpful for
the maintenance of enzyme activity. When preserved at 4 ◦C, the
enzyme activity was maintained over 70% even at the 5th day. The
residual enzyme activities were about 70 and 50%, respectively,
when preserved at 30 ◦C for 3 and 5 days. However, the enzyme
lost 90% of its initial activity when the cells were preserved at 60 ◦C
for 3 days (Fig. 4A).
Fig. 5. Time course of enantioselective hydrolysis of phenyl-1,2-ethanediol cyclic
carbonate 1 (20 mM) into diol 2 by Bacillus sp. ECU0015. Symbols: (ꢂ) concentration
of (S)-diol; (♦) concentration of (R)-diol; (ꢃ) enantiomeric excess (ee) of (S)-diol.

100
L. Chang et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 95–100
Fig. 6. Structures of substrates 3, 4 and 5.
ever, the ee and yield of residual carbonate 1 was 81.1% and 22.4%,
as determined by HPLC.
cells of Bacillus sp. ECU0015 catalyzed the enantioselective hydrol-
ysis of cyclic carbonates, producing (S)-1-phenyl-1,2-ethanediol in
high ee value (>99%) and moderate yield. It can also catalyze the
enantioselective hydrolysis of other chiral esters, such as substrate
3 and 4, affording (R)-mandelic acid (97.8% ee) and (R)-3-chloro-
mandelic acid (>99% ee) in satisfactory optical purities.
In the preparation of diol 2 in gram scale, 15 grams of rest-
ing cells of Bacillus sp. ECU0015 were suspended in 96 ml of KPB
(50 mM, pH 7.3), and 4.0 ml cyclic carbonate 1 solution (250 mM,
dissolved in methanol) was added to give a final substrate concen-
tration of 10 mM. After incubation at 30 ◦C, 160 rpm for 36 h, the
reaction was stopped and the product of (S)-2 was extracted as
described in section 2.10, with an excellent enantiomeric excess (>
99%, after refined) and a moderate yield (27.8%).
Acknowledgements
This research was financially supported by the National Natural
Science Foundation of China (Nos. 20773038 and 20902023), Min-
istry of Science and Technology, PR China (Nos. 2007AA02Z225,
2009CB724706 & 2009ZX09501-016), China National Special Fund
for State Key Laboratory of Bioreactor Engineering (No. 2060204),
Shanghai Municipal Fund for Natural Science (No. 07ZR14030) and
Shanghai Leading Academic Discipline Project (No. B505).
3.4. Substrate spectrum of Bacillus sp. ECU0015
To examin the substrate spectrum of Bacillus sp. ECU0015, sub-
strates 1, 3, 4 and 5 (Fig. 6) were tested. The strain displayed
pretty high activity and stereoselectivity for majority of the sub-
strates (10 mM) examined. In the case of 3 and 4, high yields (31.5%
and 26.5%, respectively) and almost enantiopure (R)-mandelic acid
and (R)-3-chloro-mandelic acid were obtained. While non-cyclic
dimethyl carbonate 5 could be hydrolyzed to methanol and carbon
dioxide completely.
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4. Conclusions
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activity and ideal stereoselectivity for the transformation of cyclic
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