Industrial production of S-2,2-dimethylcyclopropanecarboxamide with a novel recombinant R-amidase from Delftia tsuruhatensis

Article abstract of DOI:10.1016/j.procbio.2010.08.005

R-Stereoselective amidase, a key enzyme responsible for the formation of chiral center of cilastatin, has been cloned from Delftia tsuruhatensis and expressed in Escherichia coli under T7 promoter. This recombinant amidase exhibits strict R-selectivity towards 2,2-dimethylcyclopropanecarboxamide (DMCPCA). The amidase activity of the engineered E. coli strain reached 2963 U/L in a 5-L bioreactor, which was 8.27 times higher than that of D. tsuruhatensis, and was further increased to 5255 U/L in a 100-L bioreactor. Using cell-free extract prepared from 1 kg (wet cell weight) of recombinant E. coli cells as catalyst, 60 kg of R,S-DMCPCA was resolved into S-DMCPCA (28.6 kg) and R-2,2-dimethylcyclopropanecarboxylic acid (R-DMCPCS 31.7 kg) in 18 h, and the enantiomeric excess (ee) value of S-DMCPCA reached 99.32%. During the purification process, 24.6 kg of S-DMCPCA was eluted from adsorption resin HZ801 with 80% (v/v) acetone, and then 20.5 kg of pure S-DMCPCA was obtained after concentration and crystallization, corresponding to a total yield of 34.2% from R,S-DMCPCA. Therefore, the industrial production process of S-DMCPCA was successfully established using recombinant R-amidase from D. tsuruhatensis.

Full text of DOI:10.1016/j.procbio.2010.08.005

Process Biochemistry 46 (2011) 182–187
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Industrial production of S-2,2-dimethylcyclopropanecarboxamide with a novel
recombinant R-amidase from Delftia tsuruhatensis
Zhong-Yi Yanga,b, Ye Nia,∗, Zhong-Yuan Lub, Xiang-Ru Liaoa,∗, Yu-Guo Zhengc, Zhi-Hao Suna
a The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Rd., Wuxi 214122, China
b Zhejiang Hisun Pharmaceutical Co. Ltd., Taizhou 318000, China
c Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 June 2010
Received in revised form 30 July 2010
Accepted 4 August 2010
R-Stereoselective amidase, a key enzyme responsible for the formation of chiral center of cilastatin, has
been cloned from Delftia tsuruhatensis and expressed in Escherichia coli under T7 promoter. This recom-
binant amidase exhibits strict R-selectivity towards 2,2-dimethylcyclopropanecarboxamide (DMCPCA).
The amidase activity of the engineered E. coli strain reached 2963 U/L in a 5-L bioreactor, which was 8.27
times higher than that of D. tsuruhatensis, and was further increased to 5255 U/L in a 100-L bioreactor.
Using cell-free extract prepared from 1 kg (wet cell weight) of recombinant E. coli cells as catalyst, 60 kg
of R,S-DMCPCA was resolved into S-DMCPCA (28.6 kg) and R-2,2-dimethylcyclopropanecarboxylic acid
(R-DMCPCS 31.7 kg) in 18 h, and the enantiomeric excess (ee) value of S-DMCPCA reached 99.32%. During
the purification process, 24.6 kg of S-DMCPCA was eluted from adsorption resin HZ801 with 80% (v/v)
acetone, and then 20.5 kg of pure S-DMCPCA was obtained after concentration and crystallization, cor-
responding to a total yield of 34.2% from R,S-DMCPCA. Therefore, the industrial production process of
S-DMCPCA was successfully established using recombinant R-amidase from D. tsuruhatensis.
© 2010 Elsevier Ltd. All rights reserved.
Keywords:
R-Amidase
S-(+)-2,2-
Dimethylcyclopropanecarboxamide
Industrial production
Delftia tsuruhatensis
1. Introduction
Rhodococcus erythropolis (ATCC 25544) [8] and Rhodococcus
sp. AJ270 [9] harboring S-stereoselective amidase have been
S-(+)-2,2-Dimethylcyclopropanecarboxamide (S-DMCPCA) is a
key intermediate of cilastatin [1] and also a resolving agent for
secondary alcohols [2]. S-DMCPCA can be hydrolyzed into S-2,2-
dimethylcyclopropanecarboxylic acid (S-DMCPCS), which is an
important precursor for the preparation of insecticide pyrethroid
[3].
S-DMCPCA can be obtained either by asymmetric synthesis or
resolution of racemic DMCPCS or DMCPCA. In the reported asym-
metric synthesis [4], expensive chiral ligands are required, and
the enantiomeric excess (ee) of the product is lower than 95%,
which is not adequate for the industrial production of S-DMCPCA.
The production of S-DMCPCA by resolution of R,S-DMCPCS usu-
ally involves diastereomer formation with resolving agents such
as chiral amine [5], chiral alcohol [6], and chiral heterocyclic com-
pounds [7]. These chiral resolving agents are costly and the yield of
single resolution step is low, sometimes requiring several recrys-
tallization steps. The bio-resolution of R,S-DMCPCA is a promising
approach for the production of S-DMCPCA, which can be catalyzed
by S- or R-stereoselective amidase.
reported for the biotransformation of S-isomers in R,S-DMCPCA
to S-DMCPCS. These two strains also have hydratase activity,
capable of converting R,S-dimethylcyclopropanecarbonitrile into
R,S-DMCPCA. S-DMCPCS can therefore be obtained directly from
R,S-dimethylcyclopropanecarbonitrile in reactions catalyzed by
one strain. However, the reported ee of S-DMCPCS was around 90%,
which was not adequate for the industrial synthesis of cilastatin.
Biotransformation of R,S-DMCPCA with R-specific amidase has
also been reported. Comamonas acidovorans A:18 (DSM No 6315),
C. acidovorans TG308 (DSM No 6552), Pseudomonas sp. NASK:42
(DSM No 6433), and Bacterium sp. VIII:II (DSM No 6316) have
been reported by Lonza Ltd. [10] to transform the R-isomer in R,S-
DMCPCA to R-DMCPCS, with ee of unreacted S-DMCPCA reaching
99%. Biotransformation with cell-free enzymes and immobilized
cells of these microorganisms were also reported. Expression of
the R-amidase gene from C. acidovorans A:18 can greatly increase
the hydrolysis activity toward R-DMCPCA [11].
With
a colorimetric screening method [12], two novel
R-stereospecific amidase-producing strains, Brevibacterium epi-
dermidis [13] and Delftia tsuruhatensis [14], were previously
isolated. Additionally, a hydratase-producing strain Rhodococcus
boritolerans FW815 was isolated for the conversion of R,S-2,2-
dimethylcyclopropanecarbonitrile (R,S-DMCPCN) to R,S-DMCPCA
[15]. Using above mentioned amidase- and hydratase-producing
∗
Corresponding authors.
E-mail addresses: yni@jiangnan.edu.cn (Y. Ni), liaoxiangru@163.com (X.-R. Liao).
1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.08.005

Z.-Y. Yang et al. / Process Biochemistry 46 (2011) 182–187
2.5. Fermentation of E. coli BL21(DE3)/pET-dam in 5-L and 100-L bioreactors
183
strains, a mild, cost-effective, and environmentally friendly proce-
dure for the conversion of R,S-DMCPCN to S-DMCPCA via a two-step
biotransformation process was established.
Here, we report the hetero-expression of R-specific amidase
gene (dam) from D. tsuruhatensis in E. coli and the industrial process
for the production of S-DMCPCA using this recombinant amidase.
Fermentation was carried out in 5- and 100-L bioreactors (Bioengineering AG).
A single colony of E. coli BL21(DE3)/pET-dam strain was inoculated into 5 mL LB with
30 ␮g/mL kanamycin in a 15-mL tube and was incubated at 37 ◦C in a reciprocator.
After overnight cultivation, 1 mL of the cells was inoculated into 60 mL of LB broth
with 30 ␮g/mL kanamycin in a 500-mL flask. When the OD600 reached 0.8–1.0, 17 mL
of the culture was transferred into 3.5 L of 2× YT medium (1% yeast extract, 1.6%
tryptone, and 0.5% NaCl) in a 5-L fermentor. The fermentation was performed at 37 ◦C
for 1–2 h until OD600 reached about 1. Then the culture was induced by lowering the
temperature to 22 ◦C and adding 35 g of lactose. Two hours later, 17.5 g of lactose was
added for the secondary induction. The aeration rate was maintained at 1.3 VVM. An
agitation cascade was selected to maintain the dissolved oxygen (DO) concentration
at above 20% through automatic adjustment of the agitation speed between 300 and
700 rpm. Two-microliter samples were taken at regular intervals, 1 mL was used for
OD600 determination and the remainder was used for enzyme activity assay.
The procedure for the 100-L fermentation was similar to that of the 5-L fermen-
tation with the exception that 300 mL of culture with an OD600 of 0.8–1.0 was used
to inoculate 60 L of 2× YT medium in the 100-L fermentor.
2. Materials and methods
2.1. Bacterial strains, plasmids, and enzymes
D. tsuruhatensis CCTCC M 205114 was provided by Professor Yuguo Zheng (Zhe-
jiang University of Technology) [14]. D. tsuruhatensis CCTCC M 205114 was cultured
at 30 ◦C, 150 rpm for 24 h in a 250-mL Erlenmeyer flask containing 40 mL of medium
(pH 7.5) that consisted of (per liter) 8.4 g of glucose, 3.56 g of acetamide, 7.0 g of yeast
extract, 0.7 g of peptone, 1 g of NaCl, 1 g of KH2PO4, and 1 g of K2HPO4. For fermen-
tation in a 5-L bioreactor (Bioengineering AG), 3.5 L of medium was inoculated with
200 mL of flask culture of D. tsuruhatensis. The fermentation was performed at 30 ◦C
with aeration rate of 1.0 VVM. Agitation cascade was selected to maintain the dis-
solved oxygen (DO) concentration at above 20% through automatic adjustment of
the agitation speed between 300 and 500 rpm.
Fermentation broth from the 100-L fermentor was then harvested by centrifu-
gation (continuous flow) at 10,000 × g and flow rate of 50 L/h. Cells were stored at
−20 ◦C or used fresh.
Pyrobest DNA polymerase, Blunting Kination Ligation Kit (including pUC118
Hinc II/BAP vector), and restriction enzymes were purchased from Takara (Dalian,
China). Plasmid pET24a and E. coli strain BL21 (DE3) were purchased from Novagen.
Gene sequencing was conducted by Shanghai Sangon Biotech Co. Ltd. All other
reagents were analytical grade commercial products from standard suppliers.
2.6. Production of S-DMCPCA with E. coli BL21(DE3)/pET-dam
One kilogram of E. coli BL21(DE3)/pET-dam cells was suspended in 3 L of water,
and the mixture was homogenized at 15,000 bar for three times (Avestin EmulsiFlex-
C55, Canada). Then, the homogenized solution was centrifuged at 12,000 × g for
20 min (Hitachi, Japan), and the supernatant was used for S-DMCPCA production.
The resolution of R,S-DMCPCA was performed in a 15,000-L tank. Sixty kilograms of
R,S-DMCPCA (Zhejiang Hisun Pharmaceutical, Taizhou, China) was added to 10,000 L
of water. The temperature of the solution was then maintained at 35 ◦C using a
coil pipe, and the pH was maintained at 7.0 with 1 mol/L NaOH and 1 mol/L HCl.
The reaction was initiated by adding homogenized E. coli BL21(DE3)/pET-dam cell
extract with agitation speed of 100 rpm. Samples were taken every 2 h and analyzed
by HPLC or GC.
When the reaction was completed, the pH of the reaction solution was adjusted
to 10.5 with 2N NaOH. The solution was then loaded onto 1.0 m3 of HZ801 adsorbent
resin (Shanghai Huazhen Sci & Tech Co., Ltd., China). After washing with 1 m3 of
water, the adsorbed S-DMCPCA was eluted with 2 m3 of 80% acetone. The eluate was
concentrated at 60 ◦C under vacuum, and then cooled to 4 ◦C to allow crystallizing
overnight. Then product was obtained after filtration and washing with water.
2.2. Cloning and DNA sequencing of dam gene
The genomic DNA of D. tsuruhatensis CCTCC
M 205114 was used as
the template. Cells were harvested in the late log phase from the culture
medium at 30 ◦C for 24 h, and genomic DNA was extracted. The dam gene
was PCR amplified by Pyrobest DNA polymerase using
2
primers: DAM1, 5ꢀ-
TCTCATATGATGAACGACAGCGAACTGCACCATCTCGAACT-3ꢀ (forward); and DAM2,
5ꢀ-CTAGAATTCTCAGGCAGCAGGGTGCTGTCTGTGCCAGT-3ꢀ (reverse). PCR amplifica-
tion program consists of 30 cycles at 94 ◦C for 45 s, 58 ◦C for 45 s, and 72 ◦C for 2 min.
The PCR product was electrophoresed on a 0.8% agarose gel and the 1.4-kb fragment
was purified using a gel extraction kit (Watson Biotechnologies, Shanghai). The puri-
fied dam gene fragment was ligated into pUC118-Hinc II/BAP vector using T4 DNA
ligase and then transformed into E. coli DH5␣-competent cells. Positive clones were
selected using the blue/white screening method. The cloned DNA was sequenced by
Shanghai Sangon Biotech Co. Ltd. This subcloned plasmid was named pUC-dam.
2.7. Analytical methods
The amounts of amide and the corresponding acid were assayed by HPLC
using an Agilent 1100 HPLC system. The column was a C18 reversed-phase col-
umn Hypersil ODS2, and the column temperature was 40 ◦C. The mobile phase was
50% 50 mmol/L sodium phosphate buffer (pH 3.0) and 50% methanol, and the flow
rate was 0.5 mL/min. The detection wavelength was 210 nm. Samples were diluted
5–10 times with the mobile phase and then centrifuged at 18,800 × g for 2 min, and
the supernatant was used for injection.
The ee value of DMCPCA in the reaction solution was analyzed with Agilent
6890 GC system equipped with a flame ionization detector and an enantioselec-
tive capillary column BGB-175 (BGB Analytik, Switzerland). Helium was used as the
carrier gas at a flow rate of 1.6 mL/min and the column temperature was 170 ◦C.
Samples were extracted with an equal volume of ethyl acetate after centrifugation
at 18,800 × g for 2 min, and the supernatant layer was dried over anhydrous Na2SO4
before injection. The retention time of S-DMCPCA and R-DMCPCA was 6.19 and
6.32 min, respectively.
2.3. Expression of D. tsuruhatensis dam gene in E. coli
The pUC-dam plasmid and pET24a vector were digested with NdeI and EcoRI.
The digested products were separated on a 0.8% agarose gel and the 1.4-kb band
containing the dam gene and 5.3-kb band of the digested pET24a vector were
purified using a gel extraction kit. These two fragments were ligated with T4
DNA ligase and then transformed into E. coli BL21(DE3)-competent cells. Positive
clones were selected on LB agar plate supplemented with 30 ␮g/mL kanamycin,
and confirmed by digestion with NdeI and EcoRI. The resulted plasmid was named
pET-dam. A positive E. coli BL21(DE3)/pET-dam colony was selected and incubated
in 50 mL Luria–Bertani (LB) medium (0.5% yeast extract, 1.0% tryptone, and 0.5%
NaCl) containing 30 ␮g/mL kanamycin in a 250-mL Erlenmeyer flask with shaking
at 200 rpm and 37 ◦C. When OD600 reached 0.6, a final concentration of 1 mM of
isopropyl-1-thio-␤-D-galactopyranoside (IPTG) was added for induction. Follow-
ing a 3-h induction period, the cells were centrifuged and the pellet was washed
with resuspension buffer (20 mM Tris–HCl, pH 7.5). The cells were then disrupted
by sonication, and the supernatant fraction was recovered by centrifugation at
14,000 × g for 30 min at 4 ◦C. The supernatant was subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
3. Results and discussion
3.1. Cloning and sequence analysis of the dam gene
The dam gene was amplified from D. tsuruhatensis CCTCC M
205114 by PCR. Primers were designed according to the upstream
and downstream sequences of the putative amidase gene of Delf-
tia acidovorans SPH-1 (CP000884.1). The dam gene was cloned into
pUC118 and sequenced. Comparison of the gene sequence of D.
tsuruhatensis CCTCC M 205114 amidase (HM165184), D. acidovo-
rans SPH-1 putative amidase (CP000884.1), and D. acidovorans D
amino acid amidase (AB154822.1) with amidase from C. acidovo-
rans A:18 [11] revealed high sequence identity. The gene sequences
of D. tsuruhatensis CCTCC M 205114 amidase and D. acidovorans
SPH-1 putative amidase were 100% identical. The identity between
2.4. Enzyme activity assay
Amidase activity was assayed using racemic 2,2-dimethylcyclopropane car-
boxamide as the substrate. One milliliter of fermentation broth was centrifuged,
resuspended in 1 mL of 50 mM potassium phosphate buffer (pH 7.0), and then mixed
with 3 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 70 mmol/L R,S-
DMCPCA. The reaction mixture was incubated at 35 ◦C for 10 min in a shaker with
a rotation speed of 150 rpm. The reaction was stopped by addition of 4 volumes of
HPLC mobile phase (see Section 2.6). After centrifugation at 12,000 × g for 2 min, the
supernatant was collected and analyzed by high-performance liquid chromatogra-
phy (HPLC). One unit of amidase activity was defined as the amount of cells required
to liberate the equivalent of 1 ␮mol of 2,2-dimethylcyclopropane carboxylic acid per
min at 35 ◦C and pH 7.0.

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Z.-Y. Yang et al. / Process Biochemistry 46 (2011) 182–187
Fig. 1. Amino acid sequence alignment of Delftia tsuruhatensis CCTCC M 205114 amidase (Dt), D. acidovorans SPH-1 putative amidase (Da), D. acidovorans D amino acid
amidase (Dd) and amidase from Comamonas acidovorans A:18 (Ca) using the ClustalW alignment program. (ꢀ): identical sequences; ( ): similar sequences.
gene sequences of D. tsuruhatensis CCTCC M 205114 amidase and
D. acidovorans D amino acid amidase was 97%. Though the amidase
gene sequence of C. acidovorans A:18 was 123 bp shorter than that
of D. tsuruhatensis, the identity between them was as high as 97%.
The ORF of the amidase gene in pUC-dam encodes a protein
comprised of 467 amino acids with a predicted molecular weight of
50.0 kDa and an estimated isoelectric point (pI) of 6.73. Fig. 1 shows
the alignment of deduced amino acid sequence of D. tsuruhaten-
sis CCTCC M 205114 amidase with its homologues. The deduced
amino acid sequence of amidase from D. tsuruhatensis CCTCC M
205114 was highly homologous to the putative amidase sequence
of the D. acidovorans SPH-1 (100% identity) and d-amino acid ami-
dase sequence of D. acidovorans (98% identity). More information is
needed to determine the occurrence of deletions or other mutations
in the genetic sequence of C. acidovorans A:18 amidase.
Amidases from D. tsuruhatensis CCTCC M 205114 and C. acidovo-
rans A:18 were reported to show strict R-stereoselectivity towards
R,S-DMCPCA [10,14]. Since the amidase gene sequences of the
above mentioned 4 Delftia species are highly similar, especially
within nucleotides 1–1286 bp, the stereoselectivity of amidase
from other Delftia species is expected to be high.
3.2. Expression of the recombinant amidase
Vector pET-24a was used for the expression of D. tsuruhaten-
sis CCTCC M 205114 amidase. The expression of amidase was
Fig. 2. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) of R-amidase expression in E. coli after isopropyl
␤-d-1-thiogalactopyranoside (IPTG) induction. 1: negative control. E. coli BL21(DE3)
transformed with empty plasmid pET24a; 2: engineered strain. E. coli BL21(DE3)
transformed with plasmid pET-dam; M: protein molecular weight marker.
Fig. 3. Fermentation process of E. coli BL21(DE3)/pET-dam and Delftia tsuruhatensis
CCTCC M 205114 in a 5-L bioreactor.

Z.-Y. Yang et al. / Process Biochemistry 46 (2011) 182–187
185
estimated to be about 50 kDa, which is consistent with the size
(49.99 kDa) calculated from its amino acid sequence.
3.3. Fermentation in 5-L and 100-L bioreactors
Fermentation of E. coli BL21(DE3)/pET-dam was performed with
a lactose induction strategy. In a 5-L bioreactor, the highest amidase
activity of 2958–2963 U/L was observed at 15–17 h, and the high-
est OD600 level of 33–34 was also reached in this period (Fig. 3).
The highest amidase activity in 5 L fermentation process of D. tsu-
ruhatensis CCTCC M 205114 was 358 U/L, and at that point, the
OD600 was 10.3. Thus, in 5 L fermentation, the amidase activity
of the recombinant E. coli in this study was 8.27 times higher
than that of D. tsuruhatensis CCTCC M 205114, and the specific
amidase activity of the recombinant E. coli (89.8 U/OD600) was
2.69 times higher than that of D. tsuruhatensis CCTCC M 205114
(34.6 U/OD600).
Fig. 4. R,S-DMCPCA resolution process by recombinant amidase in a 15,000-L reac-
tor.
In
a 100-L bioreactor, the enzyme activity of E. coli
BL21(DE3)/pET-dam was enhanced to 5255 U/L, which was almost
doubled compared with that of 5-L process. Improved oxygen and
mass transfer efficiency may be the main reason for the increased
enzyme activity. A slightly lower cell density OD600 29 was however
observed, indicating the elevated specific amidase activity instead
of cell density was responsible for the markedly enhanced amidase
induced by adding a final isopropyl ␤-d-1-thiogalactopyranoside
(IPTG) concentration of 1 mM to the culture media. Cells were
then harvested after 3 h of induction, and disrupted by sonication.
The supernatant of the lysate was analyzed by SDS-PAGE. Fig. 2
shows that the molecular weight of the recombinant amidase was
Fig. 5. HPLC and GC analysis of R,S-DMCPCA resolution process at different time points (0, 7.5, and 18 h).

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Z.-Y. Yang et al. / Process Biochemistry 46 (2011) 182–187
Table 1
hydrolization of R-DMCPCA [14]. In this work, we reported the
recombinant expression and industrial application of R-amidase
from D. tsuruhatensis CCTCC M 205114.
Yield and ee of purification process for S-DMCPCA.
Steps
DMCPCA (kg)
Yield (%)
ee (%) (S-DMCPCA)
Firstly, the biocatalyst cost was greatly decreased since the
enzyme activity of amidase was increased from 358 U/L to 5255 U/L.
The amidase cloned from D. tsuruhatensis CCTCC M 205114 showed
strict R-stereoselectivity towards R,S-DMCPCA, and it hydrolyzed
the R-enantiomer of the racemic R,S-DMCPCA thoroughly with
very little S-DMCPCA converted. This ensured high yield and enan-
tiomeric excess of the final product. Finally 60 kg of R,S-DMCPCA
was resolved to S-DMCPCA (28.6 kg, 99.32% ee) and R-DMCPCS
(31.7 kg).
Secondly, the production process was established with fewest
steps. With limited load of enzyme activity, no extra separation
step was needed for downstream processing. After purification
with adsorption resin HZ801 and crystallization, S-DMCPCA was
obtained from R,S-DMCPCA in an yield of 34.2%.
Though the R-amidase is efficient for the bio-resolution of
R,S-DMCPCA, the better approach for commercial production of
S-DMCPCA is to simplify the process by conducting the trans-
formation of R,S-DMCPCN to R,S-DMCPCA and bio-resolution of
R,S-DMCPCA in one reactor, so as to avoid the purification process of
R,S-DMCPCA. More feasible process is to clone the nitrile hydratase
and R-amidase in one strain, or to find an efficient streoselective
nitrilase for direct hydrolyzing S-DMCPCN to S-DMCPCA.
This work demonstrates a successful example for the industrial
production of an important pharmaceutical chiral intermediate,
S-DMCPCA. The result of study could potentially be useful for estab-
lishing a platform technology for the industrial biotransformation
process involving a series of valuable enzymes such as hydrolase,
lipase, oxidoreductase, etc.
Input
Reaction
Resin adsorbtion
Crystallization
60
100
50
99.32
100
28.6
24.6
20.5
47.7
86.0
83.3
100
Total
20.5
34.2
100
activity level in 100-L fermentation process. And 3.2 kg of cells were
harvested from 60 L fermentation broth.
3.4. Production of S-DMCPCA in a 15,000-L reactor
One kilogram of cells was used for the enzymatic resolution
reaction, which was calculated to be equivalent to a 19 L fermen-
tation volume. Cells were resuspended in water and homogenized,
and the debris was removed by centrifugation. The reaction was
initiated at 35 ◦C by addition of this amidase lysate to 10,000 L
of 6% R,S-DMCPCA solution with agitation speed of 100 r/min in a
15,000-L reaction tank. The concentration of R-DMCPCS increased
linearly during the first 14 h of the resolution reaction. At 18 h, the
DMCPCS concentration increased at a much slower rate and started
to approach its maximum, while the ee value of the unreacted S-
DMCPCA reached 99.32% (Figs. 4 and 5). The reaction was stopped
by adjusting the pH to 10.5. The yield of the resolution reaction was
47.7% (Table 1).
In this study, limited enzyme activity was applied to enlarged
reaction volume to compensate the number of reaction batches
using immobilized enzyme. With limited enzyme, filtration was
not necessary and the reaction solution could be subjected to
downstream processing directly, even if the cell suspension was
applied without homogenization. Despite the strict stereoselec-
tivity of recombinant amidase, it could hydrolyze S-DMCPCA if
R-DMCPCA has been completely hydrolyzed into R-DMCPCS. Thus,
with limited enzyme activity, this unwanted reaction could be kept
at low level during the time required for downstream process-
ing.
Acknowledgments
This work was supported by National Key Technology
R&D Program during the 11th Five-Year Plan Period of China
(2007BAQ00155), the National Natural Science Foundation of China
(30900030), and Research Fund for the Doctoral & Youth Scholars
Program of Higher Education of China (20090093120008).
Purification of the reaction solution was performed by resin
adsorption directly after reaction. R-DMCPCS was not adsorbed and
flowed through the resin, while S-DMCPCA was adsorbed by the
HZ801 resin. The reaction product R-DMCPCS and the unreacted
S-DMCPCA were thus fully separated after water wash. Following
elution with 80% (v/v) acetone, the recovery yield of S-DMCPCA in
resin separation step was 86%. The eluate was further decolorized
and concentrated, and 20.5 kg of pure S-DMCPCA was obtained after
crystallization. The ee value of the final product S-DMCPCA was
100% and the total yield (from R,S-DMCPCA) was 34.2% (Table 1).
The final S-DMCPCA yield could be further increased in a real
industrial process by additional procedures. The mother liquor of
the crystallization step could be further purified, and the R-DMCPCS
could be racemized and amidated to R,S-DMCPCA [16] for reuse in
enzymatic resolution.
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