Kinetic resolution of N-acetyl-DL-alanine methyl ester using immobilized Escherichia coli cells bearing recombinant esterase from Bacillus cereus

Article abstract of DOI:10.1002/chir.22863

D-alanine is widely used in medicine, food, additives, cosmetics, and other consumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high hydrolytic activity and stereoselectivity. In this study, we expressed the esterase gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of N-acetyl-DL-alanine methyl ester by immobilized whole E.?coli BL21 (DE3) cells, which were prepared through embedding and cross-linking. We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0, temperature of 40°C and substrate concentration of at 700?mM with an enantiomeric excess of 99.99% and e.e._p of 99.50%. The immobilized recombinant B.?cereus esterase E.?coli BL21 (DE3) cells exhibited excellent reusability and retained 86.04% of their initial activity after 15 cycles of repeated reactions. The immobilized cells are efficient and stable biocatalysts for the preparation of N-acetyl-D-alanine methyl esters.

Full text of DOI:10.1002/chir.22863

Received: 22 December 2017
DOI: 10.1002/chir.22863
Revised: 4 March 2018
Accepted: 14 March 2018
R E G U L A R A R T I C L E
Kinetic resolution of N‐acetyl‐DL‐alanine methyl ester
using immobilized Escherichia coli cells bearing
recombinant esterase from Bacillus cereus
Jianyong Zheng
| Xing Lan | Lijuan Huang | Yinjun Zhang | Zhao Wang
Key Laboratory of Bioorganic Synthesis of
Zhejiang Province, College of
Biotechnology and Bioengineering,
Zhejiang University of Technology,
Hangzhou, China
Abstract
D‐alanine is widely used in medicine, food, additives, cosmetics, and other con-
sumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high
hydrolytic activity and stereoselectivity. In this study, we expressed the esterase
gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of
N‐acetyl‐DL‐alanine methyl ester by immobilized whole E. coli BL21 (DE3)
cells, which were prepared through embedding and cross‐linking.
We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0,
temperature of 40°C and substrate concentration of at 700 mM with an enan-
tiomeric excess of 99.99% and e.e.p of 99.50%. The immobilized recombinant
B. cereus esterase E. coli BL21 (DE3) cells exhibited excellent reusability and
retained 86.04% of their initial activity after 15 cycles of repeated reactions.
The immobilized cells are efficient and stable biocatalysts for the preparation
of N‐acetyl‐D‐alanine methyl esters.
Correspondence
Zhao Wang, Key Laboratory of Bioorganic
Synthesis of Zhejiang Province, College of
Biotechnology and Bioengineering,
Zhejiang University of Technology,
Address No 18, Chaowang Road,
Hangzhou, China.
Email: hzwangzhao@163.com
Funding information
National Natural Science Foundation of
China, Grant/Award Numbers: 31660247
and 31600639; Natural Science Founda-
tion of Zhejiang Province, Grant/Award
Number: LY18B020021
KEYWORDS
Bacillus cereus, enantioselectivity, esterase, reusability, whole‐cell
1 | INTRODUCTION
for D‐Ala production presents several problems, such as
low product concentration, high cost, and complicated
D‐alanine (D‐Ala) is an essential amino acid that is
mainly used in food, medicine, additives, and auxil-
iaries.1-3 It is the sweetest amino acid and is widely used
as a functional food sweetener, such as a dipeptide
sweetener (2,2,4,4‐tea‐methylthietanylamine).4 Moreover,
D‐Ala is the raw material for the production of vitamin
B6, which has analgesic effects.5 Recent studies have also
shown that D‐Ala exhibits the new physiological activi-
ties, such as such as inducing the ectopic expression of
amino acid oxidase in the cytoplasm of tumor cells,
preventing the oxidation of lipids in vivo.6
separation. Two general chemical synthesis methods are
used to produce D‐Ala: One is direct asymmetric synthe-
sis. The other invovles the chemical synthesis of DL‐ala-
nine compounds, which are then subjected to various
optical resolution methods to yield D‐Ala. The
aminoacylase‐catalyzed resolution method is the most
commonly used method for the industrial production of
D‐Ala. Nevertheless, it is only used on a small‐scale
because of its high input requirements and low enantio-
meric excess (e.e.) value.9-11 Enzyme immobilization has
been recently applied to produce D‐Ala because this
method is highly stable and reliable. Co‐immobilized
enzymes have been used to prepare optically pure D‐Ala
from sodium pyruvate.12
D‐Ala is mainly prepared through the following pro-
cesses: microbial fermentation, chemical synthesis, and
enzymatic resolution.7,8 However, microbial fermentation
Chirality. 2018;1–6.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

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ZHENG ET AL.
Esterase (EC 3.1.1.1) is a carboxylesterase that cata-
(unless otherwise stated) and were obtained from com-
mercial sources.
lyzes ester hydrolysis. Specifically, esterases are lipases
that hydrolyze lower fatty‐acid esters.13 Esterases have
been applied in numerous fields and can be obtained
from bacteria, such as Streptomyces sp., Pseudomonas
sp., and Aspergillus sp., as well as from plants and ani-
mals.14 Bacterial estersases have been derived from mem-
bers of the Bacillus genus, including Bacillus licheniformis
S‐86, Bacillus sp. 4 and Bacillus amyloliquefaciens
WZZ002.15-17 The present work investigated the effects
of different reaction parameters, such as pH, temperature,
and substrate concentration, on the enzymatic‐catalyzed
enantio‐selective hydrolysis of N‐acetyl‐DL‐alanine
methyl ester by immobilized recombinant E. coli BL21
(DE3) cells expressing Bacillus cereus esterase. Further-
more, the reusability of the immobilized recombinant
cells as biocatalysts was evaluated. Results indicated that
the whole recombinant E. coli BL21 (DE3) cells exhibit
both high reusability and remarkable stereoselectivity
toward N‐acetyl‐DL‐alanine methyl ester, as shown in
Scheme 1.
2.2 | Microorganisms and cultivation
conditions
B. cereus WZZ001 was isolated from soil and deposited in
the China Center for Type Culture Collection (CCTCC M
2012403). The B.cereus esterase gene (GenBank accession
number: MF111093) was ligated into pEASY‐E1 vector
and transformed into E. coli BL21 (DE3) by our labora-
tory. The transformed E. coli BL21 was cultured at 37°C
in sterile medium containing 25 g/L glycerinum, 17.5 g/
L peptone, 7.5 g/L (NH4)2SO4, 0.5 g/L NaCl, 11.4 g/L
K2HPO4, 6.8 g/L KH2PO4, 2.0 g/L citric acid
monohydrate, 3.7 g/L MgSO4, and 100 mg/L ampicillin
sodium. After 12 hours of incubation at 25°C when the
optical density of fermentation solution at 600 nm
(OD600) had reached 0.6. Protein expression was induced
by final concentration of 0.1 mM IPTG. Subsequently,
cells were collected through centrifugation (10000 rpm
for 10 min at 4°C).
2 | MATERIALS AND METHODS
2.1 | Materials
2.3 | Immobilization of the whole cells of
recombinant E. coli by embedding and
cross‐linking
N‐acetyl‐DL‐alanine methyl ester was synthesized in our
laboratory and confirmed through gas chromatography
(GC)–mass spectrometery (Figure 1). N‐acetyl‐DL‐alanine
was purchased from Aladdin Chemistry Co Ltd (Shang-
hai, China). All other chemicals were of reagent grade
Recombinant E. coli cells were firstly immobilized
through embedding in carrageenan. Carrageenan 2.5%
(w/v) was dissolved in distilled water at 60°C for 1 hour,
then added 10% (w/v) of the cells of E. coli BL21 and
mixed at 45°C for 5 minutes. The mixture was incubated
in (3%, w/v) KCl solution at 4°C for 1 hour, and then cut
into sections with dimensions of 4 mm × 4 mm × 4 mm.
The sections were cross‐linked with 1% (w/v)
polyethylenepamine and 0.5% (w/v) glutaraldehyde for
1 hour. The immobilized cells were washed two times
with distilled water. The immobilized cells were main-
tained in phosphate buffer (pH 7.0) at 4°Cand recovered
via filtration for use as biocatalysts.
SCHEME 1 Bacillus cereus esterase catalyzed resolution of N‐
acetyl‐DL‐alanine methyl ester
FIGURE 1 GC‐MS spectrum of N‐acetyl‐DL‐alanine methyl ester

ZHENG ET AL.
3
structure of the enzyme.19 Figure 3 illustrates the effect
of pH on the conversion of N‐acetyl‐DL‐alanine methyl
ester under 40°C and pH of 6.5 to 9.0. Intensive and spon-
taneous hydrolysis occurred in the reaction system under
alkaline pH conditions (pH > 7.0). As pH increased from
6.0 to 7.0, the conversion increased from 36.8% to 46.9%.
Therefore, pH 7.0 was selected as the optimal pH for
the reaction.
2.4 | Biocatalytic resolution of N‐acetyl‐
DL‐alanine methyl ester
Biocatalysis was performed by adding 0.07 to 1.0 M sub-
strate and 0.1 g immobilized cells in 10 mL (50 mL flask)
of phosphate buffer solution (0.2 M, pH 7.0‐9.5) at 35°C to
60°C. The solution was stirred at 400 rpm. pH was con-
trolled through automatic titration using NaOH (1 M).
Samples were withdrawn at regular intervals and imme-
diately acidified with HCl (2 M) to stop hydrolysis. The
sample was extracted using ethyl acetate. The organic
phase was separated and dried using anhydrous Na2SO4
for GC analysis. All experiments were conducted in tripli-
cate, unless specified.
3.2 | Effect of temperature on the
biocatalytic resolution of N‐acetyl‐DL‐
alanine methyl ester
Temperature is an important factor of enzyme‐catalyzed
reactions in industrial application.20,21 The appropriate
values of conversion was observed at 40°C (Figure 4). Bio-
catalytic activity improved when temperature was
increased from 35°C to 40°C. Gradually, increasing tem-
perature to beyond 40°C mildly decreased conversion.
2.5 | GC analysis
The e.e. of N‐acetyl‐DL‐alanine methyl ester (e.e.s) and
conversion (c) were determined through GC (Agilent
7890A) using
a chiral capillary column BGB‐174
(30 m × 0.25 mm × 0.25 μm). Highly pure N2 was adopted
as carrier gas and applied at a pressure of 75 kPa. Inlet
port and FID detector temperatures were both 250°C,
and air flow and makeup flow were 300 and 25 mL/
min, respectively. The column temperature was increased
from 100 (maintained for 3 min) to 200°C (maintained for
5 min). The split ratio was 50:1, and the injection volume
was 1 μL. The retention times of N‐acetyl‐D‐alanine
methyl ester and N‐acetyl‐L‐alanine methyl ester were
14.3 and 14.7 minutes, respectively, as shown by the
results of GC (Figure 2). The enantiomeric ratio (E) was
calculated on basis of the conversion and e.e.s.18
3 | RESULTS AND DISCUSSION
FIGURE 3 Effect of pH on enantioselective hydrolysis of N‐
acetyl‐DL‐alanine methyl ester. Reaction conditions 0.1 g
immobilized cells, 70 mM N‐acetyl‐DL‐alanine methyl ester in
10 mL buffer solution, 400 rpm, 45°C, for 10 min, and titrating
with1 M NaOH. Phosphate buffer solution: pH 7.0~8.5; Tris‐HCl
buffer solution: pH 9.0. Symbols: spontaneous hydrolysis rate (●);
enzymatic conversion (◼)
3.1 | Effect of buffer pH on the
biocatalytic resolution of N‐acetyl‐DL‐
alanine methyl ester
pH is a key parameters of enzymatic activity because it
can affect the properties of amino acid residues and the
FIGURE 2 GC analysis of N‐acetyl‐DL‐alanine methyl ester and N‐acetyl‐DL‐alanine

4
ZHENG ET AL.
FIGURE 4 Effect of temperature on enantioselective hydrolysis
of N‐acetyl‐DL‐alanine methyl ester. Reaction conditions 0.1 g
immobilized cells, 100 mM N‐acetyl‐DL‐alanine methyl ester in
10 mL phosphate buffer solution (0.2 M, pH 7.0), 400 rpm,
35~60°C, for 10 min, and titrating with 1 M NaOH. Symbols:
conversion (◼)
Under optimized conditions, the reaction rate increased,
and conversion rates were maintained at a relatively high
level. Moreover, the spontaneous hydrolysis of the sub-
strate was maintained at a low level.
3.3 | Effect of substrate concentration on
the biocatalytic resolution of N‐acetyl‐DL‐
alanine methyl ester
FIGURE 5 Effect of substrate concentration on enantioselective
hydrolysis of N‐acetyl‐DL‐alanine methyl ester. A, The time
course of product concentration at different substrate concentration.
B, The initial reaction rate of different substrate concentration.
Reaction conditions 0.1 g immobilized cells, 70~1000 mM N‐acetyl‐
DL‐alanine methyl ester in 10 mL phosphate buffer solution (0.2 M,
pH 7.0), 400 rpm, 40°C, and titrating with 1 M NaOH. Symbols:
70 mM (◼); 150 mM (●);300 mM (▴); 500 mM (▾); 700 mM (⧫);
1000 mM (◂)
Substrate concentration is an essential factor of biocat-
alytic reaction because it may result in substrate
inhibition, which can decrease low substrate concentra-
tion and production efficiency. Moreover, the tolerance
of the substrate is a crucial index of an enzyme's
potential for the industrial application.22,23 The time
course of product conversion by the immobilized cells
is shown in Figure 5A. When substrate concentration
decreased to less than 700 mM, the enzymatic hydroly-
sis of N‐acetyl‐DL‐alanine methyl ester was completed
within 1 hour, with conversion rates of exceeded 40%
and an e.e.s value of 99%. When the substrate concen-
tration was 700 mM, e.e.s and conversion were 99.99%
and 49.95% after 1 hour of reaction, respectively. The
initial reaction rate increased when substrate concen-
tration was increased from 70 mM to 700 mM and
reached the maximum value of 22.75 mM/min at the
substrate concentration of 700 mM (Figure 5B). The
initial reaction rate decreased from 22.75 mM/min to
17.75 mM/min as substrate concentration increased
from 700 mM to 1 M. Therefore, 700 mM is the most
suitable substrate concentration for the biocatalyst
loading of 10 g/L. This result indicated that the
immobilized recombinant esterase has potential appli-
cations in the industrial biocatalytic resolution of N‐
acetyl‐DL‐alanine methyl ester.
3.4 | Time course of biocatalytic
resolution
The time course of the enantio‐selective hydrolysis of
N‐acetyl‐DL‐alanine methyl ester catalyzed by
immobilized cells is shown in Figure 6. pH was con-
trolled through automatic titration using 1 M NaOH
as the neutralizer. The reaction was conducted in a
100 mL reaction system under the optimal process

ZHENG ET AL.
5
exceeded 99.50%. Thus, the immobilized recombinant
esterase has potential industrial applications.
3.5 | Reusability of immobilized whole
E. coli cells
Reusability is one of the most crucial criteria for evaluat-
ing the technological and economic feasibility production
of immobilized enzyme.24 The reusability of immobilized
E. coli cells was studied under optimized conditions to
validate the stability and recyclability of the enzyme
(Figure 7). After each cycle of biocatalytic resolution,
the immobilized cells were recovered from the reaction
mixture through filtration and directly used in the next
cycle. The residual activity of immobilized cells main-
tained 92.76% of its initial activity after 10 cycles of reac-
tion, and 86.04% after 15 cycles of reaction. These results
suggest that the immobilized cells can be reused for more
than 10 cycles.
FIGURE 6 Time course of enantioselective hydrolysis of N‐
acetyl‐DL‐alanine methyl ester. Reaction conditions 1 g
immobilized cells, 700 mM N‐acetyl‐DL‐alanine methyl ester in
100 mL phosphate buffer solution (0.2 M, pH 7.0), 400 rpm, 40°C,
and titrating with 1 M NaOH. Symbols: conversion (◼); e.e.s (●);
e.e.p (▴)
4 | CONCLUSION
We investigated the enzyme‐catalyzed enantio‐selective
hydrolysis of N‐acetyl‐DL‐alanine methyl ester by
immobilized recombinant E. coli BL21 (DE3) cells
expressing B. cereus esterase. Moreover, we investigated
the effects of reaction conditions, specifically, pH, tem-
perature, and substrate concentration, on the reaction.
We found that the optimal reaction conditions were
pH 7.0 and 40°C. The optimum substrate concentration
at which the initial reaction rate reached its maximum
value of 22.75 mM/min was 700 mM. Under the substrate
concentration of 700 mM, the e.e.s of the biocatalytic
reaction exceeded 99.99%, and 49.95%conversion was
achieved after approximately 1 hour of enzymatic reac-
tion. We also investigated the reusability of immobilized
recombinant esterase E. coli cells, which retained 86.04%
of their initial activity after 15 cycles. Therefore, recombi-
nant B. cereus esterase, which has high catalytic activity
and enantio‐selectivity, has potential industrial applica-
tions in the resolution of N‐acetyl‐DL‐alanine methyl
ester.
FIGURE 7 Reusability of immobilized cells on enantioselective
hydrolysis reaction. Reaction conditions 1 g immobilized
recombinant esterase, 700 mM N‐acetyl‐DL‐alanine methyl ester in
100 mL phosphate buffer solution (0.2 M, pH 7.0), 400 rpm, 40°C,
and titrating with 1 M NaOH
conditions of 0.2 M phosphate buffer solution at 40°C
and pH 7.0. The substrate concentration and
immobilized cell loading were 700 mM and10 g/L,
respectively. The conversion rate and e.e.s increased
sharply during the first 1 minute of reaction. The con-
version rate and e.e.s gradually increased when the
reaction duration exceeded 10 minutes. After 1 hour
of reaction, the conversion rate reached 49.95% with
e.e.s of 99.99%, e.e.p of 99.50%, and E ratio greater
than 700. During the reaction, the e.e.p always
ACKNOWLEDGMENTS
This research was financially supported by National Nat-
ural Science Foundation of China (grant nos. 31600639
and 31660247) and Natural Science Foundation of Zhe-
jiang Province (LY18B020021).
ORCID
Jianyong Zheng http://orcid.org/0000-0001-9461-6305

6
ZHENG ET AL.
15. Zheng JY, Liu YY, Luo WF, Zheng RC, Ying XX, Wang Z. Bio-
catalytic resolution of rac‐α‐ethyl‐2‐oxo‐pyrrolidineacetic acid
methyl ester by immobilized recombinant Bacillus cereus ester-
ase. Appl Biochem Biotechnol. 2016;178(8):1471‐1480.
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