Enantioselective production of (S)-1-phenyl-1,2-ethanediol from dicarboxyesters by recombinant Bacillus subtilis esterase

Article abstract of DOI:10.1016/j.molcatb.2011.07.022

The whole cells of recombinant Escherichia coli BL21 overexpressing a Bacillus subtilis esterase (BsE) were utilized to sequentially hydrolyze the dicarboxyester of 1-phenyl-1,2-ethanediol for production of (S)-1-phenyl-1,2- ethanediol (PED), exhibiting high hydrolytic activity, excellent regio- and enantioselectivities towards the dicarboxyester of PED. Among the dicarboxyesters with different acyl chains (e.g., acetyl, n-butyl, and n-hexyl), the best enantioselectivity (E = 176) was observed when PED diacetate was employed as the initial substrate. Various reaction conditions were systematically investigated for enantioselective hydrolysis of PED diacetate. Under the optimal reaction conditions, kinetic resolution of 100 mM PED diacetate resulted in 49% conversion within 1 h, affording (S)-PED in 96% ee. A 150-ml scale reaction was performed, affording (S)-PED in 49% yield and 95% ee. After recrystallization in chloroform, the optical purity of (S)-PED was improved up to >99% ee, with a total yield of 45%. These results imply that this recombinant esterase (BsE) is a potentially promising biocatalyst for bioproduction of (S)-PED, an important chiral building block with wide application in pharmaceutical industry and liquid-crystal display materials.

Full text of DOI:10.1016/j.molcatb.2011.07.022

Journal of Molecular Catalysis B: Enzymatic 73 (2011) 80–84
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enantioselective production of (S)-1-phenyl-1,2-ethanediol from
dicarboxyesters by recombinant Bacillus subtilis esterase
Xin Tiana, Gao-Wei Zhenga,∗, Chun-Xiu Lia, Zhi-Long Wangb, Jian-He Xua,∗
a Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering,
East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
b School of Pharmacy, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The whole cells of recombinant Escherichia coli BL21 overexpressing a Bacillus subtilis esterase (BsE) were
utilized to sequentially hydrolyze the dicarboxyester of 1-phenyl-1,2-ethanediol for production of (S)-1-
phenyl-1,2-ethanediol (PED), exhibiting high hydrolytic activity, excellent regio- and enantioselectivities
towards the dicarboxyester of PED. Among the dicarboxyesters with different acyl chains (e.g., acetyl, n-
butyl, and n-hexyl), the best enantioselectivity (E = 176) was observed when PED diacetate was employed
as the initial substrate. Various reaction conditions were systematically investigated for enantioselective
hydrolysis of PED diacetate. Under the optimal reaction conditions, kinetic resolution of 100 mM PED
diacetate resulted in 49% conversion within 1 h, affording (S)-PED in 96% ee. A 150-ml scale reaction
was performed, affording (S)-PED in 49% yield and 95% ee. After recrystallization in chloroform, the
optical purity of (S)-PED was improved up to >99% ee, with a total yield of 45%. These results imply
that this recombinant esterase (BsE) is a potentially promising biocatalyst for bioproduction of (S)-PED,
an important chiral building block with wide application in pharmaceutical industry and liquid-crystal
display materials.
Received 9 January 2011
Received in revised form 29 July 2011
Accepted 30 July 2011
Available online 6 August 2011
Keywords:
Bacillus subtilis esterase
(S)-1-Phenyl-1,2-ethanediol
Dicarboxyester
Enantioselective hydrolysis
Kinetic resolution
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
synthesis of enantiomerically pure 1,2-diols. However, microbial
reduction involves in complicated cofactors regeneration, since
Optically pure 1,2-diols are important precursors and inter-
mediates for variety of synthetic applications [1]. Among
most of oxidoreductases are cofactor-dependent. Although epoxide
hydrolases are cofactor-independent, they usually produce (R)-
diols, which leads to less variety. Optically active (S)-PED can also
be obtained by lipase-mediated transesterification in organic sol-
vents, though direct transesterification of 1,2-diols is less effective
in most organic solvents, giving the corresponding monoacetate
with poor enantioselectivity in the case of a number of lipases
tested [20–22].
Esterases (E.C. 3.1.1.1) and lipases (E.C. 3.1.1.3) are a class
of versatile biocatalysts for the preparation of chiral compounds
in industry as they are stable, frequently exhibit high activi-
ties as well as chemo-, regio- or stereoselectivity, and do not
require expensive cofactors [23,24]. In the work described herein,
whole cells of recombinant Escherichia coli BL21 overexpressing an
esterase from Bacillus subtilis ECU0554 (BsE) were evaluated for
(S)-PED formation in aqueous medium through sequential hydrol-
ysis of corresponding dicarboxyester. The recombinant BsE showed
high hydrolytic activity, excellent regio- and stereoselectivity for
the production of optically pure (S)-PED. Some dicarboxyesters
with different acyl chains (acetyl, n-butyl and n-hexyl) were
employed as substrate. Various reaction conditions were sys-
tematically investigated for enantioselective hydrolysis of PED
diacetate.
a
these 1,2-diols, enantiomerically pure 1-phenyl-1, 2-ethanediol
(PED) is widely used in synthesis of chiral catalysts [2,3],
macrocyclic polyether [4], pharmaceutically active com-
pounds [5] and liquid crystals [6]. Thus, preparation of such
chiral entities has triggered modern researchers to investi-
gate various synthetic methods, including chemocatalysis and
biocatalysis.
As PED has two hydroxyl groups, it needs selective protection
of one hydroxyl to prepare optically pure PED with chemical meth-
ods, which takes more synthetic steps and leads to more pollution.
Therefore, bioproduction of optically pure (R) or (S)-PED becomes
more and more attractive. Biocatalytic methods, such as biore-
duction of ␣-hydroxy ketones [7–10], hydrolysis of epoxides with
epoxide hydrolases [11–13] and lipase-mediated kinetic resolu-
tion of vicinal diols or esters [14–19], have been applied for the
∗
Corresponding authors. Tel.: +86 21 6425 2498; fax: +86 21 6425 0840.
E-mail addresses: gaoweizheng@ecust.edu.cn (G.-W. Zheng),
jianhexu@ecust.edu.cn (J.-H. Xu).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.07.022

X. Tian et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 80–84
81
2. Experimental
2.1. Microorganisms and materials
The wild-type strain, B. subtilis ECU0554, was isolated and
identified in our previous work [25]. Recombinant E. coli BL21 over-
expressing BsE was constructed as described previously [26].
(R,S)-PED was purchased from Yueyang Yetop Fine Chemicals
Co. (Hunan, China). Acetic anhydride, n-butyric anhydride and
n-hexanoic anhydride were purchased from Alfa Aesar (Tianjin,
China). All other chemicals were also commercially available, with
purity of analytic grade.
2.2. General procedure for chemical synthesis of dicarboxyesters
To a certain anhydride (3 equiv.), was added 5.52 g (0.04 mol) of
(R,S)-PED, and the mixture was refluxed. The reaction was stopped
until (R,S)-PED was completely consumed as monitored by TLC (a
mixed solvent of petroleum ether and ethyl acetate was employed
as the mobile phase. The volume ratios of petroleum ether to ethyl
acetate for 1a, 2a and 3a were respectively 10:1, 15:1, 20:1, and
their corresponding Rf values were 0.7, 0.85, 0.9).
Scheme 1. Sequential hydrolysis process of 1-phenyl-1,2-ethanediol diacetate by
recombinant BsE. The dotted line indicates spontaneous reaction.
2.5. Analytical methods
The product was extracted into ethyl acetate (100 ml 3×), then
washed with an aqueous solution of saturated sodium bicarbonate
(100 ml 3×) and dried over anhydrous sodium sulfate. The organic
solvent was evaporated under reduced pressure and the product
was dried under vacuum.
Samples (250 ␮l each) were withdrawn at different time inter-
vals and immediately extracted with ethyl acetate (500 ␮l). After
centrifugation (8500 × g, 5 min), the samples were dried over anhy-
drous sodium sulfate for 12 h and filtered with 0.45 ␮m membrane
filter.
After filtration, samples from the hydrolysis reaction mixture
were analyzed on a HPLC (Agilent-1100) equipped with a chiral
column Chiracel OB-H (˚ 0.46 cm × 25 cm, Daicel, Japan), and were
isocratic eluted with hexane:isopropanol = 90:10 (v/v) at a flow
rate of 1 ml/min and detected at 210 nm. 20 ␮l of each sample was
injected at room temperature. The retention times for compounds
were shown as follows: (R)-PED, 7.0 min; (S)-PED, 8.8 min; (R,S)-1a,
11.2 min; (S)-1b, 10.3 min; (R)-1b, 14.5 min; (R,S)-2a, 43.6 min; (S)-
2b, 20.7 min; (R)-2b, 29.1 min; (R,S)-3a, 71.2 min; (S)-3b, 42.8 min;
(R)-3b, 55.4 min.
(R,S)-1-Phenyl-1,2-ethanediol diacetate (1a): 1H NMR (300 MHz,
CDCl3), ı/ppm: 2.10 (s, 6H), 4.10 (dd, 1H, J = 11.89, 8.68 Hz), 4.20
(dd, 1H, J = 11.89, 3.39 Hz), 4.90 (dd, 1H, J = 8.25, 3.39 Hz), 7.2–7.4
(m, 5H).
(R,S)-1-Phenyl-1,2-ethanediol butyrate (2a): 1H NMR (300 MHz,
CDCl3), ı/ppm: 0.92 (t, J = 7.4 Hz, 3H), 0.94 (t, J = 7.3 Hz, 3H),
1.71–1.59 (m, 4H), 2.29 (t, J = 7.3 Hz, 2H), 2.36 (t, J = 7.4 Hz, 2H),
4.36–4.26 (m, 2H), 6.04 (dd, J = 7.5, 4.7 Hz, 1H), 7.38–7.27 (m, 5H).
(R,S)-1-Phenyl-1,2-ethanediol hexanoate (3a): 1H NMR
(300 MHz, CDCl3), ı/ppm: 0.88 (t, J = 6.6 Hz, 3H), 0.89 (t, J = 6.8 Hz,
3H), 1.34–1.26 (m, 8H), 1.67–1.56 (m, 4H), 2.29 (t, J = 7.6 Hz, 2H),
2.36 (t, J = 7.5 Hz, 2H), 4.35–4.27 (m, 2H), 6.02 (dd, J = 7.5, 4.2 Hz,
1H), 7.36–7.01 (m, 5H).
3. Results and discussion
2.3. Recombinant E. coli cell production
3.1. Sequential hydrolysis of diacetate for (S)-PED formation
Production of recombinant E. coli BL21 cells was performed as
described previously [26]. The cells were harvested by centrifuga-
tion at 8500 × g for 10 min and washed twice with physiological
saline and stored at 4 ◦C for further use. The recombinant cell can
be stored at 4 ◦C for 2 weeks without any enzyme activity decrease,
and still retains 96% of its initial activity for 2 months.
The BsE in whole-cell system was more stable than the isolated
enzyme. The half-time of the esterase in whole-cell system was
11-fold higher than that of the isolated enzyme [26]. We therefore
employed recombinant whole cell of E. coli BL21 as biocatalyst for
production of (S)-PED.
The catalytic performance of biocatalyst was studied using
(R,S)-1a as a model substrate. As shown in Scheme 1, during
the hydrolysis process of diacetate, a secondary ester 1b was
first detected. It was liberated in a highly regioselective but non-
enantioselective manner, occurring far from the stereocentre. This
process proceeded very fast and the initial reaction rate of the first
step is 36.8 mM/min, and 1b was formed without any enatioselec-
tivity. Then 1b was transformed to (S)-PED by further enzymatic
hydrolysis, and the amount of (S)-PED increased with the reaction
time. Compared with the first step, the initial reaction rate of the
second step was 6.2 mM/min, which is much slower, but the over-
all reaction could also complete within 1 h (Fig. 1). (S)-PED was
obtained in 49% yield and 96% ee (Table 2, entry 1). As we know,
the use of (R,S)-1b as substrate is a chemical way to force the hydrol-
ysis to occur at merely the stereocentre rather than elsewhere in
the molecule of (R,S)-1a. However, the chemical synthesis of 1b
needs protecting groups which increases synthetic steps and for
2.4. Enantioselective hydrolysis of dicarboxyesters
The resting cells of recombinant E. coli BL21 were resuspended
in 0.9 ml KPB (potassium phosphate buffer, 200 mM, pH 7.0).
The enantioselective hydrolysis reaction was initiated by adding
racemic dicarboxyester dissolved in 0.1 ml ethanol into a shaking
incubator to give a final substrate concentration of 100 mM at 30 ◦C,
180 rpm. Samples (250 ␮l each) were withdrawn at different time
intervals and immediately extracted with ethyl acetate (500 ␮l).
After centrifugation (8500 × g, 5 min), the samples were analyzed
by HPLC. Data are means of at least three parallel reactions.
Enantiomeric excess of PED (eep) and that of correspond-
ing secondary ester (ees) were determined by HPLC. Conversion
of corresponding secondary ester (c) was calculated as
c
(%) = ees/(ees + eep) × 100. Enantioselectivity (E) was calculated as
E = ln[(1 − c)(1 − ees)]/ln[(1 − c)(1 + ees)].

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X. Tian et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 80–84
100
80
60
40
20
0
100
100
80
60
40
20
0
100
80
60
80
60
40
20
0
40
20
0
5
6
7
8
9
10
pH
0
10
20
30
40
50
60
70
Reaction time (min)
Fig. 2. Effects of pH on rac-1a sequential hydrolysis catalyzed by recombinant E. coli
BL21 overexpressing BsE. Reaction condition: 4.5 ml buffer with pH ranging from
6.0 to 9.0 (200 mM KPB, pH 6.0∼8.0 and 200 mM glycine–NaOH, pH 8.0∼9.0), 0.5 ml
ethanol dissolving 1 M rac-1a, wet cells of 10 mg ml−1, 30 ◦C, 180 rpm. (ꢀ) Relative
activity in KPB buffer; (ꢁ) relative activity in glycine–NaOH buffer; (ꢂ) ee in KPB
buffer; (ꢁ) ee in glycine–NaOH buffer.
Fig. 1. Time course of rac-1a sequential hydrolysis catalyzed by recombinant E. coli
BL21 overexpressing BsE. Reaction conditions: 0.9 ml KPB buffer (200 mM, pH 7.5),
0.1 ml ethanol dissolving 1 M rac-1a, wet cells of 10 mg ml−1, 35 ◦C, 180 rpm. (ꢀ)
rac-1a; (ꢁ) 1b; (•) PED; (ꢀ) ee of PED.
pH 7.5 and 35 ◦C were chosen as the optimal reaction conditions
and were used in the subsequent experiments.
this reason it should be avoided when other possibilities are at
hand. Therefore, direct hydrolysis of PED diacetate (1a), instead
of PED monoacetate (1b), seems more reasonable both technically
and economically.
The cost of biocatalyst is also a limitation for practice. To find the
most economical cell concentration for the biotransformation, we
examined the effect of cell concentration on the kinetic resolution
of 1a. With the decrease of cell concentration, the reaction time
increased. Unexpectedly, the ee of PED also declined correspond-
ingly, as shown in Table 1. When the reaction lasted for a longer
time, 1c had been detected. Further study showed that 1b was not
stable in aqueous medium, if the reaction was hold for a longer
period, a part of rac-1b could be transformed into rac-1c, which was
hydrolyzed without stereoselectivity, thus leading to the decrease
of ee value. Nevertheless, due to the high activity of recombinant
BsE towards 1a, the enzymatic hydrolysis can completely finish
within 1 h. During this period, the acyl transfer from the secondary
position to the primary position was not detected. When the cell
concentration was increased up to 20 mg (wet cell) ml−1, the reac-
tion rate did not change so much. So 10 mg ml−1 was selected as the
favorable cell concentration for kinetic resolution. In initial explo-
rative experiments, more than 100 wild-type strains were isolated
from the soil. Unfortunately, the results were not so satisfactory,
3.2. Optimization of reaction conditions for (S)-PED
bioproduction
It is well known that the buffer pH plays an important role in
enzymatic reaction. The variation of pH will alter the ionic state
of the substrate and the stereochemical configuration of enzyme
in the neighborhood of active sites, which in turn influence the
enzymatic activity and enantioselectivity. In order to determine
the optimum pH for the hydrolysis of 1a, the reaction was carried
out at different pH ranging from 6.0 to 9.0 at 30 ◦C. As shown in
Fig. 2, the optimum pH was around 7.5, which was similar to the
optimum pH (7.4) of the esterase from B. subtilis (BS2) and its dou-
ble mutant E188W/M193C used for removal of carboxyl protecting
groups of glutamate diesters [27]. Moreover, the ee of PED was not
sensitive to pH changes and high ee values could be obtained under
all conditions, being kept at >95%.
Effect of temperatures on the biocatalytic resolution of 1a was
also studied. The ee of PED and relative activities at different tem-
peratures (20−50 ◦C) are shown in Fig. 3. The relative activity
of the enzyme increased when the temperature was raised from
20 ◦C to 35 ◦C. But at higher temperatures, activities of the enzyme
decreased dramatically. Only very slight changes in the ee of (S)-PED
were observed as the temperature changed. It declined only slightly
to 94.5% ee at 50 ◦C. Therefore, based on the results obtained above,
120
100
100
80
60
40
20
80
60
40
Table 1
Effect of cell concentration on sequential hydrolysis of rac-1a catalyzed by recom-
binant E. coli BL21 overexpressing BsE.a
20
Cell concentration (mg ml−1
)
t (min)
ees (%)b
eep (%)b
c (%)c
2
5
10
20
300
120
60
75
77
96
97
72
87
96
97
51
47
50
50
0
0
15
25
35
45
55
45
Temperature (oC)
a
Reactions were carried out in 0.9 ml KPB buffer (200 mM, pH 7.5) with 0.1 ml
ethanol dissolving 100 mM rac-1a and different concentration of wet cells at 35 ◦C,
180 rpm.
Fig. 3. Effects of temperature on rac-1a sequential hydrolysis catalyzed by recombi-
nant E. coli BL21 overexpressing BsE. Reaction condition: 4.5 ml KPB buffer (200 mM,
pH 7.5), 0.5 ml ethanol dissolving 1 M rac-1a, wet cells of 10 mg ml−1, temperature
ranging from 30 ◦C to 50 ◦C, 180 rpm. (ꢃ) Relative activity, (ꢁ) ee of PED.
b
ee of 1b (ees) and ee of PED (eep) were determined by HPLC.
Conversion of rac-1b (c) was calculated as c (%) = ees/(ees + eep) × 100.
c

X. Tian et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 80–84
83
Table 2
Enantioselectivity of BsE-producing recombinant E. coli BL21in kinetic resolution of
1a–3a.
Substrate
t (min)
ees (%)a
eep (%)a
c (%)b
Ec
rac-1a
rac-2a
rac-3a
60
60
160
96
44
20
96
95
91
50
32
18
176
65
26
Reactions were carried out in 0.9 ml KPB buffer (200 mM, pH 7.5) with 0.1 ml ethanol
dissolving 100 mM different substrates and 10 mg ml−1 of wet cells at 35 ◦C, 180 rpm.
a
ee of PED (ees) and eep (ee of corresponding secondary ester) were determined
by HPLC.
b
Conversion of corresponding secondary ester (c) was calculated as
c
Scheme 2. Dicarboxyesters of PED and their correponding primary monoesters and
(%) = ees/(ees + eep) × 100.
secondary monoesters.
c
Enantioselectivity (E) was calculated as E = ln[(1 − c)(1 − ees)]/ln[(1 − c)(1 + ees)].
70
60
50
40
30
20
10
0
50
40
30
20
10
0
0
30
60
90
120
150
180
210
240
270
Time (min)
Fig. 5. Time courses of sequential hydrolysis of dicarboxyesters with increasing lin-
ear acyl chains catalyzed by recombinant E. coli BL21 overexpressing BsE. Reaction
conditions: 0.9 ml KPB buffer (200 mM, pH 7.5), 0.1 ml ethanol dissolving 1 M dif-
ferent dicarboxyesters, wet cells of 10 mg ml−1, 35 ◦C, 180 rpm, different substrate:
(ꢃ) rac-1a; (•) rac-2a; (ꢁ) rac-3a.
0
12
24
36
48
60
72
84
96
Reaction time (min)
Fig. 4. Time courses of rac-1a hydrolysis at different concentrations catalyzed by
recombinant E. coli BL21 overexpressing BsE. Reaction conditions: 0.9 ml KPB buffer
(200 mM, pH 7.5), 0.1 ml ethanol dissolving rac-1a, wet cells of 10 mg ml−1, 35 ◦C,
180 rpm, final concentration of rac-1a: (ꢃ) 150 mM; (ꢀ) 100 mM; (ꢁ) 50 mM.
enantiomeric ratio (E) were determined, as listed in Table 2. The
recombinant E. coli cells exhibited the highest hydrolytic activ-
ity and most satisfactory enantioselectivity (E = 176) towards 1a
among the three substrates tested. As compared with 1a, longer
reaction time was needed for 2a and 3a to achieve an ideal conver-
sion of 49%, especially for 3a (Fig. 5). The reason might be attributed
to the longer chain length of acyl group which may hinder the
movement of substrates into active site of the enzyme [27]. What
was worse, the ee of (S)-PED decreased to 78% when 3a was used
as substrate. Therefore, 1a was selected as the best substrate and
was used for the biopreparation of (S)-PED.
and only moderate yield and ee were obtained. The activities of
these isolated strains from the soil were not as high as that of
recombinant BsE so that the hydrolysis of 1a proceeded for more
than 10 h. In a putative experiment, during such a period (10 h),
about 60% of 1b was transformed into 1c. This is one possibility to
explain why the ee of PED is always low.
Substrate concentration affects not only the reaction rate but
also the enantioselectivity due to the possible existence of sub-
strate inhibition. The effect of substrate concentration of 1a on the
product formation was investigated at a fixed ratio of substrate
to catalyst (S/C). Different substrate concentrations ranging from
50 mM to 150 mM were employed for the kinetic resolution under
the optimal conditions obtained above. As shown in Fig. 4, when
the substrate concentration was increased from 50 mM to 100 mM,
the rate of (S)-PED formation increased accordingly. When the sub-
strate concentration increased from 100 mM to 150 mM, a longer
reaction time was needed to reach 50% substrate conversion. More-
over, it also caused the problem of acyl migration which will reduce
the ee value of PED to 90.7%. Therefore, 100 mM was selected as the
optimal substrate concentration of reaction.
3.4. Kinetic resolution of 1a in preparative scale
To further evaluate the potential of whole cells of recombinant
E. coli BL21 for practical use, a preparative scale resolution of 1a was
conducted. A total amount of 1.8 g wet cells of recombinant E. coli
BL21 and 3.3 g substrate (100 mM) were employed in a volume of
150 ml. The reaction was monitored by HPLC analysis, and it was
stopped at 1 h. After extraction and normal work-up, 0.98 g of (S)-
PED was obtained (49% yield, 95% ee). After simple recrystallization
in CHCl3, the ee of (S)-PED was enhanced up to >99%, with a total
yield of 45%.
3.3. Effect of acyl groups with varied carbon chain length
4. Conclusions
Three dicarboxyesters with varied acyl chains 1a–3a (acetyl, n-
butyl, n-hexyl) were prepared (Scheme 2). The hydrolysis of these
dicarboxyesters by recombinant E. coli cells was performed and
compared. Conversion of substrate (c), ee value of PED (eep) and
The preparation of (S)-PED by kinetic resolution of dicar-
boxyesters with the whole cells of recombinant E. coli BL21 overex-
pressing BsE was investigated. The recombinant strain exhibits high

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X. Tian et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 80–84
esterase activity and excellent regio-, enantioselectivity towards
dicarboxyesters, meanwhile, the best enantioselectivity (E = 176)
was observed for diacetate among three dicarboxyesters with var-
ied acyl chains (acetyl, n-butyl, n-hexyl). Then the reaction condi-
tions were optimized in aqueous system, giving the optimal condi-
tions as follows: pH 7.5, 35 ◦C, 10 mg ml−1 of cell concentration and
100 mM substrate. In this work, (S)-PED can be obtained through
direct hydrolysis of rac-1a instead of rac-1b which needs protecting
groups in its chemical synthesis process. Decrease of synthetic steps
of substrate makes the process more reasonable both technically
and economically. The preparative reaction (150-ml scale) was suc-
cessfully performed, indicating a potentially promising biocatalyst
for practical production of (S)-PED in the future.
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Acknowledgements
This work was financially supported by the National Natural Sci-
ence Foundation of China (Nos. 20902023 and 31071604), Ministry
of Science and Technology, P.R. China (Nos. 2009CB724706 and
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