Whole-cell biocatalytic of Bacillus cereus WZZ006 strain to synthesis of indoxacarb intermediate: (S)-5-Chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester

Article abstract of DOI:10.1002/chir.23124

In this study, a newly isolated strain screened from the indoxacarb-rich agricultural soils, Bacillus cereus WZZ006, has a high stereoselectivity to racemic substrate 5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester. (S)-5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester was obtained by bio-enzymatic resolution. After the 36-hour hydrolysis in 50-mM racemic substrate under the optimized reaction conditions, the e.e._s was up to 93.0% and the conversion was nearly 53.0% with the E being 35.0. Therefore, B cereus WZZ006 performed high-level ability to produce (S)-5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester. This study demonstrates a new biocatalytic process route for preparing the indoxacarb chiral intermediates and provides a theoretical basis for the application of new insecticides in agricultural production.

Full text of DOI:10.1002/chir.23124

Received: 18 March 2019
DOI: 10.1002/chir.23124
Revised: 18 July 2019
Accepted: 4 August 2019
R E G U L A R A R T I C L E
Whole‐cell biocatalytic of Bacillus cereus WZZ006 strain to
synthesis of indoxacarb intermediate: (S)‐5‐Chloro‐1‐oxo‐2,3‐
dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester
Yinjun Zhang
Zhao Wang
| Hongyun Zhang | Feifei Cheng | Ying Xia | Jianyong Zheng
|
Department Key Laboratory of Bioorganic
Synthesis of Zhejiang Province, Institution
College of Biotechnology and
Abstract
In this study, a newly isolated strain screened from the indoxacarb‐rich agricul-
tural soils, Bacillus cereus WZZ006, has a high stereoselectivity to racemic sub-
Bioengineering, Zhejiang University of
Technology, Hangzhou, China
strate
5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic
acid
Correspondence
methyl ester. (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic
acid methyl ester was obtained by bio‐enzymatic resolution. After the 36‐
hour hydrolysis in 50‐mM racemic substrate under the optimized reaction con-
ditions, the e.e.s was up to 93.0%
Zhao Wang, Department Key Laboratory
of Bioorganic Synthesis of Zhejiang
Province, Institution College of
Biotechnology and Bioengineering,
Zhejiang University of Technology, No 18
Chaowang Road, Hangzhou, China.
Email: hzwangzhao@163.com
and the conversion was nearly 53.0% with the E being 35.0. Therefore, B cereus
WZZ006 performed high‐level ability to produce (S)‐5‐chloro‐1‐oxo‐2,3‐
dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester. This study dem-
onstrates a new biocatalytic process route for preparing the indoxacarb chiral
intermediates and provides a theoretical basis for the application of new insec-
ticides in agricultural production.
Funding information
Zhejiang Provincial Natural Science
Foundation, Grant/Award Number:
LY18B020021
KEYWORDS
chiral intermediate, enantioselectivity, enzymatic resolution, indoxacarb, whole cell biocatalyst
1 | INTRODUCTION
channels, which is associated with high insecticidal activ-
ity.6-8 The 5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐
Chiral pesticides with specificity and environmental
safety have captured continuously with wide attention
in agriculture,1,2 because of the emphasis on environmen-
tal pollution and the advocacy of green chemistry.
Indoxacarb (see Figure 1 for structure), a new chiral
oxadiazine pro‐insecticide,3 being converted into the
active compound by removal of a CO2Me in the insect
gut (discovered and developed by the E.I. DuPont Com-
pany), has shown to possess high activity, commercial
availability, and environmental compatibility to adminis-
tered to lepidopteran and hemipteran pests.4,5 An essen-
tial characteristic of indoxacarb is its novel bioactivation
mechanism by blocking insect voltage‐dependent sodium
indene‐2‐carboxylic acid methyl ester is a crucial chiral
intermediate for the preparation of indoxacarb. Studies
have shown that the S‐enantiomer of indoxacarb is active
as the racemate in insects, while the R‐enantiomer is
inactive.3 Therefore, the synthesis of (S)‐5‐chloro‐1‐oxo‐
2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic
acid
methyl ester has sparked heated interest in the pesticide
industry.
Currently, the standard procedure for synthesizing (S)‐
5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐car-
boxylic acid methyl ester is limited to organic synthetic
methods. Although these chemical methods are popularly
used due to mature technology and can achieve a rapid
Chirality. 2019;1–10.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

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ZHANG ET AL.
subtilis 168 whole cells for efficient production of 2,3‐
butanediol. As far as we know, only a few studies have
reported the whole‐cell biocatalytic process for producing
(S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐
carboxylic acid methyl ester. Therefore, it would be valu-
able to develop its bioprocess.
FIGURE 1 The structure of indoxacarb
Herein, we aimed to explore a convenient and efficient
bioprocess to obtain (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐
hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester by a
newly isolated strain Bacillus cereus WZZ006.
and efficient synthesis, the reactions are still complex,
toxic, and not conducive to large‐scale industrial produc-
tion.9 The purity of the obtained (S)‐5‐chloro‐1‐oxo‐2,3‐
dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl
ester is only about 90%. McCann et al10 found that the
(S)‐isomer of 54% enantiomeric excess (e.e.) was obtained
with the Sharpless asymmetric hydroxylation reagent
AD‐mix‐β. With the chiral N‐camphorsulfonyl
oxaziridine reagents developed by Davis et al,11 the yield
could be increased to 85%, but the e.e. value was only
35%. Under the condition of using the chiral alkaloid cin-
chonine as a catalyst and tert‐butyl hydroperoxide
(TBHP) as an oxidant,12 85% yield and 50% e.e. were
obtained. The level of e.e. directly affects the quality of
the final product and the activity of pesticides,13-15 so
exploring its new synthetic method has a long‐term appli-
cation significance.
2 | MATERIALS AND METHODS
2.1 | General
Indoxacarb‐rich agricultural soils samples used to screen
bacteria with hydrolytic activity were collected from var-
ious places of Zhejiang, Anhui, and Henan Provinces,
China. (R,S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐
indene‐2‐carboxylic acid methyl ester (98%) was prepared
from 5‐chloro‐1‐indanone in our laboratory, according to
reported procedures.29-31 All chemicals used were analyt-
ical reagents.
Nowadays, the biocatalytic approach using microor-
ganisms or isolated enzymes, from the perspective of
environmental and industrial application, has increas-
ingly to be a recent focus on chemical, agricultural, and
pharmaceutical industries. Because of the potential for
the preparation of chiral compounds with high optical
purity, the biocatalytic approach has gradually become a
research hotspot for the synthesis of chiral compounds.
In comparison with chemical synthesis, the biocatalytic
approach has many superiorities because of high selectiv-
ity, broad substrate range, environment‐friendly process,
and mild reaction conditions. In particular, dynamic
kinetic resolution (DKR) that couples a racemization
reaction with a conversion of the unwanted enantiomer
into the product, can increase the yield up to 100%.16-19
Also, whole‐cell biocatalysts compared with isolated
enzyme shows the advantages of cheap, stable, less labo-
rious, and accessible. The whole microbial cells as excel-
lent catalysts,20 avoiding cofactor addition and enzyme
purification, have the advantages of high substrate speci-
ficity, good stereoselectivity, mild reaction conditions,
and low pollution. The whole cell biocatalytic synthesis
has continually emerged as an outstanding method to
replace traditional chemical methods in some fields.21-25
Few bacterial strains like, Lactobacillus paracasei BD101
were used as whole cell biocatalyst for pure (S)‐
cyclohexyl (phenyl)methanol.26 Also, Vitale et al27 used
Lactobacillus reuteri DSM 20016 whole cells for the pro-
duction of (S)‐rivastigmine. Samuel et al28 used Bacillus
2.2 | Analytical methods
Nuclear magnetic resonance (NMR) was recorded on
Bruker Ultrashield spectrometer (Bruker Ltd.,Swissland)
operating at 500 MHz (1H NMR) and 125 MHz (13C
NMR) in CDCl3. Gas chromatography mass spectrometry
(GC‐MS) analysis was performed on an Agilent 7980A
GC system equipped with an HP‐5MS Agilent 19091S‐
433 column.
The progress of the hydrolysis reaction was monitored
by high‐performance liquid chromatography (HPLC,
Waters, 1525), and the biotransformation yield and
enantioselectivity were quantitatively determined. The
determination was carried out by using an HPLC method
on an Ultimate Cellud‐Y (5 μm, 4.6 × 250 mm, Yuexu,
China) column with a mobile phase of chromatographic
grade n‐hexane and isopropanol (80:20, v/v) at 0.5
mL/min flow rate, UV detection wavelength was set to
254 nm, column temperature was 25°C and injection vol-
ume was 10 μL. The retention time of 5‐chloro‐1‐
indanone, (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐
indene‐2‐carboxylic acid methyl ester, and (R)‐5‐chloro‐
1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid
methyl ester was 17.49, 18.86, and 23.50 minutes, respec-
tively (Figure 2).
One unit of esterase activity was defined as the
amount of enzyme required to catalyze the hydrolysis of
1 μmol of substrate per minute at 30°C, pH 7.0. The peak

ZHANG ET AL.
3
FIGURE 2 High‐performance liquid
chromatography (HPLC) of 5‐chloro‐1‐
indanone and (R,S)‐5‐chloro‐1‐oxo‐2,3‐
dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic
acid methyl ester
areas of the chromatograms based on the (S)‐ and (R)‐
configuration substrates and reaction products were used
to calculate the substrate optical purity and
enantioselectivity. The percentage enantiomeric excesses
of the substrate (e.e.s), conversion (C), and
enantioselectivity (E) were calculated according to equa-
tions as below32:
oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid
methyl ester, 3.0‐g NaNO3, 1.0‐g KH2PO4, 0.5‐g KCl, 0.5‐
g MgSO4·7H2O, and 0.01‐g FeSO4·7H2O (pH 7.0). The
reaction mixtures were incubated at 30°C for 5 days on
a rotary shaker at 200 rpm. Then, when the optical den-
sity at 600 nm (OD600) reached 4 to 5, 1‐mL cultures were
transferred to fresh medium with the same ingredients
after the evidence of microbial growth. The procedure
above had three replications. Then, 100‐μL enrichment
cultures were diluted and spread onto agar medium
plates with the same composition except the agar to iso-
late pure colonies. The single colonies were grown aerobi-
cally at 30°C for 24 hours at 200 rpm in the medium that
composed (per liter) 10.0‐g glucose, 10.0‐g peptone, 6.0‐g
KH2PO4·3 H2O, 3.0‐g beef extract, 3.0‐g KH2PO4, 0.5‐g
MgSO4·7H2O, and 0.5‐g NaCl (pH 7.0). Following cultiva-
tion, wet cells were centrifuged at 10,000 rpm for 10
minutes and resuspended in 950‐μL phosphate buffer
(200 mM, pH 7.0). 20‐mM (R,S)‐5‐chloro‐1‐oxo‐2,3‐
dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl
ester was added to the reaction and stirred at 200 rpm
and 30°C for 24 hours. Samples were acidified with 4‐M
hydrochloric acid (HCl), extracted with ethyl acetate,
dried by a vacuum rotary evaporator, and combined with
HPLC analys is to measure the substrate conversion and
enantiomeric excess (e.e.s).
½Sꢀ − ½Rꢀ
e:e:s ¼
C ¼
× 100% ;
(1)
½Sꢀ þ ½Rꢀ
C0 − Ct
× 100% ;
(2)
(3)
C0
ln½ð1 − CÞð1 − e:e:sÞꢀ
ln½ð1 − CÞð1 þ e:e:sÞꢀ
E ¼
In the formula, [S] and [R] represent the peak areas of
the (S)‐ and (R)‐configuration substrates, respectively; C0
represents the initial concentration of the substrate (mM),
and Ct represents the substrate concentration remaining
after the reaction (mM).
2.3 | Screening of strains for
enantioselective hydrolysis of
5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐
indene‐2‐carboxylic acid methyl ester
2.4 | Identification of the isolated strain
Bacteria with enzymatic resolution activity of 5‐chloro‐1‐
oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid
methyl ester were isolated from indoxacarb‐rich agricul-
tural soils through two rounds of screening. In the first
round, strains with ester hydrolysis activity were
screened, and then strains were further screened for high
enantioselectivity. An enrichment culture procedure was
carried out previous to screening as follows: a little fresh
soil sample was suspended in 50‐mL enrichment medium
with a composition (per liter) of 0.5‐g (R,S)‐5‐chloro‐1‐
The isolated WZZ006 strain, was grown on beef extract
peptone agar medium at 30°C for 24 hours, was identified
according to its cell morphology and genus information.
Then, the single colony was picked for observing the mor-
phological characterization via an optical microscope
(Nikon Eclipse E100). For genotypic identification, the
chromosomal DNA of strain WZZ006 was extracted
according to the procedure of Wilson33 with the SK8255
Column Bacterial Genomic DNA Extraction Kit (Sangon

4
ZHANG ET AL.
Biotech [Shanghai] Co., Ltd.). The 16S ribosomal DNA
(rRNA) gene was polymerase chain reaction (PCR) ampli-
fied using the 27F (5′‐AGTTTGATCMTGGCTCAG‐3′)
cooled to room temperature after the reaction was com-
pleted. 2‐M HCl solution was added to adjust the pH to
7. The organic phase was separated and collected, then
dried with anhydrous sodium sulfate, and filtered and
evaporated to give a brown solid. The creamy‐white
solid was obtained by recrystallization with isopropyl
alcohol.
and
1492R
(5′‐GGTTACCTTGTTACGACTT‐3′)
primers.34,35 The reaction was cycled with an initial dena-
turation for 94°C for 4 minutes and followed by 30 cycles
of denaturation (94°C for 45 s), annealing (55°C for 45 s),
elongation (72°C for 90 s), and then a final elongation
step at 72°C for 10 minutes. The PCR products were puri-
fied with SanPrep Column DNA Gel Extraction Kit
(Sangon Biotech [Shanghai] Co., Ltd.). The sequencing
was carried out by Sangon Biotech (Shanghai) Co., Ltd.
The final gene sequence data were analyzed with the
BioEdit program and compared with the BLAST network
services provided by the National Center for Biotechnol-
ogy Information (NCBI). The Biolog OmniLog Identifica-
tion System (Biolog GENIII)36 was assessed for its ability
to identify genospecies of strain WZZ006.
2.6.2 | Synthesis of methyl
5‐chloro‐2‐hydroxy‐1‐oxo‐2,3‐dihydro‐1H‐
indene‐2‐carboxylate (compound III)
Compound II (5.0 g, 22 mmol) was dissolved in 50 mL of
1,4‐dioxane, and 150 mL of water and potassium hydro-
gen persulfate complex salt (Oxone) (15 g, 24.5 mmol)
was added at 60°C. The solution slowly became clear
and monitored by HPLC.
After the reaction was completed, it was cooled to
room temperature and extracted with 200 mL of dichloro-
methane. The organic phase was washed once with satu-
rated sodium thiosulfate and brine (200 mL), and the
organic phase was collected, dried with anhydrous
sodium sulfate, and filtered and evaporated at reduced
pressure (0.1 MPa). The crude product was recrystallized
from cyclohexane to give a brown solid. The structure
was determined by NMR data interpretation.
2.5 | Production of an esterase from B
cereus WZZ006
The B cereus WZZ006 was cultivated in a 250‐mL shake
flask to produce an esterase. The medium included the
following constituents (per liter): 10‐g glucose, 10‐g pep-
tone, 3‐g beef extract, 6‐g K2HPO4·3H2O, 3‐g KH2PO4,
0.5‐g MgSO4·7H2O, and 0.5‐g NaCl, pH 7.The reaction
mixture was incubated at 180 rpm, 30°C for 24 hours.
The cultures were withdrawn periodically to evaluate
the
biomass
(OD600),
enzyme
activity
and
enantioselectivity of B cereus WZZ006. The whole cells
were harvested after fermentation by centrifugation
under 4°C at 12,000 rpm for 10 minutes and were lyoph-
ilized for further use.
2.7 | Enzymatic hydrolysis of (R,S)‐5‐
chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐
indene‐2‐carboxylic acid methyl ester
The enzymatic resolution of (R,S)‐5‐chloro‐1‐oxo‐2,3‐
dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl
ester was carried out in a 250‐mL three‐necked flask,
which contained 2 g of wet cells of B cereus WZZ006,
200 mg of the racemic substrate, and 40 mL of phosphate
buffer (200 mM, pH 7.0). The reaction was stirred at 30°C,
180 rpm for 48 hours. The hydrolysis reaction scheme is
shown in Scheme 1. Aliquots of the reaction sample were
withdrawn at a fixed time interval, acidified with 4‐M
HCl, extracted with ethyl acetate, dried by a vacuum
rotary evaporator, and further determined by HPLC. At
the end of the reaction period, the aqueous phase was
acidified with 4‐M HCl then extracted with ethyl acetate.
Ethyl acetate was removed at reduced pressure. A prepar-
ative thin‐layer chromatographic (TLC) method on silica
gel was applied to separate and purify the crude product
with petroleum ether and ethyl acetate (3:2, v/v) as
solvent.
2.6 | General procedure for chemical
synthesis of racemic
5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐
indene‐2‐carboxylic acid methyl ester29-31
2.6.1 | Synthesis of
5‐chloro‐2,3‐dihydro‐1oxan‐1‐indanone‐
carboxylic acid methyl ester (compound II)
In
a
250‐mL four‐necked flask, 6.12‐g sodium
methoxide, and 60‐mL dimethyl carbonate (DMC) were
added in sequence. The reaction was stirred at 90°C
under nitrogen. When the fraction (DMC) was eluted,
60‐mL 5‐chloro‐1‐indanone (9.17 g, 0.0552 mol) of
DMC solution was slowly added dropwise. After the
titration was completed, the reaction was carried out
for 1 hour. The reaction was monitored by HPLC, and

ZHANG ET AL.
5
SCHEME 1 Route of resolution of (R,S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester by Bacillus cereus
WZZ006
The comprehensive analysis and identification of
strain 1 by 16S rDNA and BIOLOG (Table S1) showed
that the strain was in line with the characteristics of B
cereus. Therefore, it was named B cereus WZZ006 and
deposited in the China Center for Type Culture Collec-
tion under the accession number of CCTCC M 2017621.
3 | RESULTS AND DISCUSSION
3.1 | Identification of stereoselective ester
hydrolase producing bacteria
In this experiment, seven strains with superior
stereoselective hydrolysis activity were screened from
the freeze‐dried bacteria powder, which were isolated
and purified from more than 500 indoxacarb‐rich agricul-
tural soil samples collected from different places of Zhe-
jiang, Anhui, and Henan Provinces, China. Among
them, strains WZZ006, C26, Pt9, X225, S14, and X075
selected to hydrolyze the R‐isomer of the substrate, and
CM008 hydrolyzed S‐isomer. As showed in Table 1, strain
WZZ006 exhibited the highest hydrolysis activity and
enantiomeric activity to the racemic substrate, so strain
WZZ006 was selected for activation and preservation as
a subsequent experimental strain.
The selected strains were inoculated into a beef paste
peptone solid medium and cultured at 30°C for 24 hours
to observe the colony morphology. As shown in Figure
S1, a single cell is rod shaped, with a square end, and
multiple cells can be connected to a growing chain. The
colonies were grayish white, large, rough, flat, irregular,
and with spores. After Gram staining, the cells were pur-
ple, indicating that the strain was a gram‐positive bacte-
rium. After full‐length, sequencing, and analysis by
Shanghai Shenggong Bioengineering Co., Ltd., the 16S
rDNA sequence of this strain was 1,455 bp in length.
The specific sequence is as Figure S2.
3.2 | Effects of pH, temperature, rotating
speed, cosolvent, and substrate
concentration on enzymatic resolution of
racemic indoxacarb intermediates
A change in the surface charge density and molecular
structure of the cell or enzyme molecule is accomplished
by changes in pH. This has led to the changes in the rate
of entry and exit of the substance followed by activity and
selectivity of the enzyme change.37,38 Therefore, it is
essential to regulate the pH of the reaction medium. As
shown in Figure 3, the enzyme activity of the cells
increased within the pH range of 5.0 to 7.0; the conver-
sion rate increased from 0.4 0.4% to 44.6 0.7%, and
e.e.s also increased sharply. When the pH at 7.0, the opti-
cal purity of the enzyme activity and the substrate
TABLE 1 The screening of microorganism for enantioselective
hydrolysis of the substrate
Number
Strain Code
e.e.s, %
Conversion, %
E
1
2
3
4
5
6
7
WZZ006
C26
80.1
52.2
41.6
38.7
21.6
18.5
73.3
55.5
58.9
53.1
46.2
40.5
37.9
60.7
11.0
3.5
6.8
3.8
2.4
2.2
5.9
Pt9
FIGURE 3 Effects of pH on the enzymatic resolution of racemic
indoxacarb intermediate. Reaction conditions: 0.05‐g Bacillus cereus
WZZ006 freeze‐dried powder, 50‐μL 20‐mM racemic substrate, 180
rpm, 30°C, 24 hours; 50‐mM citrate‐sodium citrate buffer, pH 5.0;
50‐mM phosphate buffer, pH 6.0 to 8.0; 50‐mM tris‐HCl buffer, pH
9.0; 50‐mM Na2CO3‐NaHCO3 buffer, pH 10.0.The hydrolysis of the
substrate was determined by high‐performance liquid
X225
S14
X075
CM008
Abbreviations: E, enantioselectivity; e.e.s, enantiomeric excesses of the
substrate.
chromatography (HPLC)

6
ZHANG ET AL.
reached the maximum; the conversion rate was 44.6
0.7%, and the e.e.s was 78.1 1.9%. When pH > 8.0, e.e.s
gradually decreased with the increase of pH, and the
enantioselectivity of the enzyme was minimized. There-
fore, pH 7.0 is the optimum pH for the reaction.
Temperature is a principle element affecting the activ-
ity of biocatalysts and the thermodynamic equilibrium of
the reaction.39 The enzymatic resolution of the racemic
substrate at different temperatures is shown in Figure 4.
As the temperature increases, the enzyme activity first
rises and then decreases. Under low‐temperature condi-
tions (20°C‐25°C), the enzyme activity is weak, in an
inactive catalytic state. When the temperature is 25°C to
30°C, the activity of the enzyme increases significantly.
At 30°C temperature, the enzyme activity was maximum;
the conversion rate is 52.7 1.2%, and the e.e.s is 88.7
1.9%. As the temperature is further increased, the activity
of the enzyme is rapidly reduced, especially when the
temperature is higher than 40°C, the activity of the
enzyme is only 10%, which may be due to the high‐
temperature affects the molecular structure of the
enzyme, resulting in a substantial reduction in enzyme
FIGURE 6 Effects of cosolvent on the enzymatic resolution of
racemic indoxacarb intermediate. Reaction conditions: 0.05‐g
Bacillus cereus WZZ006 freeze‐dried powder, 0.05 g of the substrate
and pH 7.0 PB buffer; 5% different cosolvents (isooctanol, DMSO,
DMF, acetone, ethanol) were added to 30°C, 180 rpm, 24 hours.
Take no cosolvent as a control. Determine the hydrolysis of the
reaction by high‐performance liquid chromatography (HPLC), and
define the highest enzyme activity under different cosolvent
conditions to be 100%. Calculate relative vitality
FIGURE 4 Effects of temperature on the enzymatic resolution of
racemic indoxacarb intermediate. Reaction conditions: 0.05‐g
Bacillus cereus WZZ006 freeze‐dried powder, 20‐mM substrate and
pH 7.0 PB buffer, under different temperature gradients (20°C,
25°C, 30°C, 35°C, 40°C, 45°C), 180 rpm, 24 hours. The hydrolysis of
the substrate was determined by high‐performance liquid
chromatography (HPLC)
FIGURE 5 Effects of rotation speed on the enzymatic resolution
of racemic indoxacarb intermediate. Reaction conditions: 0.05‐g
Bacillus cereus WZZ006 freeze‐dried powder, 20‐mM substrate and
pH 7.0 PB buffer, 30°C, different speed (120, 150, 180, 210, 240, 270
rpm), 24 hours. High‐performance liquid chromatography (HPLC)
determined the hydrolysis of the substrate
FIGURE 7 Effects of substrate concentrations on the enzymatic
resolution of racemic indoxacarb intermediate. Reaction
conditions: 0.05‐g Bacillus cereus WZZ006 freeze‐dried powder,
different concentrations of substrate (5‐165 mM) and pH 7.0 PB
buffer, 30°C, 180 rpm, 24 hours. The hydrolysis of the substrate was
determined by high‐performance liquid chromatography (HPLC)

ZHANG ET AL.
7
activity. In light of this, the temperature of the reaction
should be controlled at 30°C.
subsequent researches, and the conversion rate and e.e.s
at 52.7 0.5% and 88.7 1.4%, respectively.
The rotational speed has a tendency to affect the diffu-
sion of the compound in the reaction system and the con-
tact time of the cells or enzymes with the substrate. The
experiment explored the effect of different rotation speed
on the enzyme‐catalyzed enzymatic resolution reaction.
The results are presented in Figure 5. The conversion rate
and e.e.s were significantly improved at speeds ranging
from 120 to 210 rpm, while the e.e.s dropped when the
speed increased. When the speed is 180 rpm, the conver-
sion rate and e.e.s are the highest. Probably, it can show
that high rotational speed can alter the internal structure
of cells by shear stress.40 Therefore, 180 rpm was used for
For the bioconversion of ester compounds, the addition
of a cosolvent can accelerate the reaction rate, and also
some enzymes can exhibit better enantioselectivity in
water‐organic systems. The results in Figure 6 show that
the addition of a cosolvent facilitates the reaction and
accelerates the reaction rate. The effect of adding DMSO
is the best, while isooctanol, DMF, and ethanol have little
effect on the catalytic reaction. Therefore, DMSO was cho-
sen as a cosolvent for subsequent experiments.
When the amount of biocatalyst added is constant, it is
necessary to investigate the maximum saturated substrate
concentration catalyzed by the enzyme. The effect of the
substrate with a concentration range of 5 to 165 mM on
the catalytic reaction was investigated. The results are
shown in Figure 7. When the biocatalyst amount is 0.05
g and the concentration is 5 to 45 mM, the conversion
rate remains unchanged, substantially higher than 50.0
0.8%, and e.e.s reaches 88.0 1.8%. When the substrate
concentration is greater than 45 mM, the e.e.s and conver-
sion rates drop sharply. This indicates that the high con-
centration may cause substrate inhibition, the catalysis of
enzyme reached a saturation point, the reaction speed
became slow, and the high concentration may destroy
the molecular structure of the enzyme to affect enzyme
activity. In summary, under certain conditions of
enzymes, the appropriate substrate concentration should
be selected; the concentration of 45 mM was used for sub-
sequent experiments.
FIGURE 8 Time course of enzymatic resolution of racemic
indoxacarb intermediate under optimized conditions
FIGURE 9 1H NMR of 5‐chloro‐2,3‐dihydro‐2‐hydroxy‐1‐oxo‐1H‐indene‐carboxylic acid methyl ester (III)

8
ZHANG ET AL.
FIGURE 10 13C NMR of 5‐chloro‐2,3‐dihydro‐2‐hydroxy‐1‐oxo‐1H‐indene‐carboxylic acid methyl ester (III)
product was not acid, and its polarity was greater than
that of the substrate ester (III) by observed the retention
time. Based on the substrate structure, it was speculated
that the product might be decarboxylated and converted
into an alcohol compound: 5‐chloro‐2‐hydroxy‐1‐
indolone (VI). Also, the resulting product was separated
by TLC and analyzed with AD‐H chiral column (Figure
S3). There were two close and equal peaks with similar
retention times, which inferred that the product was a
racemic compound, and it also indicated that the sub-
strate was not ring opened or formed into other structures
after being hydrolyzed, just decarboxylated. Furthermore,
the product structure was then characterized by GC‐MS
3.3 | Time course of whole‐cell
biocatalytic reaction
The time course of enantioselective hydrolysis of 5‐
chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carbox-
ylic acid methyl ester using B cereus WZZ006 is shown in
Figure 8. The reaction was carried out under the follow-
ing optimized conditions: 0.05‐g B cereus WZZ006
freeze‐dried powder, 45‐mM substrate, solution pH 7.0,
reaction temperature 30°C, rotation speed of 180 rpm,
cosolvent DMSO, respectively. After 36 hours, the hydro-
lysis was basically completed, and the e.e.s and conversion
rates reached the highest, respectively 93.0
53.0 1.6%.
1.4% and
3.4 | Analysis of the hydrolysis product
In this study, a simple chemical reaction was made to
prepare a racemic substrate 5‐chloro‐1‐oxo‐2,3‐dihydro‐
2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester (III,
yield: 75%) with a purity of 98% using 5‐chloro‐1‐
1
indanone as a raw material. The H and 13C NMR spec-
trum were recorded in Figures 9 and 10.
Then, the enzymatic resolution of the racemic sub-
strate was carried out and analyzed by HPLC. The three
peaks in Figure 2 represented the product (VI), the S‐
isomer (IV, unreacted), and the R‐isomer (V, trans-
formed) of the substrate, respectively. The results showed
that there was only a single reaction in the hydrolysate
and no other side effects. Besides, it was found that the
FIGURE 11 Analysis of the product by gas chromatography
mass spectrometry

ZHANG ET AL.
9
SCHEME 2 The production of the hydrolysis product 5‐chloro‐2‐hydroxy‐1‐indanone
(Figure 11) and NMR (Figures S4 and S5). MS (ESI) m/z:
182 [M + H]+, 1H NMR (500 MHz, DMSO‐d6): δ = 7.65 (t,
J = 7.65, 1H), 7.49 (d, J = 7.49,1H), 5.89 (m, J = 5.89, 1H),
4.42 (m, J = 4.42, 1H), 3.49 (m, J = 3.49, 1H), 2.82 (m, J =
2.82, 2H); 13C NMR (125 MHz, DMSO‐d6): δ = 205.0 (C),
152.9 (C), 140.2 (C), 133.2 (C), 128.2 (CH), 127.0 (CH),
125.1 (CH), 72.9 (CH), 35.4 (CH2). Finally, the GC‐MS
and NMR results also illustrated the product had a fur-
ther decarboxylation after hydrolysis (Scheme 2) and
was identified as 5‐chloro‐2‐hydroxy‐1‐indanone (VI).
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How to cite this article: Zhang Y, Zhang H,
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biocatalytic of Bacillus cereus WZZ006 strain to
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