A novel method of producing the key intermediate ASI-2 of ranirestat using a porcine liver esterase (PLE) substitute enzyme

Article abstract of DOI:10.1080/09168451.2019.1580139

(R)-2-amino-2-ethoxycarbonylsuccinimide (ASI-2) is a key intermediate used in the pharmaceutical industry and is valuable for the industrial synthesis of ranirestat, which is a potent aldose reductase inhibitor. ASI-2 was synthesized in a process combining chemical synthesis and bioconversion. Bioconversion in this study is a key reaction, since optically active carboxylic acid derivative ((R)-1-ethyl hydrogen 3-benzyloxycarbonylamino-3-ethoxycarbonyl-succinate, Z-MME-AE) is synthesized from a prochiral ester, diethyl 2-benzyloxycarbonyla-mino-2-ethoxycarbonylsuccinate, Z-MDE-AE, at a theoretical yield of 100%. Upon screening for microorganisms that asymmetrically hydrolyze Z-MDE-AE, Bacillus thuringiensis NBRC13866 was found. A novel esterase EstBT that produces Z-MME-AE was purified from Bacillus thuringiensis NBRC13866 and was stably produced in Escherichia coli JM109 cells. Using EstBT rather than porcine liver esterase (PLE), ASI-2 was synthesized with a 17% higher total yield by a novel method, suggesting that the esterase EstBT is a PLE substitute enzyme and therefore, may be of interest for future industrial applications.

Full text of DOI:10.1080/09168451.2019.1580139

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20
A novel method of producing the key intermediate
ASI-2 of ranirestat using a porcine liver esterase
(PLE) substitute enzyme
Ei-Tora Yamamura, Kazuya Tsuzaki & Shinji Kita
To cite this article: Ei-Tora Yamamura, Kazuya Tsuzaki & Shinji Kita (2019): A novel method of
producing the key intermediate ASI-2 of ranirestat using a porcine liver esterase (PLE) substitute
enzyme, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2019.1580139
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1580139
A novel method of producing the key intermediate ASI-2 of ranirestat using a
porcine liver esterase (PLE) substitute enzyme
Ei-Tora Yamamura , Kazuya Tsuzaki and Shinji Kita
Technical Department, Kyowa Pharma Chemical Co., Ltd., Takaoka, Japan
ABSTRACT
ARTICLE HISTORY
Received 27 December 2018
Accepted 1 February 2019
(R)-2-amino-2-ethoxycarbonylsuccinimide (ASI-2) is a key intermediate used in the pharma-
ceutical industry and is valuable for the industrial synthesis of ranirestat, which is a potent
aldose reductase inhibitor. ASI-2 was synthesized in a process combining chemical synthesis
and bioconversion. Bioconversion in this study is a key reaction, since optically active
carboxylic acid derivative ((R)-1-ethyl hydrogen 3-benzyloxycarbonylamino-3-ethoxycarbonyl-
succinate, Z-MME-AE) is synthesized from a prochiral ester, diethyl 2-benzyloxycarbonyla-
mino-2-ethoxycarbonylsuccinate, Z-MDE-AE, at a theoretical yield of 100%. Upon screening
for microorganisms that asymmetrically hydrolyze Z-MDE-AE, Bacillus thuringiensis
NBRC13866 was found. A novel esterase EstBT that produces Z-MME-AE was purified from
Bacillus thuringiensis NBRC13866 and was stably produced in Escherichia coli JM109 cells.
Using EstBT rather than porcine liver esterase (PLE), ASI-2 was synthesized with a 17% higher
total yield by a novel method, suggesting that the esterase EstBT is a PLE substitute enzyme
and therefore, may be of interest for future industrial applications.
KEYWORDS
Bioconversion; PLE; prochiral
molecule; asymmetric
hydrolysis; ranirestat (AS-
3201)
ASI-2
[(R)-2-amino-2-ethoxycarbonylsuccinimide],
PLE, which is a type of the valuable esterases [9–13]
used in manufacturing industries, is one of the most
useful esterases for process chemistry and organic
synthesis. Numerous reports [6,8,14–18] have high-
lighted various methods producing a wide range of
optically active molecules using commercial PLE.
However, commercial PLE has the drawback of being
derived from porcine liver tissue extracts, which are
obtained from an animal organ. In addition, commer-
cial PLE has the drawback that its activity differs
between various batches, since commercial PLE
extracted from porcine liver tissue has different ratios
of esterases [19] and contains a plurality of isoenzymes
(α, β, and γ-PLE) [20].
To solve the problems associated with heteroge-
neous commercial PLE, many studies involving the
production of recombinant γ-PLE have been
reported. Brüsehaber et al. [21] have described the
expression of the γ-PLE gene with the Thioredoxin-
tag (Trx-tag) in Escherichia coli. Böttcher et al. [22]
have described co-expression with the molecular
chaperones GroEL and GroES in E. coli to produce
γ-PLE. These methods have the drawback that the
activity in whole cell is low for industrial applica-
tions. Böttcher et al. [22] have also described that
the γ-PLE gene was expressed with ompA (outer
membrane protein A secretion signal) and relB (pec-
tate lyase B signal sequence for periplasmic translo-
cation) for periplasmic expression of the γ-PLE
gene; however, the majority of γ-rPLE (recombinant
which is a key intermediate used in the pharmaceutical
industry, is valuable for the industrial synthesis of
ranirestat
((R)-(−)-2-(4-bromo-2-fluorobenzyl)-
1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-4-spiro-3′-
pyrrolidine-1,2′,3,5′-tetrone) [1]. Ranirestat is a potent
aldose reductase inhibitor that was initially developed
as a therapeutic agent for diabetic complications [1].
Aldose reductase is the rate-limiting enzyme of the
polyol pathway and catalyzes the reduction reaction
from glucose to sorbitol. Increases in aldose reductase
activity and accumulation of sorbitol in diabetic
patients have been reported [2]. Toyoda et al. [3]
have described that ranirestat reduced the area of
stained glial fibrillary acidic protein and retinal thick-
ness in Spontaneously Diabetic Torii (SDT) rats.
Several methods for synthesizing the key inter-
mediate of ranirestat, ASI-2, using the optical reso-
lution method have been reported [1,4–7]. These
methods have the drawback that recovery and race-
mization of enantiomer are needed after optical
resolution. Kasai et al. [8] have described a method
of synthesizing ASI-2 without optical resolution.
The method includes a hydrolysis step of a pro-
chiral ester derivative to an optically active car-
boxylic acid derivative using a porcine liver
esterase (PLE). There is an advantage that optically
active carboxylic acid derivatives can be obtained
with a theoretical yield of 100%; however, this
method has the drawback of using PLE.
CONTACT Ei-Tora Yamamura
eitora.yamamura@kyowa-kirin.co.jp
© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
E.-T. YAMAMURA ET AL.
γ-PLE) exists in an insoluble form as inclusion
bodies in the cytoplasm. In the study with yeast as
a host, Lange et al. [23] have described that active γ-
rPLE was secreted into the culture supernatant using
recombinant Pichia pastoris expressing the γ-PLE
gene. This method is limited by the fact that the
activity of the culture supernatant is low for indus-
trial applications.
Recently, in the manufacturing industries, envir-
onmental aspects are emphasized in addition to
production costs. Many production processes
using bioconversion reactions occur at ambient
temperature and pressure [24–28], and are good
for environmental conservation. In this study, we
sought to identify an esterase, EstBT, that substi-
tutes for PLE and constructed a scheme to synthe-
size the pharmaceutical intermediate ASI-2 in a
process in which chemical synthesis and bioconver-
sion are combined (Figure 1).
Standard genetic manipulations and sequence
analysis
Cloning was performed by standard genetic manipula-
tion techniques [29]. E. coli transformations were per-
formed using the E. coli Transformation Buffer Set
(Zymo Research Corp., Irvine, CA, USA). Genomic
DNA was isolated using the Genomic DNA Buffer set
and the Genomic-tip 500/G (Qiagen, Hilden,
Germany). PCR fragments were prepared with
KOD -plus- (Toyobo Co., Ltd.). DNA sequences of
the plasmid inserts were determined using the primer-
walking method described by Yamamura [30].
Sequence assembly and analysis were performed using
GENETYX Ver.12 (GENETYX Corp., Tokyo, Japan)
and DNASIS Pro Ver.2.0 (Hitachi Software Corp.,
Tokyo, Japan).
Synthesis of diethyl 2-benzyloxycarbonylamino-2-
ethoxycarbonylsuccinate (Z-MDE-AE)
Materials and methods
A suspension of diethyl 2-benzyloxycarbonylamino-
malonate (5.0 g), potassium carbonate (2.7 g),
potassium iodide (0.27 g), and ethyl chloroacetate
(2.6 g) in N,N-dimethylformamide (DMF, 50 mL)
was stirred at 50°C for 1 h. The reaction mixture
was cooled, and then poured into diluted hydro-
chloric acid, and the mixture was extracted with
ethyl acetate. The extract was washed with satu-
rated brine, and then dried over anhydrous magne-
sium sulfate. The solvent was evaporated under
reduced pressure, and the residue was crystallized
from ethyl acetate/n-hexane to obtain diethyl 2-
benzyloxycarbonylamino-2-ethoxycarbonylsuccinate
(Z-MDE-AE).
Strains and plasmids
Bacillus thuringiensis NBRC3951, NBRC101235, and
NBRC13866 were obtained from the National
Institute of Technology and Evaluation (Tokyo,
Japan). E. coli JM109 was obtained from Toyobo Co.,
Ltd. (Osaka, Japan). The E. coli expression vector
pQE70 was obtained from Qiagen (Hilden, Germany).
Chemicals and enzymes
All chemicals were purchased from FUJIFILM Wako
Pure Chemical Corp. (Osaka, Japan) and Sigma-
Aldrich (St. Louis, MO, USA). PLE was purchased
from Sigma-Aldrich. All restriction enzymes and
DNA modification enzymes were purchased from
Toyobo Co., Ltd. (Osaka, Japan) and New England
Biolabs, Inc. (Ipswich, MA, USA). The synthetic oli-
gonucleotides used in this study were purchased from
Hokkaido System Science Co., Ltd. (Hokkaido, Japan).
Screening of a bacterial library for asymmetric
hydrolyzing activity of Z-MDE-AE
The actinomycetes and bacteria were inoculated into
5 mL of GPY broth (1% (w/v) glucose, 0.5% (w/v)
Bacto peptone, and 0.3% (w/v) Bacto yeast extract)
Figure 1. Scheme for the synthesis of ASI-2 via the bioconversion of Z-MDE-AE to Z-MME-AE.
Z-MDE-AE is synthesized by condensation. Z-MME-AE is synthesized by bioconversion of Z-MDE-AE. ASI-2 is synthesized from Z-MME-AE by cyclization,
amidation, and deprotection. Z, Z-MDE-AE, Z-MME-AE, and ASI-2 indicate benzyloxycarbonyl group (Cbz), diethyl 2-benzyloxycarbonylamino-2-
ethoxycarbonylsuccinate, (R)-1-ethyl hydrogen 3-benzyloxycarbonylamino-3-ethoxycarbonylsuccinate, and (R)-2-amino-2-ethoxycarbonylsuccinimide,
respectively.

PRODUCING THE KEY INTERMEDIATE USING A PLE SUBSTITUTE ENZYME
3
and incubated at 30°C with shaking for 3–7 days.
The cells were harvested, freeze-dried, and sus-
pended in 0.5 mL of 100 mM sodium phosphate
buffer (pH 7.0) containing 0.1% (w/v) Z-MDE-AE
and 10% (v/v) acetonitrile. The mixtures were incu-
bated at 30°C with shaking for 72 h. The reactions
were stopped by the addition of 1.5 mL of ethanol.
Cell suspensions were centrifuged at 10,000 × g for
10 min at 4°C, and the resulting supernatants were
analyzed by high-performance liquid chromatogra-
phy (HPLC) as described below.
0.3 M NaCl in buffer A. The active fraction was
dialyzed to remove CHAPS, combined with polyox-
yethylene lauryl ether (Brij 35) at a final concentra-
tion of 0.05% (w/v) and NaCl at a final concentration
of 0.3 M. Samples were then applied to Phenyl
Sepharose High Performance (GE Healthcare UK
Ltd., Buckinghamshire, England) equilibrated with
10 mM Tris buffer (pH 8.0) containing 0.05% (w/v)
Brij 35, 1 mM DTT, and 0.3 M NaCl. The proteins
were eluted with a linear gradient of 0 to 0.3 M NaCl
in 10 mM Tris buffer (pH 8.0) containing 0.05%
(w/v) Brij 35 and 1 mM DTT. The active fraction
was dialyzed against buffer A, concentrated by ultra-
filtration, and applied to Mono Q (GE Healthcare UK
Ltd., Buckinghamshire, England) equilibrated with
buffer A. The proteins were eluted with a linear
gradient of 0 to 0.5 M NaCl in buffer A. The active
fraction was applied to Benzamidine Sepharose 4 Fast
Flow (GE Healthcare UK Ltd., Buckinghamshire,
England) equilibrated with buffer A and then pro-
teins were eluted with a linear gradient of 0 to 0.5 M
NaCl. Protein purity was analyzed on SDS-PAGE
(5–20% gradient gel) by the method described by
Laemmli [31] and was visualized by staining with
Coomassie Brilliant Blue R250 and Silver Stain 2 kit
purchased from FUJIFILM Wako Pure Chemical
Corp. (Osaka, Japan). The SDS-PAGE standards
(Broad) purchased from Bio-Rad Laboratories, Inc.
(Hercules, CA, USA) were used as molecular weight
markers. The purified enzyme was designated as
EstBT. The amino acid sequence of the purified
EstBT was determined by automated Edman degra-
dation and the analysis was carried out at APRO
Science Inc. (Tokushima, Japan).
Analysis of reaction mixtures by HPLC
The hydrolysis rate of Z-MDE-AE and optical purity of
the product 1-ethyl hydrogen 3-benzyloxycarbonyla-
mino-3-ethoxycarbonylsuccinate were determined by
HPLC on a CHIRALCEL OJ-RH (ϕ 4.6 × 150 mm;
Daicel Corp., Tokyo, Japan). The mobile phase was acet-
onitrile/aqueous perchloric acid [pH 2.0, 30:70 (v/v)], the
flow rate was 1.0 mL/min, the column temperature was
20°C, and the detection wavelength was at 254 nm.
Purification of an esterase EstBT
B. thuringiensis NBRC13866 cells were cultivated in
GPY broth as described by Kozono et al. [27]. An
esterase EstBT was purified as described by
Yamamura [10]. B. thuringiensis NBRC 13866 was
aerobically cultured in the GPY broth at 30°C for
16 h. Wet cells were collected from the culture
broth by centrifugation (12,000 × g, 10 min) and
suspended in 10 mM Tris buffer (pH 8.0). The sus-
pension was disrupted by ultrasonication with an
ultrasonic homogenizer US300 (Nihonseiki Kaisha
Ltd., Tokyo, Japan) on ice, and then centrifuged at
14,000 × g for 20 min. The resulting supernatant was
used as the crude extract. The crude extracts were
added with ammonium sulfate at 20% (w/v) saturated
concentration on ice. The mixture was stirred and
centrifuged (12,000 × g, 10 min), and the supernatant
was removed. The precipitates were suspended in
10 mM Tris buffer (pH 8.0) containing 1 mM dithio-
threitol (DTT), and dialyzed against the same buffer.
This solution was added with DEAE Sepharose Fast
Flow (GE Healthcare UK Ltd., Buckinghamshire,
England), the samples were mixed, and the mixture
was filtered to collect an active solution. The solution
was added with 3-[(3-chloroamidopropyl)dimethy-
lammonio]-propanesulfonate (CHAPS) at a final con-
centration of 1% (w/v) and mixed. The mixture was
subjected to treatment with an ultrasonic homogeni-
zer for 30 min on ice, and then applied to DEAE
Sepharose equilibrated with 10 mM Tris buffer
(pH 8.0) containing 1 mM DTT and 1% (w/v)
CHAPS (henceforth referred to as buffer A). The
proteins were eluted with a linear gradient of 0 to
Enzyme and protein assays
The reaction mixture consisting of 1% (w/v) Z-MDE-
AE, 100 mM phosphate buffer (pH 7.0), 10% (v/v)
acetonitrile, and enzyme was incubated at 30°C for
2 h. The reactions were stopped by addition of three
volumes of ethanol and were centrifuged at 12,000 × g
for 5 min at 4°C. The resulting supernatants were
analyzed by high-performance liquid chromatography
(HPLC) as described above. The activity for converting
1 µmol of Z-MDE-AE per 1 min was defined as 1 unit
(U). Proteins were measured using a Bio-Rad Protein
Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA)
with bovine serum albumin as a standard.
Construction of estBT expression vectors
The estBT gene that encodes EstBT, which contains a
deletion of 40 amino acids at the N-terminus, was
amplified using the following primers: P-F120: 5′-
GTACAAAGCATGCAAAAGAAACCAGTCTCTT-
TAACGGAGCG-3′ (SphI site is underlined) and

4
E.-T. YAMAMURA ET AL.
P-R1380: 5′-CTTTTGTTGTAGATCTTTTCTTTGTC
GCATTGACCCA-3′ (BglII site is underlined), from
the genomic DNA of B. thuringiensis NBRC13866
Producing (R)-1-ethyl hydrogen 3-
benzyloxycarbonylamino-3-
ethoxycarbonylsuccinate (Z-MME-AE) by
bioconversion
with
a
Mastercycler gradient thermal cycler
(Eppendorf, Hamburg, Germany). The PCR fragment
containing estBT was digested with SphI and BglII
and then ligated into the SphI and BglII sites of
pQE70 to generate the plasmid pBTEST38, which
produces recombinant EstBT (rEstBT) with deletion
of 40 amino acids at the N-terminus. To produce the
full-length rEstBT (rEstBT-full), the full-length estBT
was amplified using the following primers: P-F1: 5′-
AAACAGCATGCAAAAAAAGAAATTACAAAAA-
TCAGCAC-3′ (SphI site is underlined) and P-R1380:
5ʹ-CTTTTGTTGTAGATCTTTTCTTTGTCGCATT
GACCCA-3ʹ (BglII site is underlined) from the geno-
mic DNA of B. thuringiensis NBRC13866. The PCR
fragment was digested with SphI and BglII, ligated
into the SphI and BglII sites of pQE70, and designated
as pBTEST12. The plasmids pBTEST38 and
pBTEST12 produce rEstBT and rEstBT-full, respec-
tively, with a His-tag attached to C-terminus.
The reaction mixture consisting of 89 mM [3.5%
(w/v)] Z-MDE-AE, 100 mM phosphate buffer (pH
7.0), and 100 mU/mL enzyme (EstBT, rEstBT, PLE,
or recombinant whole-cell) was incubated at 25°C.
The pH of the reaction mixture was maintained at
7.0 with 28% NaOH automatically, since the pH
decreases during the reaction. The reaction mixture
was centrifuged at 12,000 × g for 5 min at 4°C to
obtain the supernatant containing Z-MME-AE.
Synthesis of ASI-2 from Z-MME-AE
To cyclize Z-MME-AE, a solution of Z-MME-AE
(1.8 g) in tetrahydrofuran (THF, 20 mL) was combined
with triethylamine (0.96 mL) and isobutyl chlorofor-
mate (0.87 g) in this order at −15°C with stirring, and
the mixture was stirred for 5 min. A solution of 25%
aqueous ammonia (0.47 mL) was dropped into the
reaction mixture at the same temperature. The reaction
mixture was stirred at the same temperature for 1 h, and
then poured into diluted hydrochloric acid. The mix-
ture was extracted with ethyl acetate, and the extract
was dried over anhydrous magnesium sulfate. The sol-
vent was evaporated under reduced pressure, and the
residue was subjected to silica gel column chromato-
graphy, in which elution was performed with n-hexane/
ethyl acetate (1:1) for purification, and then crystallized
from ethyl acetate/n-hexane. For amidation, a solution
of the obtained crystals in dehydrated ethanol (75 mL)
was added with sodium ethoxide (310 mg) on ice while
stirring. The mixture was stirred at the same tempera-
ture for 2 h, then cold 1 mol/L hydrochloric acid was
added, and the mixture was extracted with ethyl acetate.
The extract was washed with saturated brine, and then
dried over anhydrous magnesium sulfate. The solvent
was evaporated under reduced pressure, and the residue
was recrystallized from ethyl acetate/n-hexane. For
deprotection, the obtained crystals were dissolved in
ethanol (140 mL), the solution was added with 5%
palladium/carbon (56 mg), and catalytic hydrogenation
was performed at room temperature under a hydrogen
atmosphere. After the catalyst was removed by filtra-
tion, the filtrate was concentrated under reduced pres-
sure. The residue was recrystallized from ethanol to
obtain ASI-2 as colorless crystals.
Cultivation of recombinant E. coli expressed
estBT and purification of rEstBT
Recombinant E. coli JM109 cells were inoculated
into 5 mL of Luria-Bertani (LB) broth (1% Bacto
tryptone, 0.5% Bacto yeast extract, and 1% NaCl)
with ampicillin (100 μg/mL) and were incubated at
25°C with shaking for 16 h. Four milliliters of seed
broth was transferred into 200 mL of LB broth with
ampicillin (100 μg/mL) and isopropyl-β-D-thioga-
lactopyranoside (0.1 mM) and was incubated at
25°C with shaking for 24 h. With the Ni-NTA
Fast Start Kit obtained from Qiagen (Hilden,
Germany), the His-tagged rEstBT and the His-
tagged rEstBT-full were purified from recombinant
E. coli JM109 harboring the plasmids pBTEST38
and pBTEST12, respectively.
Activity measurements of recombinant E. coli
JM109 cells
The reaction mixture consisting of 89 mM [3.5%
(w/v) Z-MDE-AE, 100 mM buffer [standard buffer
was phosphate buffer (pH 7.0)], and a portion of the
cultured recombinant E. coli JM109 cells was incu-
bated at 20–40°C (standard reaction condition was
25°C) for 2 h. The reaction was stopped by the addi-
tion of three volumes of ethanol and samples were
centrifuged at 12,000 × g for 5 min at 4°C. The
resulting supernatant was analyzed by HPLC as
described above. The activity for converting 1 µmol
of Z-MDE-AE per 1 min was defined as 1 U.
Nucleotide sequence accession number
The sequence of the estBT gene has been submitted to
the DDBJ/EMBL/GenBank databases under accession
number LC434454.

PRODUCING THE KEY INTERMEDIATE USING A PLE SUBSTITUTE ENZYME
5
Results
added to the buffer. In the Phenyl Sepharose step,
0.05% (w/v) Brij 35 was added to the buffer, since the
specific activity of the active fraction increased. In
this step, the addition of 1% (w/v) CHAPS hardly
increased the specific activity. Figure 2 shows that
proteins in the Benzamidine Sepharose step displayed
a single protein band with a molecular weight of
49 kDa via SDS-PAGE. The purified protein was
designated as EstBT. The specific activity of the pur-
ified EstBT was 160 mU/mg, and the optical purity of
the product was 99.9% ee of the (R)enantiomer.
Screening for Z-MDE-AE-asymmetric hydrolyzing
microorganisms
E. coli is the most useful host for the expression of
various genes and has many advantages, including
easy handling, short generation times, various genetic
tools, and high transformation efficiency. To express
a gene in recombinant E. coli, prokaryotes were
screened. A total of 495 strains from our bacterial
library were used, consisting of 151 strains of actino-
mycetes and 344 strains of bacteria. Among the
microbial strains, only one strain, B. thuringiensis
NBRC13866, exhibited Z-MDE-AE-asymmetric
hydrolyzing activity, and the optical purity of the
product was 99.9% enantiomeric excess (ee) of the
(R)-enantiomer.
Amino acid and nucleotide sequence of EstBT
The amino acid sequence at the N-terminus of pur-
ified EstBT was determined by automated Edman
degradation. Homology search for the obtained
sequence (EKKPVSLTERTSLFF) was performed
using the BLASTP 2.2.26 program (protein–protein
Basic Local Alignment Search Tool, https://www.
ddbj.nig.ac.jp/index-e.html) [32]. The obtained
sequence was homologous to α/β hydrolase
(UniProt accession no. A0A243J741) derived from
B. thuringiensis serovar pirenaica. Primers were pre-
pared based on the sequence of the α/β hydrolase; the
estBT gene that encodes EstBT was amplified by PCR
from B. thuringiensis NBRC13866, and the sequence
of the estBT gene was determined using the primer
walking method [30]. The estBT gene is 1380 bp in
length and encodes a protein of 460 amino acid
residues (Figure 3). According to the TBLASTN
2.2.26 program (protein–translated nucleotide Basic
Local Alignment Search Tool) [32], EstBT is homo-
logous to the α/β esterase (Table 2). EstBT contains a
conserved serine protease motif (G-X-S-X-G) [33],
and the serine protease belongs to the superfamily
of proteins with α/β hydrolase fold. Analysis of the
amino acid sequence presumed from the sequence of
the obtained estBT gene revealed that the N-terminal
sequence (EKKPVSLTERTSLFF) of EstBT that was
purified from B. thuringiensis NBRC13866 was not
at the N-terminal presumed from the obtained estBT
gene sequence but rather at 41 amino acid residues
(Figure 3). The predicted molecular weight from the
Identification of a Z-MDE-AE-asymmetric
hydrolyzing enzyme from B. thuringiensis
NBRC13866
In enzyme purifications, the target enzyme can be
efficiently purified by the addition of a surfactant to
the buffer. Therefore, 12 different types of surfactants
were screened. With two surfactants, CHAPS and Brij
35, the enzyme showed high specific activities in the
active fraction in the purification steps. Without sur-
factant, no activity of EstBT was detected in fractions.
For these reasons, CHAPS or Brij 35 was added to the
buffer in the purification steps. On the other hand, in
the enzyme reaction, the activity of EstBT was
detected with no surfactant. For this reason, the
activity measurements were performed without add-
ing surfactants to the reaction solution.
To identify the enzyme that hydrolyzes Z-MDE-
AE to Z-MME-AE asymmetrically, the crude extracts
of B. thuringiensis NBRC13866 were subjected to six
purification steps (Table 1). In the first step, the
ammonium sulfate precipitation step, the specific
activity did not increase. However, when this step
was omitted, EstBT could not be purified in next
steps. For all purification steps, with the exception
of the Phenyl Sepharose step, 1% (w/v) CHAPS was
Table 1. Purification of esterase from Bacillus thuringiensis NBRC13866.
Total
Activity (mU)
4740
Total
Protein (mg)
7400
Specific
Purification step
Activity (mU mg−1
)
Yield (%)
100
Purification Fold
Crude extructs
0.641
0.641
0.794
1.65
1.00
1.00
1.24
2.57
12.4
45.7
250
Ammonium sulfate precipitation (0–20%)
DEAE Sepharose Fast Flow (through)
DEAE Sepharose Fast Flow (0–0.3 M NaCl)
Phenyl Sepharose High Performance
Mono Q
4700
7330
99.1
4160
5240
87.7
287
174
6.05
98.1
12.3
7.97
2.07
52.9
1.81
29.3
1.12
Benzamidine Sepharose 4 Fast Flow
12.1
0.0756
160
0.255
U = μmol min−1
.

6
E.-T. YAMAMURA ET AL.
Figure 2. SDS-PAGE of EstBT from Bacillus thuringiensis NBRC13866.
Lane 1, size marker; lane 2, the active fraction of Mono Q; lane 3, the active fraction of Benzamidine Sepharose.
amino acid sequence EKKPVSLTERTSLFF to the C-
terminus of EstBT was 47 kDa, which is agreement
with the results of SDSPAGE in Figure 2.
52 kDa on SDS-PAGE, which is in agreement with the
predicted molecular weight. The production level of
rEstBT-full was 5.5 0.7% DCW.
The relative activities of native EstBT, rEstBT,
rEstBT-full, and PLE were determined to be
Expression of estBT in E. coli cells
168
8 mU/mg, 164
6 mU/mg, 161
12 mU/
For overexpression of the estBT gene in E. coli, two
types of estBT expression vectors were constructed.
The first is an expression vector that produces full-
length EstBT with His-tag (rEstBT-full), designated as
pBTEST12. The other is an expression vector that
produces the His-tagged EstBT (rEstBT), which is
truncated at 40 amino acid residues and resembles
the EstBT purified from B. thuringiensis NBRC13866,
designated as pBTEST38.
The plasmid pBTEST38 was transformed into E.
coli JM109. rEstBT was overproduced in the recombi-
nant E. coli JM109 and was detected in a soluble form
with a molecular weight of 49 kDa on SDS-PAGE,
which is in agreement with the predicted molecular
weight (Figure 4). The rEstBT was purified from the
recombinant E. coli JM109 with the Ni-NTA Fast Start
Kit (Figure 4, lane 4). Analysis of the expression level
of rEstBT by protein assay revealed 5.7 0.5% rEstBT
per dry cell weight (DCW). Moreover, the recombi-
nant E. coli JM109 harboring the plasmid pBTEST12
also overproduced rEstBT-full. The rEstBT-full was
detected in a soluble form with a molecular weight of
mg, and 288 18 mU/mg, respectively (Table 3). The
relative activities between native EstBT, rEstBT, and
rEstBT-full were nearly identical, while that of PLE
being approximately 1.7 times higher than those of
the other enzymes. Additionally, the optical purities
of (R)-enantiomer of native EstBT, rEstBT, rEstBT-
full, and PLE were 99.9% ee, 99.9% ee, 99.9% ee, and
93.6% ee, respectively (Table 3). The optical purities
between native EstBT, rEstBT, and rEstBT-full were
the same, while those of EstBT, rEstBT, and rEstBT-
full were higher than that of PLE.
In the pH stability, incubation at pH 6, pH 7,
and pH 8 for 2 h at 4°C did not reduce the enzyme
activity of native EstBT, rEstBT, rEstBT-full,
and PLE.
Characterization of recombinant E. coli JM109
producing rEstBT
The optimal reaction conditions of recombinant E.
coli JM109 producing rEstBT, which harbors the
plasmid pBTEST38, were investigated to efficiently

PRODUCING THE KEY INTERMEDIATE USING A PLE SUBSTITUTE ENZYME
7
Figure 3. Nucleotide sequence of estBT from Bacillus thuringiensis NBRC13866 and deduced amino acid sequence of full-length
EstBT.
The conserved G-X-S-X-G motif of serine type proteases is boxed. The amino acid sequence at the N-terminus of EstBT purified from Bacillus thuringiensis
NBRC13866 is underlined. Directing arrows indicate primers used to construct estBT expression vectors.
produce Z-MME-AE from Z-MDE-AE. The optimal
pH of the activity of the recombinant E. coli JM109
was examined from pH 4.0 to 10.0 and was found to
be 7.0 (Figure 5(a)). The effects of reaction tempera-
ture and stable temperature were also examined. The
maximum activity was observed at 30°C (Figure 5(b))
and the stable temperature was 30°C or lower
(Figure 5(c)). Therefore, the bioconversion described
below was performed at 25°C.
Bioconversion of Z-MDE-AE to Z-MMD-AE
Figure 6(a) shows bioconversion of Z-MDE-AE to Z-
MMD-AE with 100 mU/mL of the enzymes EstBT,

8
E.-T. YAMAMURA ET AL.
Table 2. Features and identities between EstBT and known proteins.
GenBank accession no.
AJA23081
Product
Organism
Bacillus thuringiensis serovar galleriae
Bacillus mycoides KBAB4
Bacillus sp. E25
Identity
95%
Refs.
Hydrolase
-
[34]
-
ABY46486
Putative hydrolase
92%
AXR22762
α/β fold hydrolase
92%
AXR17068
α/β fold hydrolase
Bacillus sp. CR71
92%
-
AJH17030
α/β hydrolase fold family protein
Bacillus mycoides strain ATCC 6462
91%
[35]
Figure 4. SDS-PAGE of crude extracts from recombinant Escherichia coli JM109 expressing the estBT gene and the purified
rEstBT.
Lane 1, size marker; lane 2, the crude extracts from recombinant E. coli JM109 harboring pQE-70; lane 3, the crude extracts from recombinant E. coli
JM109 harboring pBTEST38; lane 4, the rEstBT purified from the recombinant E. coli JM109 harboring pBTEST38.
Table 3. Comparison of the relative activity between EstBT,
rEstBT, rEstBT-full, and PLE.
rEstBT, and PLE. The sequential changes in the pro-
duction of Z-MME-AE with EstBT, rEstBT, and PLE
were almost the same, and 89 mM [3.5% (w/v)]
Z-MDE-AE was converted to approximately 61 mM
Z-MME-AE (conversion rate 69%) in 33 h. On the
other hand, Figure 6(b) shows bioconversion of
Z-MDE-AE to Z-MMD-AE with 100 mU/mL recom-
binant whole-cell (wet-cell paste), which contains
Enzyme
EstBT
Relative activity (mU/mg)
Optical purity (% ee)
168
164
8
6
99.9
99.9
99.9
93.6
rEstBT
rEstBT-full
PLE
161 12
288 18
U = μmol min−1
.
Figure 5. Determination of optimal pH and temperature by recombinant Escherichia coli JM109 producing rEstBT.
(a) Effect of pH. Activity was assayed under standard reaction conditions, except for the buffer. Open circle, open triangle, open square, and closed circle
indicate acetic acid-sodium acetate buffer (AS), sodium phosphate buffer (NaP), Tris buffer (Tris), and borate buffer (borate), respectively. (b) Effect of
reaction temperature. Activity was assayed under standard reaction conditions, except for temperature. (c) Temperature stability of rEstBT. Activity was
assayed under standard reaction conditions. Reaction mixture was incubated for 30 min at 20–70°C before adding substrate. Open square indicates no
temperature treatment. Open circle indicates temperature treatment at 20–70°C.

PRODUCING THE KEY INTERMEDIATE USING A PLE SUBSTITUTE ENZYME
9
Figure 6. Bioconversion of Z-MDE-AE to Z-MME-AE with enzymes at a final activity concentration of 100 mU/mL.
(a) Bioconversion with purified enzymes. Substrate is 89 mM Z-MDE-AE. Closed circle, closed triangle, and closed square indicate Z-MDE-AE
concentrations of native EstBT, recombinant EstBT, and commercial PLE, respectively. Open circle, open triangle, and open square indicate Z-MME-AE
concentrations of native EstBT, recombinant EstBT, and commercial PLE, respectively. Vertical bars indicate standard deviation (SD) from three
independent experiments. (b) Bioconversion using recombinant Escherichia coli JM109 producing rEstBT. Substrate is 89 mM Z-MDE-AE. Closed circle
and open circle indicate Z-MDE-AE and Z-MME-AE concentrations, respectively. Vertical bars indicate standard deviation (SD) from three independent
experiments.
recombinant E. coli JM109 cells producing rEstBT.
For 4 hours from the start of the reaction, the con-
version rate with recombinant whole-cell was almost
the same as those with enzymes of EstBT, rEstBT,
and PLE. From the fourth hour onwards, the conver-
sion rate with recombinant whole-cell was higher
than those with EstBT, rEstBT, and PLE. In 33 h,
89 mM Z-MDE-AE was converted to approximately
81 mM Z-MME-AE (conversion rate 91%) with
recombinant whole-cell.
porcine liver tissue has different ratios of esterases
[19] and contains a plurality of isoenzymes (α, β, and
γ-PLE) [20]. To address the problems associated with
heterogeneous commercial PLE, many studies invol-
ving the production of recombinant γ-PLE have been
reported. However, the activities of whole-cell are low
for industrial applications.
We screened a PLE substitute enzyme that hydro-
lyzes Z-MDE-AE asymmetrically and is produced via
recombinant techniques in E. coli. B. thuringiensis
NBRC13866 hydrolyzed Z-MDE-AE asymmetrically
and the optical purity of the product was 99.9% ee,
while B. thuringiensis NBRC3951 and NBRC101235
from our bacterial library exhibited no hydrolysis
activity of Z-MDE-AE. This might be due to differ-
ences in the membrane permeability of the substrate,
expression levels of esterase gene, and substrate spe-
cificity of esterase among these strains. The freeze-
dried cells and crude extracts of B. thuringiensis
NBRC13866 hydrolyzed Z-MDE-AE, whereas wet
cells of B. thuringiensis NBRC13866 exhibited no
hydrolysis activity of Z-MDE-AE. Z-MDE-AE might
have poor membrane permeability in B. thuringiensis
NBRC 13866 since it is a gram-positive bacterial
species.
To identify the enzyme that hydrolyzes Z-MDE-
AE to Z-MME-AE asymmetrically, an enzyme pur-
ification protocol was performed. Figure 2 shows that
proteins in the Benzamidine Sepharose step displayed
the single protein band on the SDS-PAGE, suggesting
that EstBT was purified from the crude extracts of B.
thuringiensis NBRC13866 by six purification steps. In
the first step of enzyme purification, which is the
ammonium sulfate precipitation step, the specific
activity did not increase, although this step was indis-
pensable as biological substances other than proteins
Synthesis of ASI-2 by chemical synthesis and
bioconversion
ASI-2 was synthesized from diethyl 2-benzyloxycar-
bonylaminomalonate and ethyl chloroacetate
(Figure 1), which are readily and economically avail-
able starting materials. Via bioconversion with
recombinant whole-cell producing rEstBT, ASI-2
was synthesized with a total yield of 55%. On the
other hand, ASI-2 was synthesized with a total yield
of 38% via bioconversion with PLE. Using recombi-
nant whole-cell rather than PLE, ASI-2 was synthe-
sized with a 17% higher total yield. Through the
novel method using the PLE substitute enzyme,
which is recombinant whole-cell producing rEstBT,
ASI-2 was synthesized.
Discussion
PLE is one of several valuable esterases [9–13] used in
manufacturing industries, and the most useful ester-
ase for process chemistry, including the synthesis of
ASI-2 [8]. However, commercial PLE has the draw-
back that the activity is different between various
batches, since commercial PLE extracted from

10
E.-T. YAMAMURA ET AL.
(e.g., lipids, sugars) might be removed at this time.
The subsequent DEAE Sepharose step (through) was
required. This is likely because many enzymes that
interact strongly with DEAE were present in the
active fraction of ammonium sulfate precipitation.
In all purification steps, no activity of EstBT was
detected without surfactant. Due to the absence of
surfactant, it may be that the resin and EstBT directly
interacted and the structure of EstBT was denatured
[10]. On the other hand, in the enzyme reaction, the
activity of EstBT was detected with or without sur-
factant. This result suggests that EstBT is stable in
aqueous solution with or without surfactant.
One of our research objectives is to obtain the
gene of a PLE substitution enzyme that can be over-
expressed in E. coli. To construct estBT expression
vectors in E. coli, the nucleotide sequence of estBT
was determined (Figure 3). Homology search based
on the determined sequence suggests that EstBT is a
novel enzyme (Table 2) and contains a conserved
serine protease motif (G-X-S-X-G) [33]. During
enzyme purification (Table 1), EstBT was purified
with Benzamidine Sepharose, which is a carrier for
the purification of serine proteases, suggesting that
EstBT is a serine protease.
Two kinds of the recombinant E. coli JM109 cells
were constructed. The recombinant E. coli JM109
cells expressed the estBT gene in a soluble form,
which is consistent with the predicted molecular
weight. The production levels of rEstBT and
rEstBT-full, which were produced in recombinant E.
coli JM109 harboring pBTEST38 and pBTEST12,
were 5.9 0.5% DCW and 5.5 0.8% DCW, respec-
tively. These results suggest that the estBT gene was
overexpressed in E. coli JM109.
The relative activities and the optical purities
between native EstBT, rEstBT, rEstBT-full, and PLE
were compared. The relative activities and the optical
purities between native native EstBT, rEstBT, and
rEstBT-full were nearly identical, suggesting that
native EstBT, rPLE (truncated recombinant EstBT),
and rEstBT-full (full-length recombinant EstBT) have
the same activity. The relative activity of PLE was
only 1.7 times higher than those of the other enzymes
and the optical purity of PLE was lower than those of
the other enzymes. These results suggest that rEstBT
is the PLE substitute enzyme. However, we cannot
explain the effect due to the difference of 40 amino
acid residues between rEstBT and rEstBT-full. Due to
the low solubility of Z-MDE-AE [<0.05% (w/v)],
parameters (e.g., kcat, Km, or kcat/Km) of enzyme reac-
tion could not be determined. In addition, substrate
inhibition was not observed in 4% (w/v) Z-MDE-AE.
This may be due to the low concentration of Z-MDE-
AE dissolved in the reaction solution.
key reaction in this process, since Z-MME-AE is
synthesized at a theoretical yield of 100% (Figure 1).
For bioconversion of Z-MDE-AE to Z-MME-AE, the
optimal reaction conditions of EstBT using recombi-
nant E. coli JM109 producing rEstBT were investi-
gated. The optimal pH, maximum reaction
temperature, and stable temperature were 7.0, 30°C,
and 30°C or less, respectively, which represent neutral
pH and ambient temperature. These results suggest
that EstBT is suitable for bioconversion.
Using enzymes (EstBT, rEstBT, PLE, and recom-
binant whole-cell producing rEstBT) at a final activity
concentration of 100 mU/mL, bioconversion of Z-
MDE-AE to Z-MME-AE was performed. For
4 hours from the start of the reaction, the Z-MME-
AE concentrations among these enzymes in recombi-
nant whole-cell were almost the same. These results
suggest that there is almost no issue of membrane
permeability of Z-MDE-AE and Z-MME-AE when
recombinant whole-cell is used. From the fourth
hour onwards, the Z-MME-AE concentration with
recombinant whole-cell producing rEstBT was higher
than that with the purified rEstBT. This result sug-
gests that rEstBT is more stable in the cell than the
purified rEstBT, and there is nearly no issue of com-
pound stability of Z-MDE-AE and Z-MME-AE when
the recombinant whole-cell is used. In addition, since
cultured cells can be directly used for bioconversion,
rather than purified enzyme, the synthesis process of
Z-MME-AE is simplified. Moreover, the sequential
changes in the production of Z-MME-AE with
EstBT, rEstBT, and PLE were nearly identical. This
result suggests that enzyme stabilities of EstBT and
rEstBT are nearly identical to that of PLE. At
30 hours from the start of the reaction using
enzymes, compounds presumed to be decomposition
products of Z-MME-AE were detected, although the
structures could not be determined. If the cause of
this decomposition is clarified, it may be possible to
synthesize Z-MME-AE via bioconversion at a conver-
sion rate of 100%.
Numerous reports [6,8,14–18] have shown efficient
methods to produce a wide range of optically active
molecules using commercial PLE. In this study, we
used recombinant whole-cell produced rEstBT, rather
than PLE, to synthesize ASI-2 with a 17% higher total
yield. This may be due to the fact that the yield was
20% higher in the bioconversion of Z-MDE-AE to
Z-MME-AE (Figure 6). In addition, Z-MME-AE may
have been lost in the separation of Z-MME-AE from
the reaction solution containing recombinant cells
after bioconversion. Future studies to investigate the
range of substrate specificity of rEstBT compared to
PLE will be conducted using a wide range of
molecules.
Bioconversion of the prochiral ester Z-MDE-AE to
the optically active carboxylic acid Z-MME-AE is the
By making only effective optical isomers for phar-
maceutical products, the effect is often enhanced and

PRODUCING THE KEY INTERMEDIATE USING A PLE SUBSTITUTE ENZYME
11
[4] Tanaka D, inventor; Dainippon Sumitomo Pharma
Co., Ltd., assignee. 3-Hydrazino-2,5-dioxopyrroli-
dine-3-carboxylates, process for production of the
same, and use of the same. United States patent US
8,003,808 B2. 2011 Aug 23.
[5] Tanaka D, Negoro T, inventors; Dainippon Sumitomo
Pharma Co., Ltd., assignee. Optically active 3-amino-
2,5-dioxopyrrolidine-3-carboxylate, process for pro-
duction of the compound, and use of the compound.
United States patent US 8,058,456 B2. 2011 Nov 15.
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Industries, Ltd., Dainippon Sumitomo Pharma Co.,
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side effects are often reduced. As with PLE, EstBT is
one of the useful enzymes in the pharmaceutical
industry, since EstBT hydrolyzed Z-MDE-AE asym-
metrically and the optical purity of the product was
99.9% ee.
In conclusion, a novel esterase EstBT, which is the
PLE substitute enzyme, was identified and produced
stably in E. coli. Using a novel method that combines
chemical synthesis and bioconversion using recombi-
nant whole-cell produced rEstBT, the pharmaceutical
intermediate ASI-2 was synthesized efficiently.
Acknowledgments
The authors would like to thank Dr. Masaji Kasai for the
information on key bioconversion reactions. We would like
to thank Dr. Tadashi Ogawa of Kyowa Pharma Chemical
Co., Ltd., for the helpful information of compounds includ-
ing substrates. We gratefully acknowledge Prof. Jun Ogawa
of Kyoto University for his helpful advice regarding this
manuscript.
Author contributions
Ei-Tora Yamamura, Kazuya Tsuzaki, and Shinji Kita con-
ceived and designed the experiments. Shinji Kita investi-
gated the patents on the ASI-2 manufacturing method. Ei-
Tora Yamamura wrote the manuscript. Ei-Tora Yamamura
and Kazuya Tsuzaki performed the experiments.
[9] Hayashi Y, Onaka H, Itoh N, et al. Cloning of the
gene cluster responsible for biosynthesis of KS-505a
(longestin),
a
unique tetraterpenoid. Biosci
Biotechnol Biochem. 2007;71:3072–3081.
[10] Yamamura ET, Kita S. A novel method of producing
the pharmaceutical intermediate (R)-2-chloroman-
delic acid by bioconversion. Biosci Biotechnol
Biochem. 2019;83:309–317.
Disclosure statement
[11] Okazaki R, Kiyota H, Oritani T. Enzymatic resolu-
tion of ( )-epoxy-β-cyclogeraniol, a synthetic pre-
cursor for abscisic acid analogs. Biosci Biotechnol
Biochem. 2000;64:1444–1447.
No potential conflict of interest was reported by the
authors.
Funding
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al. Purification and characterization of organic sol-
vent and detergent tolerant lipase from thermotoler-
ant Bacillus sp. RN2. Int J Mol Sci. 2010;11:3783–
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This work was supported by Kyowa Pharma Chemical Co.,
Ltd;Kyowa Pharma Chemical Co., Ltd. [n/a].
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ORCID
Ei-Tora Yamamura
098X
http://orcid.org/0000-0002-3705-
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