Asymmetric biocatalysis of S-3-amino-3-phenylpropionic acid with new isolated Methylobacterium Y1-6

Article abstract of DOI:10.1016/j.procbio.2013.08.015

β-amino acids are widely used in drug research, and S-3-amino-3-phenylpropionic acid (S-APA) is an important pharmaceutical intermediate of S-dapoxetine, which has been approved for the treatment of premature ejaculation. Chiral catalysis is an excellent method for the preparation of enantiopure compounds. In this study, we used (±)-ethyl-3-amino-3-phenylpropanoate (EAP) as the sole carbon source. Three hundred thirty one microorganisms were isolated from 30 soil samples, and 17 strains could produce S-APA. After three rounds of cultivation and identification, the strain Y1-6 exhibiting the highest enantioselective activity of S-APA was identified as Methylobacterium oryzae. The optimal medium composition contained methanol (2.5 g/L), 1,2-propanediol (7.5 g/L), soluble starch (2.5 g/L), and peptone (10 g/L); it was shaken at 220 rpm for 4-5 days at 30 C. The optimum condition for biotransformation of EAP involved cultivation at 37 C for 48 h with 120 mg of wet cells and 0.64 mg of EAP in 1 ml of transfer solution. Under this condition, substrate ee was 92.1% and yield was 48.6%. We then attempted to use Methylobacterium Y1-6 to catalyze the hydrolytic reaction with substrates containing 3-amino-3-phenyl-propanoate ester, N-substituted-β-ethyl-3-amino-3-phenyl-propanoate, and γ-lactam. It was found that 5 compounds with ester bonds could be stereoselectively hydrolyzed to S-acid, and 2 compounds with γ-lactam bonds could be stereoselectively hydrolyzed to (-)-γ-lactam.

Full text of DOI:10.1016/j.procbio.2013.08.015

Process Biochemistry 48 (2013) 1674–1678
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Asymmetric biocatalysis of S-3-amino-3-phenylpropionic acid with
new isolated Methylobacterium Y1-6
Yi Li, Wenfu Wang, Yumian Huang, Qianwen Zou, Guojun Zheng∗
State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 May 2013
Received in revised form 25 August 2013
Accepted 26 August 2013
Available online 3 September 2013
␤-amino acids are widely used in drug research, and S-3-amino-3-phenylpropionic acid (S-APA) is an
important pharmaceutical intermediate of S-dapoxetine, which has been approved for the treatment of
premature ejaculation. Chiral catalysis is an excellent method for the preparation of enantiopure com-
pounds. In this study, we used ( )-ethyl-3-amino-3-phenylpropanoate (EAP) as the sole carbon source.
Three hundred thirty one microorganisms were isolated from 30 soil samples, and 17 strains could pro-
duce S-APA. After three rounds of cultivation and identification, the strain Y1-6 exhibiting the highest
enantioselective activity of S-APA was identified as Methylobacterium oryzae. The optimal medium com-
position contained methanol (2.5 g/L), 1,2-propanediol (7.5 g/L), soluble starch (2.5 g/L), and peptone
(10 g/L); it was shaken at 220 rpm for 4–5 days at 30 ◦C. The optimum condition for biotransformation of
EAP involved cultivation at 37 ◦C for 48 h with 120 mg of wet cells and 0.64 mg of EAP in 1 ml of transfer
solution. Under this condition, substrate ee was 92.1% and yield was 48.6%. We then attempted to use
Methylobacterium Y1-6 to catalyze the hydrolytic reaction with substrates containing 3-amino-3-phenyl-
propanoate ester, N-substituted-␤-ethyl-3-amino-3-phenyl-propanoate, and ␥-lactam. It was found that
5 compounds with ester bonds could be stereoselectively hydrolyzed to S-acid, and 2 compounds with
␥-lactam bonds could be stereoselectively hydrolyzed to (-)-␥-lactam.
Keywords:
Biocatalysis
Chiral separation
Methylobacterium sp.
S-3-amino-3-phenylpropanoate ester
Strain screening
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
( )-ethyl-3-amino-3-phenylpropanoate (EAP), but the yield was
markedly insufficient [10].
␤-amino acids are widely used in drug research [1]. One ␤-
amino acid, the chiral building block 3-amino-3-phenylpropionic
acid (APA, ␤-phenylalanine), is present in several bioactive
molecules that can be used as anticancer agents, such as Taxol
[2]. The S-APA is the intermediate of S-dapoxetine, which has been
approved for the treatment of premature ejaculation [3].
In the current research, we aimed to screen a microbial strain
with EAP as the sole carbon source that could stereoselectively
hydrolyze EAP to S-APA with high conversion and good selectivity
(Scheme 1).
2. Materials and methods
Enantiomerically pure APA can be obtained via several chem-
ical reactions, such as Lewis base-catalyzed hydrosilylation [4],
stereoselective reduction [5], and rhodium-catalyzed hydrogena-
tion [6]. But the industrialization of these reactions are not
viable because of their difficult operation, low enantiomeric excess
(ee), and generation of by-products [7]. In contrast, biotransfor-
mation has many advantages, including eco-friendliness, fewer
by-products, and high stereoselectivity [8]. Zhang et al. attempted
the biocatalytic synthesis of ethyl R-2-hydroxy-4-phenylbutyrate
by using Candida krusei, leading to the R-enantiomer with 99.7%
ee and 95.1% yield [9]. In addition, Tasnadi et al. used commercial
lipases such as Lipase PS and Lipase AK to selectively hydrolyze
2.1. Preparation of 3-amino-3-arylpropanoate ester 2a–e
EAP (1) was prepared using the one-pot synthesis method[11] from benzalde-
hyde (30 mmol) through condensation with malonic acid (1 equiv.) and ammonium
acetate (2 equivs.) in EtOH at reflux for 8 h. The white solid was filtered off and
washed with cold EtOH twice. 10 mmol 1 was dissolved in SOCl2 (10 equivs.) and
refluxed for 2 h [11]. The solvent was evaporated off and R-OH (30 equivs.) was added
into the reaction, and then heated at reflux for 6–8 h. After removing the solvent,
the yellow oil was washed with petroleum ether, and 2a–e. HCl was crystallized
(Scheme 2).
2.2. Preparation of N-substituted ethyl-3-amino-3-phenylpropanoate 4a–b
N-(Phenylacetyl)-3-amino-3-phenylpropanoic acid (3) was prepared according
to the method proposed by Giuliana Cardillo [12]; esterification from 3 to 4a was
the same as in Section 2.1. 5 mmol 2b was added to 10 ml of THF, and then pH
was adjusted between 8 and 9 with TFA; the temperature was kept under 0 ◦C [13].
To this solution, 5 ml TFA containing 5 mmol di-tert-butyl dicarbonate was added
slowly. The mixture was stirred at room temperature overnight and extracted with
ethyl acetate twice. Organic phase pH was adjusted to 6 with 10% citric acid, and
∗
Corresponding author. Tel.: +86 1064437507.
E-mail address: zhenggj@mail.buct.edu.cn (G. Zheng).
1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.08.015

Y. Li et al. / Process Biochemistry 48 (2013) 1674–1678
1675
eluent at a flow rate of 0.525 ml/min, with UV detection at 210 nm. The concentration
of each compound was calculated from the peak area.
The enantiomeric excess and yield were calculated as follows:
ꢀ
ꢀ
SA − SB
SA + SB
ee %
(
=
× 100%
)
where SA and SB are the peak areas of S-APA and R-APA, respectively, after the
reaction; and
Scheme 1. Methylobacterium Y1-6 mediated asymmetric biotransformation of EAP
to S-APA.
SA0 − SA1
yield %
=
× 100%
(
)
SA0 + SB0
then the organic phase was washed with water and saturated brine. The solvent
was evaporated off, and the resulting 4b was crystallized as white acicular crystal
(Scheme 2).
where SA0 and SB0 are the peak areas of S-EAP and R-EAP, respectively, before the
reaction, and SA1 is the peak area of S-EAP after the reaction.
2.6. Enzymatic activity assay
2.3. Soil samples and media
One unit (U) of enzyme activity is defined as the amount of wet cell required to
hydrolyze 1 ␮mol of EAP in 24 h under the assay condition.
The peak area of EAP and APA in HPLC chromatogram was chosen to calculate
the enzyme activity.
Soil samples used for screening were collected from different areas of sev-
eral regions of China. The enrichment medium consisted of 1.6 g/L NaH2PO4·2H2O,
0.04 g/L MgSO4, 1.24 g/L K2HPO4·3H2O, and 2 g/L NH4Cl. Rich medium 1 consisted
of 5 g/L olive oil, 10 g/L tryptone, 5 g/L yeast extract, 1 g/L NaCl, 3.2 mg/L MgSO4,
20 mg/L CaCl2, 2 g/L (NH4)2SO4, and 2 g/L KH2PO4·3H2O. Rich medium 2 consisted
of 7.5 g/L 1,2-propanediol, 2.5 g/L methanol, and 10 g/L tryptone.
3. Results and discussion
3.1. Screening of microorganisms
2.4. Screening methods
In this study, more than 300 strains of microorganisms, includ-
ing bacteria, yeasts, and molds, which were capable of using EAP as
the sole carbon source were isolated from the MSM agar plates
through two rounds of screening. Three-quarters of them could
convert EAP to APA through a hydrolytic reaction. As shown in Fig. 1,
22 strains were found to be potentially useful for the preparation
of S-APA, with enantioselectivity ranging from 20% to 70% (reac-
tion time: 24 h) and improving with time. Among all the 22 strains,
the strain Y1-6 showed relative stability after three-generation cul-
tivation and gave the highest yield and higher enantioselectivity.
Therefore, we chose this strain for further study.
In a 250 ml Erlenmeyer flask, 50 ml of MSM was inoculated with 0.1 g of soil
sample, and EAP was added as the sole carbon source, giving a final substrate concen-
tration of 1.6 g/L. After shaking at 200 rpm at 30 ◦C for 2–3 days, the cell supernatant
was taken from the Erlenmeyer flask, diluted to 10−9, and then plated onto MSM agar
plates, which were further incubated at 30 ◦C for 4–5 days until colonies appeared.
A single colony with different shapes was inoculated into MSM for the second round
of screening. After 3–4 days, new cells were obtained. The isolated colonies were
cultivated in rich medium 1 and shaken at 220 rpm at 30 ◦C for 48 h for conservation,
and in rich medium 2 at 220 rpm at 30 ◦C for 4–5 days for cell collection. The cells
were harvested and washed twice with 0.05 M phosphate buffer solution (PHS, pH
7) for biotransformation.
The reaction mixture, comprised of wet cells (100 mg) and a certain amount of
EAP, was added into a 1.5 ml centrifuge tube and shaken at 220 rpm at 30 ◦C for
24 h. The reaction was stopped by centrifugation, and the supernatant was used for
further analysis.
3.2. Identification of strain Y1-6
2.5. Analytical HPLC methods
Universal primers of bacteria (27F: 5ꢀ-AGAGTTTGATCCTGGC-
TCA-3ꢀ and 1492R: 5ꢀ-AGAGTTTGATCCTGGCTCA-3ꢀ) were used to
amplify the 16S rRNA gene sequence. DNA analysis was completed
using BLAST network services provided by the National Center for
Biotechnology Information (NCBI, United States). The sequence of
Y1-6 was 99% identical with the 16SrRNA sequence of Methylobac-
terium oryzae CBMB20; therefore, the strain Y1-6 was identified
The reaction from EAP to APA was monitored with an HPLC system (Shimadzu,
Japan) equipped with a Diamonsil C18 column (0.46 mm × 250 mm × 5 ␮m). The
HPLC conditions were as follows: methanol/0.05% TFA (1:2, v/v) as the mobile phase,
a flow rate of 0.6 ml/min, and a UV detection wave at 210 nm.
The enantiomeric excess analysis was performed using an HPLC sys-
tem (Shimadzu, Japan) equipped with
a Shiseido chiral CD-Ph column
(0.46 mm × 250 mm × 5 ␮m). 35 mmol KH2PO4: methanol (2:5, v/v) was used as the
Scheme 2. Synthesis of ␤-amino acid ester.

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Y. Li et al. / Process Biochemistry 48 (2013) 1674–1678
Fig. 3. The effect of temperature on the asymmetric resolution by Methylobacterium
sp. Y1-6. Reaction condition: 100 mg wet cells, 0.64 g/L EAP in 1 ml transfer solu-
tion, shaken at 220 rpm for 48 h, with 37 ◦C chosen as the optimal temperature; ꢁ
represents relative yield, and ꢁ represents relative ee.
Fig. 1. The results of the second round of screening; 22 strains of microorganisms
with obvious asymmetric hydrolytic activity for the production of EAP were found;
ꢀ represents Y1-6, and positive ee values represent S-APA absolute configuration.
as Methylobacterium sp. and subsequently marked as Methylobac-
terium sp. Y1-6. The colonies on the agar plate were pink, small, and
smooth. A rod-shaped, Gram-negative, capsule-included bacterium
based on observation via microscope, Y1-6 can use both methanol
and 1,2-propanediol as carbon sources.
to a decrease of substrate ee and yield. Therefore, all subsequent
bioreactions were carried out at 37 ◦C.
3.3.3. Optimization of reaction with pH
The enantioselectivity and activity of cells can be strongly
influenced by initial pH and reaction temperature. The pH buffer
containing 50 mM Tris, 50 mM boric acid, 33 mM citric acid, and
50 mM Na2HPO4 was adjusted to a pH value between 4 and 9 with
either HCl or NaOH [14]. As shown in Fig. 4, under an alkaline
environment (pH > 8), EAP spontaneously hydrolyzed, and the yield
was insufficient. When pH was below 5, no product was obtained.
Obviously, the optimal buffer pH for asymmetric resolution by
Methylobacterium sp. Y1-6 should be 7.
3.3. Optimization of reaction conditions of Y1-6
3.3.1. Optimization of reaction with time
Major parameters affecting the biotransformation mediated by
Methylobacterium sp. Y1-6 were investigated, including reaction
time, pH, and temperature. As shown in Fig. 2, the asymmetric
hydrolase activity of the new isolated strain increased in parallel
with the extension of reaction time, but after 50 h ee and yield of
substrate were slightly decreased. Therefore, 48 h was chosen as
the optimal reaction time.
3.3.4. Optimization of reaction with wet cell weight
Under the reaction condition of 100 mg of wet cells, substrate
ee and yield were not higher than expected. Therefore, the impact
of wet cell weight on the asymmetric resolution was tested, and
the results are presented in Fig. 5. substrate ee and yield increased
almost linearly with the increase of weight within the range of
60–120 mg. When the wet cell weight was higher than 120 mg, the
substrate ee and yield were almost at the same level, indicating that
cell weight inhibition had occurred. In the reaction system, when
3.3.2. Optimization of reaction with temperature
Generally, temperature can influence the rate and enantioselec-
tivity of biotransformation. It can be seen from Fig. 3 that substrate
ee and yield increased as temperature increased. The highest yield
and substrate ee were obtained at 42 ◦C, but over-high tempera-
ture affected the stability of the cells, which released red pigment
into the reaction buffer. Furthermore, over-high temperature led
Fig. 2. The effect of reaction time on the asymmetric resolution by Methylobacterium
sp. Y1-6. Reaction condition: 100 mg wet cells, 0.64 g/L EAP in 1 ml transfer solution,
shaken at 220 rpm at 30 ◦C, with 48 h chosen as the optimal time; ꢁ represents
relative yield, and ꢁ represents relative ee.
Fig. 4. The effect of pH on the asymmetric resolution by Methylobacterium sp. Y1-6.
Reaction condition: 100 mg wet cells, 0.64 g/L 2b in 1 ml transfer solution, shaken
at 220 rpm at 37 ◦C for 48 h, with the pH of deionized water chosen as the optimal
pH; ꢁ represents relative yield, and ꢁ represents relative ee.

Y. Li et al. / Process Biochemistry 48 (2013) 1674–1678
1677
Table 1
Biotransformation of different substrates (0.64 g/L) by Methylobacterium sp. Y1-6.
Enter
Substrate
Structure
Time
24 h
eea
Yieldb
1
Methyl-3-amino-3-phenylpropanoate
19%(S)
47%
39%
28%
2
3
Ethyl-3-(brnxoylamino)-3-phenylpropanoate
16 h
31%(S)
Benzenepropanoic
8 h
11.5%(S)
acid-␤-[[(1,1-dimethylethoxy)carbonyl]amino]-ethyl ester
4
5
Propyl-3-amino-3-phenylpropanoate
Butyl-3-amino-3-phenylpropanoate
48 h
48 h
45.4%(S)
34.7%(S)
35.2%
25.9%
6
7
8
Isopropyl-3-amino-3-phenylpropanoate
24 h
24 h
12 h
–
–
␥-Lactam
76%(+)
65.2%
19%
38%
3-(Acetyloxy)-4-phenyl-2-Azetidinone
9
2-Azabicyclo[2.2.1]heptan-3-one
24 h
24 h
–
–
–
–
10
2-(Hydroxymethyl)-2-Azabicyclo[2.2.1]hept-5-en-3-one
a
ee were determined by chiral HPLC, reaction condition: 30 ◦C, 100 mg wet cell, 220 rpm, substrates concentration, 0.6 g/L, reaction volume, 1 ml.
The yield were determined by HPLC.
b
140 or 160 mg of wet cells catalyzed 0.64 g/L of substrate, we can’t
find obvious increase of ee and yield. Therefore, the optimal wet cell
weight was chosen to be 120 mg. We chose the peak area of EAP
and APA in HPLC chromatogram to calculate the enzyme activity of
Y1-6. The enzyme activity of Y1-6 was 22.7 U/L.
using Methylobacterium sp. Y1-6. Several aqueous/organic (4:1,
v/v) reaction systems were investigated, such as n-hexane, cyclo-
hexane, methyl tert-butyl ether (MTBE), isopropyl ether, ethyl
acetate, toluene and dichloromethane. The results showed that
two-phase system had negative effect on yield and ee. Usually,
metallic ions such as Ca2+ and Mg2+ may influence the rate of enzy-
matic reaction. We chose CaCl2, MgSO4, KCl, NaCl, FeCl2 and CuCl2
to prepare 10 mmol/L buffer as the reaction condition, but no sig-
nificant increases in reaction rate were observed. Therefore, we
3.3.5. Effect of two-phase system and metallic ions
In addition, the asymmetric resolutions in an aqueous two-
phase system and in a metallic ion solution were also tested

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Y. Li et al. / Process Biochemistry 48 (2013) 1674–1678
4. Conclusions
We have succeeded in isolating a new bacterial strain with
EAP as a sole carbon source, which has high activity and ideal
stereoselectivity for preparing S-APA from soil samples. This strain
was identified as Methylobacterium sp. Y1-6. The conditions for
bioreduction catalyzed by Y1-6 were optimized. Under the opti-
mal condition (48 h, 37 ◦C, substrate concentration of 0.64 g/L with
120 mg of wet cells in 1 ml of transfer solution), 48.6% yield and
92.1% substrate ee were obtained. This strain can also catalyze the
enantioselective hydrolysis of chiral ester and ␥-lactam.
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Fig. 5. The effect of wet cell weight on the asymmetric resolution by Methylobac-
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