Utilization of deep-sea microbial esterase PHE21 to generate chiral sec-butyl acetate through kinetic resolutions

Article abstract of DOI:10.1002/chir.22983

We previously identified and characterized 1 novel deep-sea microbial esterase PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)-3-hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and yield through kinetic resolution. Herein, we further explored the potential of esterase PHE21 in the enantioselective preparation of secondary butanol, which was hard to be resolved by lipases/esterases. Despite the fact that chiral secondary butanols and their ester derivatives were hard to prepare, esterase PHE21 was used as a green biocatalyst in the generation of (S)-sec-butyl acetate through hydrolytic reactions and the enantiomeric excess, and the conversion of (S)-sec-butyl acetate reached 98% and 52%, respectively, after process optimization. Esterase PHE21 was also used to generate (R)-sec-butyl acetate through asymmetric transesterification reactions, and the enantiomeric excess and conversion of (R)-sec-butyl acetate reached 64% and 43%, respectively, after process optimization. Deep-sea microbial esterase PHE21 was characterized to be a useful biocatalyst in the kinetic resolution of secondary butanol and other valuable chiral secondary alcohols.

Full text of DOI:10.1002/chir.22983

Received: 20 February 2018
DOI: 10.1002/chir.22983
Revised: 8 April 2018
Accepted: 8 May 2018
R E G U L A R A R T I C L E
Utilization of deep‐sea microbial esterase PHE21 to generate
chiral sec‐butyl acetate through kinetic resolutions
1
,2
3
1,2
1,2
1,2,4
Yilong Wang | Yongkai Xu | Yun Zhang | Aijun Sun | Yunfeng Hu
1
CAS Key Laboratory of Tropical Marine
Abstract
Bio‐Resources and Ecology, South China
Sea Institute of Oceanology, Chinese
Academy of Sciences, Guangzhou,
Guangdong, China
We previously identified and characterized 1 novel deep‐sea microbial esterase
PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)‐3‐
hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and
yield through kinetic resolution. Herein, we further explored the potential of
esterase PHE21 in the enantioselective preparation of secondary butanol,
which was hard to be resolved by lipases/esterases. Despite the fact that chiral
secondary butanols and their ester derivatives were hard to prepare, esterase
PHE21 was used as a green biocatalyst in the generation of (S)‐sec‐butyl acetate
through hydrolytic reactions and the enantiomeric excess, and the conversion
of (S)‐sec‐butyl acetate reached 98% and 52%, respectively, after process optimi-
zation. Esterase PHE21 was also used to generate (R)‐sec‐butyl acetate through
asymmetric transesterification reactions, and the enantiomeric excess and
conversion of (R)‐sec‐butyl acetate reached 64% and 43%, respectively, after
process optimization. Deep‐sea microbial esterase PHE21 was characterized
to be a useful biocatalyst in the kinetic resolution of secondary butanol and
other valuable chiral secondary alcohols.
2
Guangdong Key Laboratory of Marine
Materia Medica, South China Sea Institute
of Oceanology, Chinese Academy of
Sciences, Guangzhou, Guangdong, China
3
Affiliated Hospital of Shandong
University of Traditional Chinese
Medicine, Jinan, Shandong, China
4
South China Sea Bio‐Resource
Exploitation and Utilization Collaborative
Innovation Center, Guangzhou,
Guangdong, China
Correspondence
Yunfeng Hu, CAS Key Laboratory of
Tropical Marine Bio‐Resources and
Ecology, South China Sea Institute of
Oceanology, Chinese Academy of
Sciences, Guangzhou 510301,
Guangdong, China.
KEYWORDS
Email: yunfeng.hu@scsio.ac.cn
biocatalysis, chiral sec‐butyl acetate, deep‐sea microorganism, kinetic resolution, novel microbial
Funding information
esterase
Strategic Priority Research Program of the
Chinese Academy of Sciences, Grant/
Award Number: XDA11030404; Scientific
and Technological Project of Ocean and
Fishery from Guangdong Province,
Grant/Award Number: A201701C12;
Guangzhou Science and Technology Plan
Projects, Grant/Award Number:
201510010012
1
| INTRODUCTION
intermediates for the synthesis of a great variety of chiral
drugs and are also used as solving agents in chemical
4,5
Chiral secondary alcohols are crucial building blocks,
intermediates, and chemicals, which have been widely
used in diverse industries such as chemical industry,
pharmaceutical industry, and material industry.
example, both enantiomers of mandelic acid are used as
industry. So, the preparation of chiral secondary alco-
hols has caused great attention from both academy and
industry. Chiral secondary alcohols should traditionally
be able to be synthesized through metallic organic
methods, and chiral chemicals represented by chiral
1-3
For
Chirality. 2018;1–9.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
WANG ET AL.
secondary alcohols were indeed mostly synthesized by
metallic organic methods. However, traditional metallic
organic synthesis involves harsh working conditions,
requires toxic heavy metallic catalysts, brings unex-
pected side products, and causes great pollution to the
butyl acetate through asymmetric hydrolytic reactions
and transesterification reactions.
2 | MATERIALS AND METHODS
2.1 | Microorganisms and reagents
6
environment. So, the adoption of environmentally
friendly biocatalytic methods, which use enzymes as
the green biocatalysts, is highly desirable, because green
biocatalysts generally work under mild working condi-
tions, require no toxic heavy metallic catalyst, and bring
The strain P. oryzihabitans HUP022 was isolated from the
sediments obtained from the deep sea of the Western
Pacific. Plasmid pET‐28a (+) (Novagen, USA) was used
as the expression vector of enzymes. Escherichia coli
DH5α and E. coli BL21 (DE3) were used as host strains
for gene cloning and protein expression, respectively.
7
no unexpected side products. Chiral secondary alcohols
can be produced through the bioreduction of keto pre-
cursors using dehydrogenases as the biocatalysts. How-
ever, the biocatalytic processes of dehydrogenases
require the involvement of expensive cofactors such as
NADH and NADPH, and an extra route should be pro-
®
TransStart FastPfu DNA PolyMerase, T4 DNA ligases,
and restriction enzymes were all obtained from TransGen
Biotech (Beijing, China). Racemic sec‐butyl acetate and
the corresponding chiral enantiomers were all purchased
from Aladdin Industrial Corporation (Shanghai, China).
Other chemicals were of analytical grade.
8,9
vided to recycle expensive cofactors. Another biocata-
lytic method for the production of chiral secondary
alcohols is through the kinetic resolutions catalyzed by
esterases. Esterases (EC 3.1.1.1) are 1 class of important
hydrolyases widely used in both laundry industry and
10-12
synthetic industry.
Chiral secondary alcohols can
2.2 | Gene cloning and expression vector
be prepared by using esterases as the green biocatalysts
through transesterifications, esterifications, or direct
construction
13-16
hydrolysis reactions.
One great advantage of the
The methods for the cloning and construction of
the expression vector for PHE21 were mentioned in
kinetic resolutions catalyzed by esterases over the
bioreductions catalyzed by dehydrogenases is that no
expensive cofactors are required during biocatalysis,
thus greatly reducing the production costs of chiral
products. Chiral secondary butanol and its ester deriva-
tives are crucial chiral chemicals and resolving agents
19
Wang et al.
2.3 | Expression and purification of
PHE21
1
and are in great demand in industry. Although there
have been many reports about the enzymatic prepara-
tion of chiral secondary alcohols, such as chiral octanol,
When the cell density (OD600) of the incubated recombi-
nant E. coli BL21 (DE3) cells reached approximately 0.6
to 0.8, isopropyl‐beta‐D‐thiogalactopyranoside (IPTG)
was added at a final concentration of 0.3 mM for protein
induction. After further 20‐hour induction at 20°C, the
cells were harvested by centrifugation at 4000 r/minute
for 10 minutes, washed twice with 50 mmol/L phosphate
buffer (pH 7.5), and then resuspended in the same buffer.
The cells were disrupted by sonication on ice for
10 minutes and centrifuged (10 000 r/minute, 20 minutes,
4°C). The recombinant protein PHE21 was purified using
nickel‐nitrilotriacetic acid agarose resin (GE Healthcare
Life Science, China) and desalted using PD‐10 desalting
columns (GE Healthcare Life Sciences, China) according
to the manufacturers' instructions. Protein concentration
was determined using the Bradford method, with bovine
serum albumin as standard. Purified PHE21 was analyzed
by SDS‐PAGE. Esterase PHE21 obtained from the
supernatant of induced cells was also lyophilized on
SCIENT‐N10 freeze dryer, and enzyme powders were
stored at −20°C for following resolution experiments.
15,17,18
by kinetic resolutions catalyzed by esterases.
But
before our study, the reports about the asymmetric syn-
thesis of chiral secondary butanols and their ester deriv-
atives through kinetic resolutions using esterases as the
biocatalysts were very rare, possibly because the chemi-
cal structure of secondary butanol is quite small and the
structural difference on both sides of the secondary
alcohols are too small to be recognized by esterases.
We previously identified and characterized 1 novel
esterase PHE21 from Pseudomonas oryzihabitans
HUP022 isolated from the deep sea of the Western
19
Pacific Ocean. Esterase PHE21 was used as a green
biocatalyst to generate chiral ethyl (S)‐3‐hydroxy-
butyrate, 1 key chiral chemical, with high enantiomeric
excess and yield, through direct hydrolysis of racemic
ethyl‐3‐hydroxybutyrate. Herein, we further explored
the potential of esterase PHE21 in enantioselective bio-
catalysis and used esterase PHE21 as a green biocatalyst
in the generation of (S)‐sec‐butyl acetate and (R)‐sec‐

WANG ET AL.
3
2
.4 | Kinetic resolution of (± ±‐sec‐butyl
termination of the enzymatic transesterification reactions,
the chiral products were extracted and analyzed by chiral
gas chromatograph (FULI GC‐9700II).
acetate by PHE21
2
.4.1 | Kinetic resolution of (± ±‐sec‐butyl
acetate through hydrolytic reactions
2
.4.4 | Optimization of the kinetic
resolution of (± ±‐sec‐butyl alcohol through
A standard hydrolytic reaction system containing 500 μL
of 50 mmol/L phosphate buffer (pH 8.0), 40 mmol/L (±)‐
sec‐butyl acetate, and 8.0 × 10 U/L purified PHE21 was
transesterification reactions
3
incubated at 35°C for 3 hours. After the termination of
enzymatic hydrolytic reactions, reaction products were
extracted with an equal volume of ethyl acetate and the
organic phase was further analyzed to evaluate the
enzymatic resolution of (±)‐sec‐butyl acetate by chiral
gas chromatograph (FULI GC‐9700II).
According to the standard reaction system, the effect of
temperature on the kinetic resolution of (±)‐sec‐butyl
alcohol was examined under temperatures ranging from
30°C to 50°C. The reaction mixture containing different
concentrations of vinyl acetate and a final concentration
of 10 mmol/L (±)‐sec‐butyl alcohol (molar ratio from
1:1 to 50:1) was performed to investigate the optimum
substrate molar ratio. A series of concentrations of (±)‐
sec‐butyl alcohol (from 5 to 100 mmol/L) were used to
determine the effects of substrate concentrations on the
kinetic resolution reaction. Different enzyme concentra-
2
.4.2 | Optimization of the kinetic
resolution of (± ±‐sec‐butyl acetate through
hydrolysis reactions
5
5
tions (from 5 × 10 to 50 × 10 U/L) were carried out to
determine the optimum enzyme concentration on the
resolution of (±)‐sec‐butyl alcohol. Under optimum
reaction conditions, products of the enzymatic reactions
were regularly monitored at different times to investigate
the optimum reaction time. All the products of reactions
were analyzed by using chiral GC.
According to the above standard reaction system, a pH
range of 6.0 to 10.0 was carried out to determine the
optimum pH on the kinetic resolution of (±)‐sec‐butyl
acetate. Under the optimum reaction pH, the optimum
reaction temperature was examined at different tempera-
tures ranging from 25°C to 50°C. The effect of organic
solvents and surfactants on enzymatic reactions was stud-
ied by adding 11 kinds of organic solvents (10%, v/v) and
5
different surfactants (0.01%) as the reaction additives
2.5 | Gas chromatograph analysis
under the optimum pH and temperature. The optimum
substrate concentration on the kinetic resolution of (±)‐
sec‐butyl acetate was investigated by adding (±)‐sec‐
butyl acetate of different concentrations (from 0.03 to
The enantiomeric excess of reaction products was ana-
lyzed by GC (FULI GC‐9700II) equipped with H flame
2
ionization detector and 112‐6632 CYCLOSIL‐B chiral
capillary column (30 m × 0.25 mm ID, 0.25 μm df). The
retention times for the R‐enantiomer and S‐enantiomer
of (±)‐sec‐butyl alcohol were 4.48 and 4.69 minutes,
respectively.
0
.06 mol/L). Various enzyme concentrations (from 0.03
to 0.06 mol/L) were carried out to determine the opti-
mum enzyme concentration on the resolution of (±)‐
sec‐butyl acetate. Under the optimum conditions, sam-
ples were periodically withdrawn to monitor the time
course of reaction to determine the optimum reaction
time for kinetic resolution through hydrolytic reactions.
The enantiomeric excess of (S)‐sec‐butyl acetate (ees),
enantiomeric excess of (R)‐sec‐butyl acetate (ee ), conver-
p
sion rate (C), and yield (Y) were calculated using the
20
equation of Chen et al, as follows:
½
ðSÞ− sec−butyl acetate− ðRÞ− sec−butyl acetateꢀ
2
.4.3 | Kinetic resolution of (± ±‐sec‐butyl
ee ¼
s
½
ðSÞ− sec−butyl acetateþðRÞ− sec−butyl acetateꢀ
1)
alcohol through transesterification
reactions
(
The transesterification of (±)‐sec‐butyl alcohol and vinyl
acetate, leading to (R)‐sec‐butyl acetate catalyzed by
PHE21, was subsequently been studied. The stan-
dard 3 mL reaction mixtures containing 10 mmol/L
½ðRÞ− sec−butyl acetate −ðSÞ− sec−butyl acetateꢀ
½ðRÞ− sec−butyl acetateþðSÞ− sec−butyl acetateꢀ
ee ¼
p
(2)
(
±)‐sec‐butyl alcohol, 30 mmol/L vinyl acetate, and
6
1
.7 × 10 U/L PHE21 enzyme powder was carried out in
ðS þ R Þ−ðS þ RÞ
an orbital shaker (220 rpm) at 45°C for 4 hours. n‐Hexane
was used as transesterification reaction medium. After the
0
0
C ¼
(3)
S0 þ R0

4
WANG ET AL.
S
Y ¼
(4)
S0
The ee represented the enantiomeric excess of (S)‐
s
sec‐butyl acetate after hydrolytic reaction. The ee repre-
p
sented the enantiomeric excess of (R)‐sec‐butyl acetate
after transesterification reaction. S and R represented
0
0
the content of (S)‐enantiomers and (R)‐enantiomers of
sec‐butyl acetate before reactions, respectively. S and R
represented the content of (S)‐enantiomers and (R)‐
enantiomers of sec‐butyl acetate after reactions,
respectively.
3
3
| RESULTS AND DISCUSSION
.1 | Kinetic resolution of (± ±‐sec‐butyl
acetate by PHE21 through hydrolytic
reactions
3
.1.1 | Effect of pH and temperature on
the kinetic resolution of (± ±‐sec‐butyl
acetate
In enzymatic kinetic resolution reactions, both the struc-
tures of enzymes and the ionic states of enzymes/sub-
strates were affected by pH and temperature, which
caused the changes of the activity, stability, and
FIGURE 1 A, Effect of pH on the kinetic resolution of (±)‐sec‐
butyl acetate by PHE21 through hydrolytic reactions. B, Effect of
temperature on the kinetic resolution of (±)‐sec‐butyl acetate by
PHE21 through hydrolytic reactions
21,22
enantioselectivity of enzymes.
As shown in Figure 1
A, B, the reaction pH and temperature for the highest
enantiomeric excess of (S)‐sec‐butyl acetate (ee ) was
s
found to be pH 8.0 and 35°C, respectively, with the ees
being over 80% and the conversion rate (C) being 45.5%.
Thus, pH 8.0 and 35°C were characterized to be the opti-
mum pH and temperature for the kinetic resolution of
X‐100 at the concentration of 0.01% greatly promoted the
kinetic resolution of (±)‐ethyl 3‐hydroxybutyrate by ester-
19
(±)‐sec‐butyl acetate by PHE21. In our previous study,
ase PHE21. The results indicated that the addition of
organic solvents and surfactant might strongly affected
both the activity and the stereoselectivity of esterase
PHE21 by affecting the substrate solubility and the struc-
the kinetic resolution of (±)‐ethyl 3‐hydroxybutyrate by
PHE21 was also found to be greatly influenced by pH
and temperature, and the process optimization of pH
and temperature greatly improved the enzymatic kinetic
23,24
ture of esterase PHE21.
Thus, no organic solvents or
19
resolution. Our results indicated that both pH and tem-
perature exhibited great influence on the enantio-
selectivity of esterase PHE21.
surfactants were added to the enzymatic reactions for
the further optimization of the kinetic resolution of (±)‐
sec‐butyl acetate in this study.
3
.1.2 | Effect of organic solvents and
surfactants on the kinetic resolution of
± ±‐sec‐butyl acetate
3.1.3 | Effect of substrate concentration
and enzyme concentration on the kinetic
resolution of (± ±‐sec‐butyl acetate
(
The effect of organic solvents and surfactants on the
kinetic resolution of (±)‐sec‐butyl acetate by PHE21 is
shown in Table 1. The results showed that both the enan-
tiomeric excess of (S)‐sec‐butyl acetate and the conver-
sion were decreased by almost all the tested organic
solvents and surfactants. However, the presence of Triton
The concentrations of substrates and enzymes could also
greatly affect the enzymatic kinetic resolution of race-
25
mates. To reduce the cost of product isolation, the adop-
tion of high concentrations of substrate could be
beneficial for practical application of an enzymatic

WANG ET AL.
5
TABLE 1 Effect of organic solvents and surfactants on the
kinetic resolution of (±)‐sec‐butyl acetate by PHE21 through
hydrolytic reactions
Organic Solvents
and Surfactants
Concentration
C (%±
s
ee (%±
Control
46.8 ± 0.8
59.7 ± 0.7
57.5 ± 1.5
56.1 ± 1.6
51.8 ± 0.6
45.4 ± 1.2
43.2 ± 1.6
40.4 ± 0.8
58.9 ± 0.6
51.8 ± 0.7
41.8 ± 0.8
51.8 ± 0.6
46.5 ± 1.8
47.5 ± 1.7
50.3 ± 0.7
69.6 ± 0.5
63.2 ± 1.6
80 ± 1
78 ± 1
75 ± 2
73 ± 2
76 ± 1
76 ± 2
68 ± 2
68 ± 1
70 ± 1
68 ± 1
56 ± 1
74 ± 1
79 ± 2
54 ± 2
64 ± 1
72 ± 1
75 ± 2
Methanol
Ethanol
10%
10%
Acetonitrile
Iso‐propyl alcohol
Butyl alcohol
Acetone
10%
10%
10%
10%
DMSO
10%
n‐Hexane
Cyclohexane
n‐Octane
10%
10%
10%
Isooctane
Triton X‐100
Tween‐20
Tween80
10%
0.01%
0.01%
0.01%
0.01%
0.01%
CTAB
Sodium
tripolyphosphate
26
process. As shown in Figure 2A, the optimum sub-
strate concentration for the kinetic resolution of (±)‐
sec‐butyl acetate by PHE21 was found to be 0.04 mol/
L, with the enantiomeric excess of (S)‐sec‐butyl acetate
being over 96% and the conversion being 48.9%, respec-
tively. Suitable enzyme loading was also important for
both reducing the production cost and increasing the
reaction productivity. The reaction profiles in Figure 2
B showed that the enantiomeric excess of (S)‐sec‐butyl
acetate was decreased when the enzyme concentration
3
3
was below or over 8.0 × 10 U/L. Thus, 8.0 × 10 U/L
was characterized to be the optimum enzyme concentra-
tion for the kinetic resolution of (±)‐sec‐butyl acetate by
PHE21, with the enantiomeric excess of (S)‐sec‐butyl
acetate being over 92% and the conversion rate being
FIGURE 2 A, Effect of substrate concentration on the kinetic
resolution of (±)‐sec‐butyl acetate by PHE21 through hydrolytic
reactions. B, Effect of enzyme concentration on the kinetic
resolution of (±)‐sec‐butyl acetate by PHE21 through hydrolytic
reactions. C, Effect of reaction time on the kinetic resolution of (±)‐
sec‐butyl acetate by PHE21 through hydrolytic reactions.
4
9.7%, respectively.
3
.1.4 | Effect of reaction time on the
kinetic resolution of (± ±‐sec‐butyl acetate
yield reached 98%, 52%, and 83%, respectively, when the
reaction time was 180 minutes. After 180 minutes, both
the enantiomeric excess of (S)‐sec‐butyl acetate and the
conversion were basically unchanged, indicating that
the optimal reaction time for the kinetic of (±)‐sec‐butyl
acetate was 180 minutes.
The effect of time course on the kinetic resolution of (±)‐
sec‐butyl acetate through hydrolytic reactions was studied
by changing the reaction time under the optimum reac-
tion conditions. As shown in Figure 2C, the enantiomeric
excess of (S)‐sec‐butyl acetate, the conversion, and the

6
WANG ET AL.
3
.1.5 | Comparation of PHE21 and other
esterases/lipases in the preparation of
secondary alcohols through hydrolysis
reactions
Previously, the reports about producing chiral sec‐butyl
acetate and sec‐butyl alcohol through direct enzymatic
hydrolysis of racemic sec‐butyl acetate or sec‐butyl alco-
hol, especially by esterases/lipases, were quite rare. Other
experiments about preparation of chiral long‐chain sec-
ondary alcohol through direct enzymatic hydrolysis were
14,17,18
reported.
The structural differences on both sides of
the chiral center in long‐chain secondary alcohols may
greatly facilitate the kinetic resolution using esterases/
lipases. In our study, esterase PHE21 could efficiently cat-
alyze the enzymatic kinetic resolutions of (±)‐sec‐butyl
acetate with both high enantiomeric excess and yield,
indicating that marine microbial esterase PHE21 was
identified as a useful biocatalyst for asymmetric synthesis
through hydrolysis reactions.
3
.2 | Kinetic resolution of (± ±‐sec‐butyl
alcohol by PHE21 through
transesterification reactions
3
.2.1 | Effect of temperature on the
transesterification reaction
FIGURE 3 A, Effect of temperature on the transesterification
reaction catalyzed by PHE21. B, Effect of substrate molar ratio on
the transesterification reaction catalyzed by PHE21
The effect of temperature on the resolution of (±)‐sec‐
butyl alcohol by PHE21 is presented in Figure 3A. The
reaction temperature for the highest enantiomeric excess
of (R)‐sec‐butyl acetate (ee ) generated from the enzymatic
p
transesterification reactions was found to be 45°C, with
the experiments. Figure 3B shows that the ee of (R)‐
p
the ee being over 56%. Thus, 45°C was characterized to
be the optimum temperature for the transesterification of
sec‐butyl acetate increased with increasing molar ratio
of (±)‐sec‐butyl alcohol/vinyl acetate, but the enantio-
meric excess of (R)‐sec‐butyl acetate decreased when
the molar ratio of (±)‐sec‐butyl alcohol/vinyl acetate
was over 1:3. So, the optimal molar ration of (±)‐sec‐
butyl alcohol and vinyl acetate was 1:3 for the
transesterification of (±)‐sec‐butyl alcohol and vinyl ace-
tate catalyzed by esterase PHE21.
p
(±)‐sec‐butyl alcohol and vinyl acetate catalyzed by ester-
ase PHE21. All the above results showed that temperature
may affect the structures and ionic states of enzymes, then
affect both the processes of asymmetric hydrolysis and
asymmetric transesterification.
3
.2.2 | Effect of substrate molar ratio on
the transesterification reaction
3.2.3 | Effect of (± ±‐sec‐butyl alcohol
concentration on the transesterification
reaction
It is widely believed that the utilization of vinyl esters
as the acylation reagent for transesterification could pro-
vide an effective solution to overcome equilibrium,
because the enol coproduct could be irreversibly trans-
formed into acetaldehyde and removed from the enzy-
matic reactions. However, the substrate molar ratio
may have a great effect on the transesterification reac-
tions. The substrate molar ratio of (±)‐sec‐butyl alcohol
to vinyl acetate was varied between 1:1 and 1:50 during
As shown in Figure 4A, the enantiomeric excess of (R)‐
sec‐butyl acetate was decreased when the (±)‐sec‐butyl
alcohol concentration was below or above 10 mmol/L.
Thus, the optimal concentration of substrate (±)‐sec‐butyl
alcohol for the transesterification reaction was chosen as
10 mM, with the highest enantiomeric excess of (R)‐sec‐
butyl acetate being 63% and a conversion of 39%.
27

WANG ET AL.
7
6
4
1%. Thus, 1.7 × 10 U/L enzyme loading was the opti-
mum enzyme concentration for the transesterification
reaction.
3
.2.5 | Effect of reaction time on the
transesterification reaction
Different reaction times (from 2 to 24 hours) were set to
investigate the optimal reaction time for the transeste-
rification reaction by esterase PHE21. The highest ee of
p
64% and a conversion rate of 43% were observed at 4 hours
(
Figure 4C). Thus, the optimum reaction time for the gen-
eration of (R)‐sec‐butyl acetate through transesterification
reactions catalyzed by PHE21 was 4 hours.
3
.2.6 | Comparation of PHE21 and
other esterases/lipases in the preparation
of secondary alcohols through
transesterification reactions
To date, there were some reports about the kinetic resolu-
tions of racemic secondary alcohols by transesterification
15,16,28
reactions catalyzed by lipases/esterases.
For exam-
ple, immobilized Pseudomonas sp. lipase (PSL) was used
to produce (S)‐2‐octanol with an ee of 99% and a conver-
s
1
5
sion of 53% through transesterification reactions. How-
ever, there were basically no reports about the kinetic
resolution of racemic short‐chain secondary alcohol. In
our study, deep‐sea esterase PHE21 was used as a green
biocatalyst in the generation of (R)‐sec‐butyl acetate
through transesterification reactions, with the ee of 64%
p
and the conversion of 43% after process optimization.
The enantiomeric excess of (R)‐sec‐butyl acetate still
needs further optimization through either process optimi-
zation or protein engineering (Figure 5).
FIGURE 4 A, Effect of (±)‐sec‐butyl alcohol concentration on
the transesterification reaction catalyzed by PHE21. B, Effect of
enzyme concentration on the transesterification reaction catalyzed
by PHE21. C, Effect of (±)‐sec‐butyl alcohol concentration on the
transesterification reaction catalyzed by PHE21
3
.2.4 | Effect of enzyme concentration on
the transesterification reaction
The enantiomeric excess and conversion rate of (R)‐sec‐
butyl acetate were increased with the increase of the con-
centration of PHE21 (Figure 4B). When the enzyme con-
centration was 1.7 × 10 U/L, the enantiomeric excess
and conversion of (R)‐sec‐butyl acetate were the highest,
FIGURE 5 Kinetic resolution of (±)‐sec‐butyl acetate and (±)‐
sec‐butyl alcohol by PHE21. A, Preparation of (S)‐sec‐butyl acetate
through direct hydrolysis reaction. B, Preparation of (R)‐sec‐butyl
acetate through transesterification reaction
6
with the ee being over 63% and the conversion rate being
p

8
WANG ET AL.
2
. Rouf A, Taneja SC. Synthesis of single‐enantiomer bioactive
molecules: A brief overview. Chirality. 2014;26(2):63‐78.
4
| CONCLUSION
Despite the fact that there were some reports about the
kinetic resolutions of long‐chain secondary alcohols by
esterases/lipases, the enzymatic kinetic resolution of
racemic sec‐butyl acetate was found to be quite chal-
lenging, possibly because the structural difference on
both sides of the ester bond of sec‐butyl acetate and
the structural difference of the 2 enantiomers are quite
small. The potential of deep‐sea microbial esterase
PHE21, 1 biocatalyst which has been used to efficiently
generate optically pure ethyl (S)‐3‐hydroxybutyrate, was
further investigated in this study. Biocatalyst PHE21
could efficiently resolve racemic sec‐butyl acetate and
generate (S)‐sec‐butyl acetate with high enantiomeric
excess (98%) and high yield (83.6%) through asymmetric
hydrolytic reactions after process optimization. Mean-
while, biocatalyst PHE21 could also generate (R)‐sec‐
butyl acetate through transesterification reactions, with
3. El Alami MSI, El Amrani MA, Agbossou‐Niedercorn F, Suisse I,
Mortreux A. ChemInform Abstract: Chiral ligands derived from
monoterpenes: Application in the synthesis of optically pure
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recognition of the 2 enantiomers by esterases were too
hard, and still needed further optimization through
either process optimization or protein engineering. So
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mer and the R‐enantiomer of racemic sec‐butyl acetate
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ACKNOWLEDGMENTS
We are grateful for the financial support from the Strate-
gic Priority Research Program of the Chinese Academy of
Sciences (XDA11030404), Scientific and Technological
Project of Ocean and Fishery from Guangdong Province
1. Ma JB, Wu L, Guo F, et al. Enhanced enantioselectivity of a car-
boxyl esterase from Rhodobacter sphaeroides by directed
evolution. Appl Microbiol Biotechnol. 2013;97(11):4897‐4906.
(A201701C12), and Guangzhou Science and Technology
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mally stable lipase from Burkholderia ambifaria YCJ01:
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of mandelic acid. J Mol Catal B‐Enzym. 2013;85‐86:105‐110.
Plan Projects (201510010012). We thank the research ves-
sel KEXUE of the Chinese Academy of Sciences for
collecting samples and WPOS sample center for providing
samples.
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Matsuno R. Lipase‐catalyzed synthesis of 6‐O‐eicosapentaenoyl
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ORCID
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Combining regio‐ and enantioselectivity of lipases for the prepa-
ration of (R)‐4‐chloro‐2‐butanol. Chirality. 2007;19(1):44‐50.
Yunfeng Hu http://orcid.org/0000-0001-9258-7205
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through kinetic resolutions. Chirality. 2018;1–9.
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