Resolution of (R, S)-ethyl-2-(4-hydroxyphenoxy) propanoate using lyophilized mycelium of Aspergillus oryzae WZ007

Article abstract of DOI:10.1016/j.molcatb.2013.07.016

The mycelium of Aspergillus oryzae WZ007 was successfully developed to kinetic resolution of (R, S)-ethyl-2-(4-hydroxyphenoxy) propanoate ((R, S)-EHPP) for production of (R)-ethyl-2-(4-hydroxyphenoxy) propanoate ((R)-EHPP). The key biocatalytic process parameters (pH, temperature, rotation speed and substrate concentration) were optimized. Under the optimum conditions, the optical purity of (R)-EHPP was improved up to >99% when the conversion was above 49%. A. oryzae WZ007 whole-cell lipase exhibited high reaction capacity, enantioselectivity and good reusability. The tolerable substrate concentration was 0.5 mol/L, and dry mycelium of A. oryzae WZ007 maintains over 80% of its initial activity after eight repeating cycles. Therefore the enzymatic preparation of (R)-EHPP route was suitable for industrial application.

Full text of DOI:10.1016/j.molcatb.2013.07.016

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 62–66
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Resolution of (R, S)-ethyl-2-(4-hydroxyphenoxy) propanoate using
lyophilized mycelium of Aspergillus oryzae WZ007
Jian-Yong Zheng, Jia-Yu Wu, Yin-Jun Zhang, Zhao Wang∗
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hang Zhou 310032, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2013
Received in revised form 24 July 2013
Accepted 24 July 2013
Available online 9 August 2013
The mycelium of Aspergillus oryzae WZ007 was successfully developed to kinetic resolution of (R, S)-ethyl-
2-(4-hydroxyphenoxy) propanoate ((R, S)-EHPP) for production of (R)-ethyl-2-(4-hydroxyphenoxy)
propanoate ((R)-EHPP). The key biocatalytic process parameters (pH, temperature, rotation speed and
substrate concentration) were optimized. Under the optimum conditions, the optical purity of (R)-EHPP
was improved up to >99% when the conversion was above 49%. A. oryzae WZ007 whole-cell lipase exhib-
ited high reaction capacity, enantioselectivity and good reusability. The tolerable substrate concentration
was 0.5 mol/L, and dry mycelium of A. oryzae WZ007 maintains over 80% of its initial activity after
eight repeating cycles. Therefore the enzymatic preparation of (R)-EHPP route was suitable for industrial
application.
Keywords:
(R)-Ethyl-2-(4-hydroxyphenoxy)
propanoate
Aspergillus oryzae
Lipase
© 2013 Elsevier B.V. All rights reserved.
Resolution
Enantioselective hydrolysis
1. Introduction
reported, but several examples of its analogs have been reported
with commercial enzymes [9–11]. Fortunately, our laboratory iso-
(R)-ethyl-2-(4-hydroxyphenoxy) propionate is key intermedi-
ate in the synthesis of aryloxyphenoxy propionate (APP) herbicides
which were used for the control of annual and perennial grasses in
broadleaf crop field [1,2]. The chiral APP herbicides have become
more attractive due to the features of safety, high effectiveness
and low toxicity. In general, the (R)-enantiomers of APP herbi-
cides are herbicidal active and are approximately twice as active
as respective racemic mixtures [3]. Recent study indicated that the
herbicidally inactive (S)-enantiomers of APP herbicides (diclofop-
methyl and diclofop) posed similar or higher toxicity to algae than
the corresponding (R)-enantiomers [4].
lated A. oryzae WZ007 exhibiting high lipase activity from soil
sample by enrichment culture. Lyophilized mycelium of A. oryzae
WZ007 was previously used as an effective biocatalyst for produc-
tion of d-biotin intermediate lactone and (R)-␣-lipoic acid in our
laboratory [12,13].
In this paper, the mycelium of A. oryzae WZ007 was applied in
the enantioselective hydrolysis of (R, S)-EHPP (Scheme 1). Major
reaction parameters affecting the enantioselective hydrolysis of (R,
S)-EHPP were investigated. The reusability of whole-cell biocata-
lysts under the optimum reaction conditions was also examined.
The use of biocatalysts for the industrial synthesis of chemi-
cals has been attracting much attention as an environment-friendly
synthetic method [5]. Compared with purified enzymes, whole-
cell biocatalysts can be much more readily and inexpensively
prepared on an industrial scale with better stability and adaptabil-
ity. Aspergillus oryzae is widely employed in industrial application
with its unique performance and higher economic benefits as a
kind of important whole-cell biocatalysts. Mycelium-bound car-
boxylesterases of lyophilized A. oryzae have been exploited for their
enantioselectivity in different applications [6–8]. Enzymatic res-
olution of (R, S)-EHPP for preparing (R)-EHPP have not yet been
2. Materials and methods
2.1. Materials
Hydroquinone and (R, S)-ethyl-2-chloropropionate were pur-
chased from Aladdin Industrial Co. (Shanghai, China). Other
chemicals were of analytical grade and purchased from various
commercial sources. (R, S)-EHPP and its analogs were synthesized
in our laboratory.
2.2. General procedures for substrates synthesis
Hydroquinone (13.2 g, 120 mmol) was dissolved in a solution
of KOH (4.48 g, 80 mmol) in 40 mL dry dimethyl sulfoxide (DMSO)
under N2 atmosphere, (R, S)-ethyl-2-chloropropionate (6.825 g,
∗
Corresponding author. Tel.: +86 0571 88320615; fax: +86 0571 88320781.
E-mail address: hzwangzhao@163.com (Z. Wang).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.07.016

J.-Y. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 62–66
63
O
O
O
O
R2
O
R2
O
O
O
OH
A. oryzae WZ007
+
R1
R1
R1
(R)
(R, S)-a-g
(S)
Scheme 1. Enantioselective hydrolysis of (R, S)-EHPP and its analogs by A. oryzae WZ007.
30 mmol) was then added dropwise over a 10–20 min period at
0 ◦C. The mixture was kept at 30 ◦C for 12 h with stirring and then
quenched with water (80 mL), extracted with dichloromethane
(3 × 40 mL). The organic phase was separated, washed with sat-
urated sodium chloride solution (100 mL), dried over Na2SO4,
filtered and concentrated under vacuum, giving the desired (R)-
EHPP (4.47 g, 21.3 mmol). All the synthesized substrates were
confirmed from the 1H NMR spectra recorded on a Bruker 500 Hz
apparatus (TMS as an internal standard).
cell growth medium consisting of 1% glucose, 0.5% peptone, 0.1%
KH2PO4, 0.05% MgSO4·7H2O, 0.001% FeSO4·7H2O, 0.05% KCl and 1%
olive oil. Cells were harvested by filtration at 4 ◦C and washed with
distilled water. Microbial cells (50 g, wet weight) were added to
20 mL phosphate buffer (0.1 M, pH 7.0) and lyophilized at −60 ◦C for
2 days, then lyophilized mycelium of A. oryzae WZ007 was obtained
as biocatalyst.
2.4. General procedure for enantioselective hydrolysis of (R,
S)-EHPP
(R, S)-Ethyl-2-phenoxy propanoate (a); 1H NMR (500 MHz,
CDCl3) ı: 7.32–7.25 (m, 2H), 6.98 (t, J = 7.4 Hz, 1H), 6.93–6.87 (m,
2H), 4.77 (q, J = 6.8 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 1.64 (d, J = 6.8 Hz,
3H), 1.26 (t, J = 7.1 Hz, 3H).
Enzymatic hydrolysis reactions were performed by adding (R,
S)-EHPP (0.1–1.6 mmol) and lyophilized mycelium of A. oryzae
WZ007 (20 mg) in 2 mL phosphate buffer. The reaction mixtures
were shaken at 200 rpm at a pH range of 4.0–10.0 and a tempera-
ture range of 20–50 ◦C. Samples of reaction solution (200 ␮L) were
taken at regular intervals and immediately acidified (pH 2.0) with
HCl (10%, v/v) to stop the reaction and enhance the extractability
of (S)-2-(4-hydroxyphenoxy) propionic acid ((S)-HPOPS). The unre-
acted (S)-EHPP and generated (S)-HPOPS were extracted by ethyl
acetate (2 × 0.5 mL) and evaporated to dryness. Then the residue
was dissolved by isopropanol for subsequent HPLC analysis.
(R, S)-Ethyl-2-(2-hydroxyphenoxy) propanoate (b); 1H NMR
(500 MHz, CDCl3) ı: 7.07–7.01 (m, 3H), 6.93 (dd, J = 18.3, 6.0 Hz,
1H), 4.70 (dd, J = 14.0, 7.0 Hz, 1H), 4.65 (q, J = 6.8 Hz, 2H), 1.69 (d,
J = 2.1 Hz, 3H), 1.33 (t, J = 7.3 Hz, 3H).
(R, S)-Ethyl-2-(3-hydroxyphenoxy) propanoate (c); 1H NMR
(500 MHz, CDCl3) ı: 7.14 (t, J = 8.3 Hz, 1H), 6.46 (ddt, J = 19.5, 10.3,
2.2 Hz, 3H), 4.70 (qd, J = 6.8, 3.4 Hz, 1H), 4.22 (qd, J = 7.1, 0.5 Hz, 2H),
1.59 (d, J = 6.8 Hz, 3H), 1.25 (t, J = 7.1 Hz, 3H).
(R, S)-Ethyl-2-(4-hydroxyphenoxy) propanoate (d); 1H NMR
(500 MHz, CDCl3) ı: 7.00 (s, 1H), 6.70 (s, 4H), 4.63 (q, J = 6.8 Hz, 1H),
4.17 (q, J = 6.6 Hz, 2H), 1.54 (d, J = 6.8 Hz, 3H), 1.20 (t, J = 7.0 Hz, 3H).
(R, S)-Methyl-2-(4-hydroxyphenoxy) propanoate (e); 1H NMR
(500 MHz, CDCl3) ı: 7.24 (s, 1H), 6.64 (s, 4H), 4.55 (q, J = 6.8 Hz, 1H),
3.62 (s, 3H), 1.46 (d, J = 6.8 Hz, 3H).
2.5. Analytical methods
The enantiomeric excess of substrate (e.e.s), and product (e.e.p)
were determined by Waters 1525 HPLC(Waters, America) with a
Daicel Chiralcel AD-H column (250 mm × 4.6 Mm, 5 ␮m) (Daicel,
Japan). The mobile phase was a mixture of n-hexane and iso-
propanol (73:7, v/v) at a flow rate of 0.8 mL/min and UV detection
was performed at 274 nm. The retention time of (S)-EHPP, (R)-EHPP,
(S)-HPOPS and (R)-HPOPS were 12.6 min, 14.2 min, 19.3 min and
21.9 min, respectively. Enantiomeric ratio (E) and conversion (c)
were calculated using the equation described by Chen et al. [14].
(R, S)-Propyl-2-(4-hydroxyphenoxy) propanoate (f); 1H NMR
(500 MHz, CDCl3) ı: 6.82 (s, 1H), 6.74–6.69 (m, 4H), 4.63 (q,
J = 6.8 Hz, 1H), 4.10 (dt, J = 3.9, 1.7 Hz, 2H), 1.61 (dd, J = 14.2, 7.1 Hz,
2H), 1.55 (d, J = 6.8 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H).
(R, S)-Butyl-2-(4-hydroxyphenoxy) propanoate (g); 1H NMR
(500 MHz, CDCl3) ı: 7.02 (s, 1H), 6.76–6.67 (m, 4H), 4.64 (q,
J = 6.8 Hz, 1H), 4.16–4.10 (m, 2H), 1.61–1.53 (m, 5H), 1.30 (dt,
J = 15.3, 7.6 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H).
3. Results and discussion
2.3. Microorganisms and culture conditions
3.1. Substrate specificity of A. oryzae WZ007 lipase
A. oryzae WZ007 was isolated in our laboratory and deposited
in China center for type culture collection (CCTCC M206105) and
maintained on Czapek’s medium (0.2% NaNO3, 0.1% K2HPO4, 0.5%
KCl, 0.001% FeSO4, 0.05% MgSO4, 3% sucrose, and 1.5% agar). The
culture was grown aerobically at 30 ◦C and 200 rpm for 48 h in
In order to study the substrate specificity of the lipase from
A. oryzae WZ007, the phenoxy propionate derivatives were sub-
jected to kinetic resolution by lyophilized mycelium of A. oryzae
WZ007 (Table 1). In all the experiments, the lipase preferentially
Table 1
Enantioselective hydrolysis of (R, S)-EHPP analogs by A. oryzae WZ007.
b
b
Entrya
Sub
R1
R2
e.e.s (%)
e.e.p (%)
cc (%)
Ed
1
2
3
4
5
6
7
a
b
c
d
e
f
H
Ethyl
Ethyl
Ethyl
Ethyl
Methyl
Propyl
Butyl
49.6
43.8
40.2
99.0
94.0
98.1
93.6
>99.0
>99.0
>99.0
>99.0
>99.0
>99.0
>99.0
33.2
30.5
28.7
49.7
48.5
49.5
48.3
>200
>200
>200
>200
>200
>200
>200
2-Hydroxy
3-Hydroxy
4-Hydroxy
4-Hydroxy
4-Hydroxy
4-Hydroxy
g
a
Reaction conditions: 20 mg lyophilized mycelium of A. oryzae WZ007, 0.1 mmol (R, S)-EHPP in 2 mL phosphate buffer (0.2 M, pH 8.0), 30 ◦C, 200 rmp, 10 min.
e.e. of the substrate and product were determined by HPLC.
Conversion: c = e.e.s/(e.e.s + e.e.p).
Selectivity: E = ln[(1 − c)(1 − e.e.s)]/ln[(1 − c)(1 + e.e.s)].
b
c
d

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J.-Y. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 62–66
100
100
80
60
40
20
50
40
30
20
10
50
80
40
60
30
40
20
20
10
15
20
25
30
35
40
45
50
55
4
5
6
7
8
9
10
pH
Temperature °C
Fig. 1. Effect of pH on enantioselective hydrolysis of (R, S)-EHPP. Reaction condi-
tions: 20 mg lyophilized mycelium of A. oryzae WZ007, 0.1 mmol (R, S)-EHPP in
2 mL phosphate buffer (0.2 M), 200 rpm, 30 ◦C, 10 min. Citric acid-sodium citrate
buffer, 4.0–5.0; sodium phosphate buffer, 6.0–8.0; glycine-sodium hydroxide buffer,
9.0–10.0. Symbols: conversion (ꢀ); e.e.p (᭹).
Fig. 2. Effect of temperature on enantioselective hydrolysis of (R, S)-EHPP. Reaction
conditions: 20 mg lyophilized mycelium of A. oryzae WZ007, 0.1 mmol (R, S)-EHPP
in 2 mL phosphate buffer (0.2 M, pH 8.0), 200 rpm, 10 min. Symbols: conversion (ꢀ);
e.e.p (᭹).
hydrolyzes (S)-enantiomer of racemic phenoxy propionate deriva-
tives and the corresponding product obtained in high enantiose-
lectivity (>99%). The result indicated that A. oryzae WZ007 showed
good enantioselectivity for phenoxy propionate derivatives. Among
the seven substrates employed, A. oryzae WZ007 exhibited the
highest hydrolytic activity and most satisfactory enantioselectivity
(E > 200) toward (R, S)-EHPP. It is noteworthy that derivatives e–g
showed the higher activity than a–c, indicating the position of phe-
nol hydroxyl has marked affect on the enantioselective hydrolysis
reaction. Meanwhile, the influence of alcohol group in ester bond
is of slight significance in terms of enzymatic kinetic resolution by
A. oryzae WZ007 lipase.
observed as the temperature changed. It indicated that the thermal
deactivation lower the activity of A. oryzae WZ007. Thus, 30 ◦C was
established to be optimum temperature. It is worth noting that high
e.e.p (>99%) was obtained between 20 ◦C and 40 ◦C, demonstrating
that A. oryzae WZ007 lipase showed good enantioselectivity in a
broad temperature range.
3.4. Effect of rotation speed
Rotation speed influenced diffusion and partition of substrate
and product in reaction system, especially in whole cell biocatalysts
systems. Rotation speed can also have a positive effect since the
low solubility of the substrate in water. It was observed (Fig. 3)
that the conversion increased with an increase of speed of rotation
from 100 to 250 rpm. There was no further change in conversion
up to 250 rpm, suggesting that there was no external mass transfer
limitation above 250 rpm. An optimal rotation speed of 250 rpm
was selected in the following experiment.
3.2. Effect of pH
The pH value is one of the critical parameters capable of influ-
encing enzymatic activities in an aqueous solution [15,16], due to
the charge density of enzyme surface and enzyme conformation
varied with different pH [17]. Fig. 1 illustrates the effect of pH on
the activity and enantioselectivity of enzymatic hydrolysis of (R,
S)-EHPP at 30 ◦C in the pH range of 4.0–10.0. Spontaneous reac-
tion occurred under harsh pH conditions (pH > 8.0 or < 5.0) which
resulted in a marked decrease of e.e.p. As pH was lifted from 4.0
to 8.0, the conversion increased sharply from 40.6% to 50.2% (e.e.s
65%–99.5%), after which it dropped as pH increased, no significant
loss of lipase activity was found after pH 8.0, suggesting that enzy-
matic activity was inhibited in acidic and strong basic solutions, and
even lost in extreme pH [18]. Buffer solution (0.2 M, pH 8.0) results
in highest activity and enantioselectivity of A. oryzae WZ007 in term
of hydrolytic reaction.
100
90
80
70
60
50
40
30
51
50
49
48
47
46
45
44
3.3. Effect of temperature
The thermal stability of biocatalysts is always regarded as a
major criterion for industrial applications [19,20]. In addition,
temperature obviously impacts the activity of biocatalyst and
the thermodynamic equilibrium of reaction as well. As shown in
Fig. 2, the appropriate values of the activity and enantioselectivity
were commonly observed at 30 ◦C (Fig. 2). The enzymatic activity
improved slightly with the increase of temperature from 20 ◦C to
40 ◦C and reached the maximum of conversion (49.6%) at 30 ◦C. Fur-
ther mildly rise in temperature beyond 40 ◦C could vastly reduce
the reaction conversion. Only very slight changes in the e.e.p were
100
150
200
250
300
350
Rotation speed (rpm)
Fig. 3. Effect of rotation speed on enantioselective hydrolysis of (R, S)-EHPP. Reac-
tion conditions: 20 mg lyophilized mycelium of A. oryzae WZ007, 0.1 mmol (R,
S)-EHPP in 2 mL phosphate buffer (0.2 M, pH 8.0), 30 ◦C, 10 min. Symbols: conversion
(ꢀ); e.e.p (᭹).

J.-Y. Zheng et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 62–66
65
14
100
80
60
40
20
100
100
80
100
12
80
80
60
40
20
10
8
60
40
20
60
6
40
4
20
2
0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Substrate concentration (mol/l)
0
1
2
3
4
8
9
10
11
Numbe5r of r6euse 7
Fig. 4. Effect of substrate concentration on enantioselective hydrolysis of (R, S)-
EHPP. Reaction conditions: 20 mg lyophilized mycelium of A. oryzae WZ007, 2 mL
phosphate buffer (0.2 M, pH 8.0), 250 rpm, 2 h. Symbols: conversion (ꢀ); e.e.p (ꢁ);
intial reaction rate (᭹).
Fig. 6. Reusability of biocatalysts on enantioselective hydrolysis of (R, S)-EHPP.
Reaction conditions: 50 mg lyophilized mycelium of A. oryzae WZ007, 0.25 mmol (R,
S)-EHPP in 5 mL phosphate buffer (0.2 M, pH 8.0), 250 rpm, 30 ◦C, 30 min. Symbols:
conversion (ꢀ); e.e.p (ꢁ).
follows: 0.2 M phosphate buffer (pH 8.0), temperature 30 ◦C, rota-
tion speed 250 rpm, the substrate concentration and the adding
amount of A. oryzae WZ007 was 0.5 mol/L and 20 g/L, respectively.
After 2 h reaction time, the yield of (R)-EHPP reached 50.1% with
e.e.s of 99.2%. Few products were gained from 2 h to 3 h, concluded
that the accumulation of the product showed a deleterious effect
on the rate of enzymatic hydrolysis.
3.5. Effect of substrate concentration
Substrate concentration is a crucial factor worthy of careful
investigation, since it may affect enzyme activity or even result in
the substrate inhibition. The conversion and e.e.s were described
in Fig. 4. It is evident that the conversion and e.e.s continu-
ously decreased when the substrate concentration was beyond
0.5 mol/L. The initial reaction rate almost increased linearly till the
highest point at the substrate concentration of 0.4 mol/L. The max-
imum conversion (50.1%) with e.e.s (99.2%) were obtained with
0.5 mol/L substrate concentration. Therefore, 0.5 mol/L is the opti-
mum substrate concentration for biocatalyst loading of 20 mg,
which indicated that lyophilized mycelium of A. oryzae WZ007 had
potential industrial value to kinetic resolution of racemic substrate
at a high concentration.
3.7. Reusability of biocatalysts
The reusability of the whole cell biocatalysts is also impor-
tant for economical use. To evaluate the reusability of biocatalysts,
lyophilized mycelium of A. oryzae WZ007 was obtained from the
water phase by simple filtration and washed with phosphate buffer
(0.2 M, pH 8.0) to remove any residual substrate. Fig. 6 shows
that the conversion ratios of A. oryzae WZ007 for (R, S)-EHPP still
retained about 45% after the eighth reuse. But the catalytic activity
of A. oryzae WZ007 decreased dramatically after the ninth repeating
circle. Furthermore, the e.e.p was not influenced after ten cycles and
retained above 98%. This is due to the inactivation of the enzyme
denaturation of protein and the leakage of lipase protein to the fil-
trate. Because the residual hydrolytic activity was detected in the
reaction and washing filtrate.
3.6. Time course of enzymatic hydrolysis reaction
The time course of enantioselective hydrolysis of (R, S)-EHPP
using lyophilized mycelium of A. oryzae WZ007 was shown as
Fig. 5. The reaction was conducted under optimization conditions as
100
80
60
40
20
100
80
60
40
20
4. Conclusion
The kinetic resolution of (R, S)-EHPP by the mycelium of A. oryzae
WZ007 enables a practical and economical synthesis of (R)-EHPP.
For optimal enzymatic activity, this reaction had to be operated
at 30 ◦C and at a pH of 8.0. Under the optimized reaction con-
ditions, (R)-EHPP was obtained in 50.1% conversion with 99.2%
e.e.s after 2 h, when the concentration of substrate and A. oryzae
WZ007 were 0.5 mol/L and 20 g/L, respectively. Furthermore, it was
demonstrated that lyophilized mycelium of A. oryzae WZ007 is
active for 8 batch reaction cycles with 80% activity and 45% con-
version. lyophilized mycelium of A. oryzae WZ007 is a potentially
promising biocatalyst for practical production of (R)-EHPP in the
future.
0
20
40
60
80
100
120
140
160
180
Time (min)
Acknowledgments
Fig. 5. Time course of enzymatic reaction under optimal condition. Reaction condi-
tions: 50 mg lyophilized mycelium of A. oryzae WZ007, 2.5 mmol (R, S)-EHPP in 5 mL
phosphate buffer (0.2 M, pH 8.0), 250 rpm, 30 ◦C. Symbols: conversion (ꢀ); e.e.s (ꢁ);
e.e.p (᭹).
This research was financially supported by National High
Technology Research and Development Program of China (No.
2011AA02A210).

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