Enantioselective resolution of (±)-1-phenylethyl acetate by extracellular proteases from deep-sea bacterium Bacillus sp. DL-2

Article abstract of DOI:10.1080/10242422.2019.1616697

Chiral 1-phenylethanol and its ester derivative are important chiral chemicals in diverse industries and the preparation of those optically pure enantiomers is of great importance. One bacterium, Bacillus sp. DL-2, whose extracellular proteases could efficiently asymmetrically hydrolyse (±)-1-phenylethyl acetate, was isolated from the deep sea of the Western Pacific. After the immobilization of extracellular proteases and the optimization of enzymatic reactions, (R)-1-phenylethanol was prepared with the enantiomeric excess (e.e.) being 97% and the yield being 41%, respectively. The optimal resolution reaction condition for the preparation of (R)-1-phenylethanol using immobilized extracellular proteases was found to be 5-mM (±)-1-phenylethyl acetate, 360 mg/mL immobilized extracellular proteases, pH 6.5, and 20 °C for 2 h. (S)-1-phenylethyl acetate was generated through enzymatic kinetic resolution with the e.e. being as high as 99% and the yield being 71%, respectively. The optimal resolution reaction condition for the preparation of (S)-1-phenylethyl acetate was found to be 2.5-mM (±)-1-phenylethyl acetate, 440 mg/mL immobilized extracellular proteases, pH 7.5, and 35 °C for 10 h. Our report is the first report about the kinetic resolution of (±)-1-phenylethyl acetate using proteases and the enantio-preference of the proteases was found to be the same as those of most other esterases/lipases. Also notably, the optical purity of (S)-1-phenylethyl acetate generated through kinetic resolution using the proteases of Bacillus sp. DL-2 was the highest report so far. Proteases from deep-sea Bacillus sp. DL-2 are new contributions to the biocatalyst library for the preparation of valuable chiral alcohols and chiral esters through kinetic resolution.

Full text of DOI:10.1080/10242422.2019.1616697

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Enantioselective resolution of (±)-1-phenylethyl
acetate by extracellular proteases from deep-sea
bacterium Bacillus sp. DL-2
Lu Dong, Yongkai Xu, Yun Zhang, Aijun Sun & Yunfeng Hu
To cite this article: Lu Dong, Yongkai Xu, Yun Zhang, Aijun Sun & Yunfeng Hu (2019):
Enantioselective resolution of (±)-1-phenylethyl acetate by extracellular proteases
from deep-sea bacterium Bacillus sp. DL-2, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2019.1616697
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2019.1616697
RESEARCH ARTICLE
Enantioselective resolution of ( )-1-phenylethyl acetate by extracellular
proteases from deep-sea bacterium Bacillus sp. DL-2
Lu Donga,b,c, Yongkai Xud, Yun Zhanga,c, Aijun Suna,c and Yunfeng Hua,b,e
aCAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of
b
c
Sciences, Guangzhou, PR China; University of Chinese Academy of Sciences, Beijing, PR China; Guangdong Key Laboratory of
d
Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, PR China; Affiliated
Hospital of Shandong University of Traditional Chinese Medicine, Jinan, PR China; South China Sea Bio-Resource Exploitation and
e
Utilization Collaborative Innovation Center, Guangzhou, PR China
ABSTRACT
ARTICLE HISTORY
Received 8 December 2018
Revised 18 April 2019
Accepted 4 May 2019
Chiral 1-phenylethanol and its ester derivative are important chiral chemicals in diverse indus-
tries and the preparation of those optically pure enantiomers is of great importance. One bac-
terium, Bacillus sp. DL-2, whose extracellular proteases could efficiently asymmetrically hydrolyse
( )-1-phenylethyl acetate, was isolated from the deep sea of the Western Pacific. After the
immobilization of extracellular proteases and the optimization of enzymatic reactions, (R)-1-phe-
nylethanol was prepared with the enantiomeric excess (e.e.) being 97% and the yield being
41%, respectively. The optimal resolution reaction condition for the preparation of (R)-1-phenyle-
thanol using immobilized extracellular proteases was found to be 5-mM ( )-1-phenylethyl acet-
ate, 360mg/mL immobilized extracellular proteases, pH 6.5, and 20 ꢀC for 2 h. (S)-1-phenylethyl
acetate was generated through enzymatic kinetic resolution with the e.e. being as high as 99%
and the yield being 71%, respectively. The optimal resolution reaction condition for the prepar-
ation of (S)-1-phenylethyl acetate was found to be 2.5-mM ( )-1-phenylethyl acetate, 440mg/mL
immobilized extracellular proteases, pH 7.5, and 35 ꢀC for 10 h. Our report is the first report
about the kinetic resolution of ( )-1-phenylethyl acetate using proteases and the enantio-prefer-
ence of the proteases was found to be the same as those of most other esterases/lipases. Also
notably, the optical purity of (S)-1-phenylethyl acetate generated through kinetic resolution
using the proteases of Bacillus sp. DL-2 was the highest report so far. Proteases from deep-sea
Bacillus sp. DL-2 are new contributions to the biocatalyst library for the preparation of valuable
chiral alcohols and chiral esters through kinetic resolution.
KEYWORDS
Asymmetric hydrolysis;
extracellular proteases;
immobilization;
marine microorganisms;
(R)-1-phenylethanol;
(S)-1-phenylethyl acetate
Introduction
synthesis and biotransformation have been the two
major technologies for the preparation of optically
pure alcohols. Compared with conventional organic
synthesis, the technology of biocatalysis has drawn
broad attention for its high efficiency, mild reaction
condition, environmental safety, outstanding stereo-
specificity, and so on (Reetz 2002; Kroutil et al. 2004;
Wu et al. 2004; Li et al. 2016). Proteases widely exist in
animals, plants, and microorganisms, and play vital
roles in many physiological and pathological processes
(Rao et al. 1998). The use of proteases derived from
microorganisms is advantageous because of fast
growth rate of microorganisms and easy formation of
multi-enzymatic systems. Microorganisms can generate
a great variety of intracellular and extracellular pro-
teases, which have potential usage in diverse
Enantiomers of chiral compounds often behave dra-
matically different or even opposite biological activ-
ities. With the increasing demands of valuable chiral
drugs and chiral fine chemicals, great attention has
been paid to the enantioselective synthesis of optically
pure compounds. Chiral alcohols are important inter-
mediates of drugs, pesticides, fine chemicals, and
functional materials. In particular, optically active aro-
matic alcohols are the precursors of antidepressants,
antiasthmatics, adrenoceptor agonists, NK1 antago-
nists, and even liquid crystals and chiral auxiliaries (Ou
et al. 2008). Chiral phenylethanol derivatives are
important chiral building blocks for the synthesis of
pharmaceuticals, agrochemicals, and complex natural
products (Habulin and Knez 2009). Nowadays, organic
CONTACT Yunfeng Hu
yunfeng.hu@scsio.ac.cn
CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of
Oceanology, Chinese Academy of Sciences, Guangzhou, PR China
Supplemental data for this article can be accessed here.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
L. DONG ET AL.
industries. Intracellular proteases are important for
various cellular and metabolic processes. Extracellular
proteases play significant roles in hydrolysing proteins
in cell-free environments for the utilization by cells
(Gupta et al. 2002; Sabotic and Kos 2012; Souza et al.
2015). Microbial proteases have also been employed
to produce chiral compounds in recent decades
(Olivier et al. 1979; Fǜlling and Sih 1987; Yamaguchi
et al. 2007; Tang et al. 2018).
Owing to the desirable advantages (reusability, bet-
ter stability, easy product separation, and continuous
operation) of immobilization techniques of industrial
enzymes, the application of immobilized enzymes in
diverse industry fields such as biotechnology and
food industry is becoming increasingly popular, and
some reports have been published in the usage of
immobilized enzymes (Huang et al. 1997; Ertan et al.
2006; Satar and Husain 2009; Cabana et al. 2009;
Contesini et al. 2012). Immobilized proteases have
been reported to exhibit better tolerance to organic
solvents and changes in pH and temperature and
improved stability and reusability (Jakubke and
et al. 2012). An alkaline extra-cellular lipase of Bacillus
cereus MTCC 8372 was immobilized on diatomaceous
earth through adsorption and further used for the syn-
thesis of ethyl acetate from vinyl acetate and ethanol
in n-nonane via transesterification reaction, which gen-
erated ethyl acetate with a yield of 73.7% after the
optimization of enzymatic reactions (Verma et al.
2009). In order to synthesize pentyl isovalerates, por-
cine pancreatic lipase and Candida cylindracea lipase
immobilized on Celite were found to be very effective
in the esterification reaction and showed better
thermal stabilities, pH stabilities, operational stabilities
than those of the free forms of the enzymes
(Sagiroglu and Telefoncu 2004). An extra-cellular lipase
of Bacillus licheniformis SCD11501 immobilized on
Celite 545 by adsorption, was successfully used to syn-
thesize some derivatives of gallic acid after sequen-
tially optimizing various reaction parameters (Sharma
et al. 2015).
The ocean is special with high salinity, high pres-
sure, low temperature, low illumination, and oligo-
trophic environment and contains huge microbial
biodiversity. Numerous enzymes with desired proper-
ties may be obtained from those extreme marine
microorganisms and further developed into valuable
biocatalysts in diverse industries (Burg 2003; Kennedy
et al. 2008). In our work, one marine microorganism
Bacillus sp. DL-2, was isolated from the environmental
sample obtained from the deep sea of the Western
Pacific. The extracellular proteases of Bacillus sp. DL-2
were immobilized and further utilized to produce (R)-
1-phenylethanol and (S)-1-phenylethyl acetate with
high enantiomeric excess (e.e.) through asymmetric
hydrolysis reactions (Scheme 1).
€
Konnecke 1987; Husain 2018).
Adsorption of industrial enzymes on kieselguhr, an
economical inorganic microporous support, is one of
the most cost-effective immobilization techniques
(Anderson et al. 1990; Mansour and Dawoud 2003;
Cho et al. 2008; Satar and Husain 2009). Some litera-
ture about the application of industrial enzymes
immobilized on kieselguhr have been previously
reported. In the study of immobilization of extracted
glucosyltransferase from Erwinia sp. by Contesini et al.,
Celite 545 was utilized as the enzyme carrier for the
conversion of sucrose into isomaltulose (Contesini
1)
360 mg/mL
O
O
immobilized extracellaluar
proteases of Bacillus sp. DL-2
(S)
(R)
O
OH
O
+
pH 6.5, 20
, 2 mM Mn2+, 2 h
°C
(S)-1-phenylethyl acetate
5 mM
(R)-1-phenylethanol
(f)-1-phenylethyl acetate
e.e.p = 97 %
yield = 41 %
2)
440 mg/mL
O
O
immobilized extracellaluar
proteases of Bacillus sp. DL-2
(S)
O
(R)
O
OH
+
pH 7.5, 35
, 2 mM Ca2+/Mg2+, 10 h
°C
(S)-1-phenylethyl acetate
(R)-1-phenylethanol
2.5 mM
)-1-phenylethyl acetate
(
e.e.s > 99%
yield = 71%
Scheme 1. Kinetic resolution of ( )-1-phenylethyl acetate using immobilized extracellular proteases from Bacillus sp. DL-2 for the
generation of (R)-1-phenylethanol (1) and (S)-1-phenylethyl acetate (2).

BIOCATALYSIS AND BIOTRANSFORMATION
3
After activation, 1-mL bacteria liquid was inoculated in
100 mL LB liquid medium, which was cultivated in a
rocking incubator at 37ꢀC and 200 r/min. A Tecan
NanoQuant Infinite M200 Pro plate reader was adopted
to determine the optical densities at the wavelength of
600 nm every 2 h during the culture process.
Subsequently, 1% bacteria liquid was added to milk
powder liquid medium (1% high protein level skimmed
milk powder) for fermentation cultivation at 37 ꢀC and
200 r/min. The enzymatic activities of extracellular pro-
teases were measured according to the SB/T 10317-1999
standard at culture times of 12, 24, 36, 48, and 60h.
Meanwhile, the resolution effects for ( )-1-phenylethyl
acetate using extracellular proteases at those five time
points were detected by chiral gas chromatograph (GC).
Materials and methods
Microorganisms and reagents
Bacillus sp. DL-2 was obtained from the sediments of
the deep sea of the Western Pacific by the Research
Vessel “KEXUE”. High protein level skimmed milk pow-
der was got from Inner Mongolia Yili Industrial Group
Co., Ltd. (Hohhot, China). Yeast extract and tryptone
were purchased from Guangdong Huankai Microbial
Science and Technology Co., Ltd. (Guangdong, China).
Kieselguhr was obtained from Tianjin Damao Chemical
Reagent Co., Ltd. (Tianjin, China). ( )-1-phenylethyl
acetate and corresponding chiral enantiomers were
purchased from Adamas Reagent Co., Ltd. (Shanghai,
China). Dodecane used as an internal standard was
gained from Aladdin Industrial Corporation (Shanghai,
China). Other chemicals were of analytical grade.
Effect of pH on the activity and stability of the
extracellular protease
PCR amplification and sequencing of 16S
rRNA gene
The crude fermentation broth containing extracellular
proteases cultivated for 24 h was freeze-dried into
crude enzyme powder by freeze drier. The optimal pH
for the activities of extracellular proteases was meas-
ured in 50 mM buffers with various pH values: pH
5.0–5.5 (sodium citrate buffer), pH 6.0–8.0 (sodium
phosphate buffer), and pH 8.0–9.0 (Tris–HCl buffer) by
using 2% casein as the substrate at 45 ꢀC. The pH sta-
bility of extracellular proteases was investigated by
incubating the extracellular proteases at 20 ꢀC for 2 h
in the buffers with different pH and then analysing
the residual activities at 45 ꢀC.
The polymerase chain reaction (PCR) was carried out to
obtain the 16S rRNA of Bacillus sp. DL-2. The forward and
reverse primers used were: 50-AGAGTTTATCCTGGCTCAG-
30 and 50-GGTTACCTTGTTACGACTT-30, respectively. The
PCR was set up using 1 lL of incubating medium as tem-
plate directly, 2 lL of both 10lM forward and reverse
primers, 25 lL of 2ꢁTaq Master Mix containing 4 mM
MgCl2 and 0.4 mM dNTP mix (Shanghai Generay Biotech
Co., Ltd.) and 20 lL aseptic ddH2O in 50lL reaction vol-
ume. The PCR program was set according to the proced-
ure as 30 cycles after an initial denaturation for 10 min at
94 ꢀC. Each PCR cycle comprised three steps: 30 s at 94 ꢀC
(denaturation), 30 s at 56 ꢀC (annealing), and 150 s at
72 ꢀC (extension). A final extension of 10 min was given
at 72 ꢀC. After PCR amplification, the 1.5-kb amplified
DNA fragment was purified from 1% agarose gel and
used directly for DNA sequencing.
Effect of temperature on the activity and stability
of the extracellular protease
The optimal temperature for the activities of extracel-
lular proteases was tested by incubating the extracel-
lular proteases at temperatures ranging from 25 to
70 ꢀC by using 2% casein as the substrate in Tris–HCl
(pH 8.0) buffer. The temperature stability of the extra-
cellular proteases was evaluated by incubating the
extracellular proteases at different temperatures rang-
ing from 25 to 70 ꢀC for 2 h and the residual activity
of the extracellular protease was measured by using
2% casein as the substrate in Tris–HCl (pH 8.0) buffer
at 45 ꢀC.
16s rRNA gene sequence analysis for molecular
identification of strains
The sequencing of the 16S rRNA of Bacillus sp. DL-2
was performed by Shanghai Majorbio Bio-Pharm
Technology Co., Ltd. The 16S rRNA nucleotide sequence
of Bacillus sp. DL-2 was blasted against the NCBI
nucleotide database using the BLASTN program.
Immobilization of extracellular proteases and
measurement of enzyme activities
Biochemical characterization of Bacillus sp. DL-2
Lysogeny broth (LB) medium was selected to culture
Bacillus sp. DL-2 was cultivated in milk powder liquid
Bacillus sp. DL-2 for determining its growth curve.
medium for 24 h. Afterwards, the crude extracellular

4
L. DONG ET AL.
proteases of Bacillus sp. DL-2 were gained by centrifu-
gation. 5-g kieselguhr was put in every 100-mL crude
fermentation broth containing extracellular proteases,
which was reacted in shaking table at 25 ꢀC, 200 r/min
for 10 h. Subsequently, the reaction mixture was fil-
trated and then the part of kieselguhr was dried at
38 ꢀC to obtain immobilized extracellular proteases.
Finally, the dried immobilized enzyme was weighed to
calculate the weight of extracellular protease adsorbed
by kieselguhr. The enzymatic activities of immobilized
extracellular proteases were measured according to
the SB/T 10317-1999 standard.
Kinetic resolution of ( )-1-phenylethyl acetate for
the preparation of (S)-1-phenylethyl acetate by
immobilized extracellular proteases
A standard 500-mL hydrolytic reaction system contain-
ing 440 mg/mL immobilized extracellular proteases,
2.5 mM ( )-1-phenylethyl acetate, and 50 mM phos-
phate buffer (pH 7.5) was incubated at 35 ꢀC for 10 h.
After the completion of the enzymatic reaction, reac-
tion samples were extracted with an equal volume of
ethyl acetate and the organic phase was further ana-
lysed by a GC.
A series of enzymatic reactions were carried out at
different pH ranging from 5.0 to 10.0 at 35 ꢀC for 10 h
to determine the optimal pH for the resolution of
( )-1-phenylethyl acetate. The effect of temperature
on the resolution of ( )-1-phenylethyl acetate was car-
ried out at different temperatures ranging from 20 to
50 ꢀC at pH 7.5 for 10 h. Immobilized extracellular pro-
teases were added to the reaction at final concentra-
tions ranging from 120 to 480 mg/mL to test the
effect of enzyme concentration on the kinetic reso-
lution of ( )-1-phenylethyl acetate. Under the optimal
pH, temperature, and enzyme concentration, different
concentrations of ( )-1-phenylethyl acetate (from 2.5
to 50 mM) were employed to determine the effect of
substrate concentration on the kinetic resolution of
( )-1-phenylethyl acetate. Under the optimal reaction
conditions, with 440 mg/mL enzyme and 2.5 mM sub-
strate ( )-1-phenylethyl acetate in the reaction, the
product of the enzymatic reaction was regularly moni-
tored at different times to study the effect of reaction
time on the e.e. of the residual 1-phenylethyl acetate.
Under the standard hydrolytic reaction system for the
preparation of (S)-1-phenylethyl acetate, thirteen
metallic ions, eight surfactants and nineteen organic
solvents were added to test the effect of various addi-
tives on the enzymatic kinetic resolution of ( )-1-phe-
nylethyl acetate.
Kinetic resolution of ( )-1-phenylethyl acetate for
the preparation of (R)-1-phenylethanol by
immobilized extracellular proteases
Standard
500-lL
reaction
system
contained
360 mg/mL immobilized extracellular proteases, 5 mM
( )-1-phenylethyl acetate in 50 mM phosphate buffer
(pH 6.5). The enzymatic reactions were carried out at
20 ꢀC for 2 h. After the termination of enzymatic reso-
lution reactions, reaction samples were extracted with
an equal volume of ethyl acetate and the organic
phase was further analysed to evaluate the enzymatic
resolution of ( )-1-phenylethyl acetate by chiral GC.
The optimal pH for kinetic resolution of ( )-1-phe-
nylethyl acetate was tested by incubating standard
enzymatic reactions at various pH ranging from 5.0 to
10.0 at 20 ꢀC for 2 h. A series standard enzymatic reac-
tion were performed at temperatures ranging from
20 to 50 ꢀC at pH 6.5 to determine the optimal tem-
perature for the kinetic resolution of ( )-1-phenylethyl
acetate. Various immobilized extracellular proteases
concentrations ranging from 120 to 480 mg/mL were
employed to confirm the effect of enzyme loading on
the kinetic resolution of ( )-1-phenylethyl acetate. The
effect of substrate concentration was investigated by
adding ( )-1-phenylethyl acetate of different concen-
trations ranging from 2.5 to 50 mM into standard reac-
tion systems. The optimal reaction time was identified
by carrying out the enzymatic reactions for different
times ranging from 1 to 12 h. Different metallic ions
and surfactants were added into standard reaction sys-
tems at final concentrations of 2 mM and 0.05% (w/v),
respectively, to test the effect of metallic ions and sur-
factants on the kinetic resolution of ( )-1-phenylethyl
acetate. The effect of organic solvents on the kinetic
resolution of ( )-1-phenylethyl acetate were studied
by incubating standard enzymatic reactions in the
presence of different organic solvents at final concen-
trations of 5% (v/v).
Operational stability of immobilized
extracellular proteases
In order to investigate the operational stability of
immobilized enzyme, the immobilized extracellular
proteases were reused to detect the enzyme activity
after repeated usage. Each reaction was considered a
single cycle and the percent of relative activity was
calculated by taking the activity of the first cycle as
100%. Meanwhile, the immobilized extracellular pro-
teases were also reused for five times to asymmetric-
ally hydrolyse ( )-1-phenylethyl acetate for the

BIOCATALYSIS AND BIOTRANSFORMATION
5
preparation of (R)-1-phenylethanol and (S)-1-phenyl-
ethyl acetate under the optimal enzymatic conditions.
Bacillus species, such as Bacillus megaterium strain
HNS68, Bacillus aryabhattai strain JH7, Bacillus sp.
CMJ1-21 and Bacillus sp. AER317-1. Therefore, the
deep-sea bacterium which can resolve ( )-1-phenyl-
ethyl acetate was named as Bacillus sp. DL-2. The
growth curve of Bacillus sp. DL-2 was determined by
detecting the optical densities (OD) at the wavelength
of 600 nm, which showed that the logarithmic phase
was 1–8 h, the stationary phase was 8–18 h, and the
decline phase was after 18 h (Figure S1).
Analytical methods
The e.e. and conversion rate (C) of the reaction prod-
ucts were analysed by GC (FULI GC-9700II) equipped
with H2 flame ionization detector and 1112-6632
CYCLOSIL-B chiral capillary column (30 m ꢁ 0.25 mm ꢁ
0.25 lm). The flow rate of carrier gas (N2) was 1.2 mL/
min and the column temperature was ranging from
80 to 210 ꢀC. The injector and detector temperature
were 210 and 210 ꢀC, respectively. The e.e. and C were
calculated by the equation as follows (Chen et al.
1982).
Comparison of the protease activity and
resolution ability of Bacillus sp. DL-2
As shown in Figure S2, the protease activity of Bacillus
sp. DL-2 reached the highest when cultured for 48 h.
However, the resolution effect towards ( )-1-phenyl-
ethyl acetate was found to be the best using the
extracellular proteases from 24-h cultures, with the e.
e.p being 70%. Bacillus is well known to produce extra-
cellular proteases and sometimes multiple proteases
can be identified from the fermentation broth.
Therefore, it is very possible that Bacillus sp. DL-2 can
product multiple extracellular proteases and the prote-
ase activities of its total extracellular proteases are not
consistent with the resolution abilities.
Â
à Â
Ã
ð Þ
ð Þ
R ꢂ1ꢂphenylethanol – S ꢂ1ꢂphenylethanol
Â
Ã
Â
Ã
Ã
e:e:p ¼
ð Þ
ð Þ
R ꢂ 1 ꢂ phenylethanol
þ
S ꢂ 1 ꢂ phenylethanol
Â
Ã
Â
Ã
ð Þ
ð Þ
S ꢂ1ꢂphenylethyl acetate ꢂ R ꢂ1ꢂphenylethyl acetate
Â
Ã
Â
e:e:s ¼
ð Þ
ð Þ
S ꢂ 1 ꢂ phenylethyl acetate þ R ꢂ 1 ꢂ phenylethyl acetate
ð
Þ
S0 þ R0ꢂ S þ R
C ¼
S0 þ R0
YR ¼ A=A0; YS ¼ B=B0
C stands for the conversion ratio of the substrate,
and e.e.p and e.e.s represent the e.e. of the product
and substrate, respectively. S0 and R0, respectively,
stand for the concentrations of the (S)- and (R)-enan-
tiomers of 1-phenylethyl acetate before reaction, while
S and R represent the concentrations of the (S)-
enantiomer and (R)-enantiomer of 1-phenylethyl acet-
ate after reaction, respectively. YR and YS stand for the
yeild of (R)-1-phenylethanol and (S)-1-phenylethyl acet-
ate, respectively. A represents the concentration of (R)-1-
phenylethanol after the reaction. A0 represent the con-
centration of (R)-1-phenylethanol when (R)-1-phenylethyl
acetate completely transform into (R)-1-phenylethanol. B
and B0 represent the concentration of (S)-1-phenylethyl
acetate after and before the reaction, respectively.
Effect of pH on the activity and stability of the
extracellular protease
The effect of pH on the activity of the extracellular
proteases was measured using different buffers with
pH 5.0–9.0 (Table S1). The results showed that the
extracellular proteases exhibited the highest hydrolytic
activity in Tris–HCl buffer of pH 8.0 (Figure S3). The pH
stability of the extracellular proteases was analysed
after incubating the enzymes in buffers with a series
of pH for 2 h. As shown in Figure S4, the extracellular
proteases were relatively stable and exhibited the
highest activity and stability in Tris–HCl buffer of pH 8.
0. The stability of the extracellular protease quickly
declined when the pH value was below 6 or above 9.
However, the lowest enzyme activity at pH 5.0 was
still 67.38 5.28% of the maximal activity.
Results and discussion
Isolation and characterization of one protease-
producing strain which can resolve ( )-1-
phenylethyl acetate
Many protease-producing strains were isolated and
purified from the sediments obtained from the
Western Pacific Ocean. One bacterium strain was iden-
tified to be able to asymmetrically hydrolyse ( )-1-
phenylethyl acetate using its extra-cellular proteases.
The 16S rRNA nucleotide sequence of this deep-sea
bacterium exhibited 99% identity with those of some
Effect of temperature on the activity and stability
of the extracellular protease
The optimal temperature of the activity of the extra-
cellular protease was investigated by measuring the
hydrolytic activities at different temperatures
(25–70 ꢀC). As shown in Figure S5, the extracellular

6
L. DONG ET AL.
proteases exhibited the highest hydrolytic activity at
45 ꢀC. When the temperature was below 40 ꢀC or over
55 ꢀC, the hydrolytic activities of the proteases
decreased rapidly. The stability of the extracellular pro-
teases was evaluated by testing the residual hydrolytic
activities of the extracellular proteases after incubation
at various temperatures for 2 h. The extracellular pro-
tease was quite stable below 45 ꢀC. When the tem-
perature was over 45 ꢀC the activities of the proteases
reduced sharply, but still exhibited 62.00 3.75% of
the maximal activity at 70 ꢀC (Figure S6), which indi-
cated that the extracellular protease exhibited relative
good tolerated to temperature.
phenylethanol. The e.e.p was the highest (95.4%) at
20 ꢀC (Figure S8). The e.e.p decreased when the tem-
perature was above 20 ꢀC. Thus, 20 ꢀC was determined
to be the optimal temperature for the kinetic reso-
lution of ( )-1-phenylethyl acetate, with the e.e.p being
95.4% and the conversion being 27.9%, respectively.
Effect of enzyme concentration on the kinetic reso-
lution of ( )-1-phenylethyl acetate
Various concentrations of immobilized enzyme ranging
from 120 to 480 mg/mL were added at pH 6.5 and
20 ꢀC to study the effect of enzyme concentration on
the resolution of ( )-1-phenylethyl acetate. As shown
in Figure S9, 360 mg/mL was characterized to be the
optimal enzyme concentration for the resolution of
( )-1-phenylethyl acetate, with the e.e.p being 96.0%
and the conversion being 25.1%, respectively. When
the enzyme concentrations were lower than 360 mg/
mL, the conversions were much lower, and when the
enzyme concentrations were above 360 mg/mL, the e.
e.p progressively decreased.
The activities of extracellular proteases
An amount of 21.1 mg extracellular protease was
adsorbed by 1 g kieselguhr. The enzymatic activity of
the extracellular proteases from the fermentation
broth cultivated for 24 h, was 104.4 U/mL. The hydro-
lytic activity of immobilized extracellular protease was
1469.5 U/g.
Process optimization for preparation of (R)-1-
phenylethanol through the kinetic resolution of
( )-1-phenylethyl acetate by immobilized
extracellular proteases
Effect of substrate concentration on the kinetic
resolution of ( )-1-phenylethyl acetate
The concentration of substrate could also greatly
affect the enzymatic kinetic resolution of racemic 1-
phenylethyl acetate. The effect of substrate concentra-
tion on the kinetic resolution of ( )-1-phenylethyl
acetate by immobilized extracellular proteases of
Bacillus sp. DL-2 was shown in Figure S10. With the
increase of substrate concentration, the conversion of
( )-1-phenylethyl acetate decreased gradually, while
the e.e.p remains basically constant. The optimal reso-
lution results were obtained when the substrate con-
centration was 5 mM, with the e.e.p and the
conversion being 96.0 and 22.4%, respectively.
Effect of pH on the kinetic resolution of ( )-1-phe-
nylethyl acetate
pH is a key factor which can the stereo-selectivity,
activity, and stability of biocatalysts. Standard reac-
tions were performed at different pH ranging from 5.0
to 10.0 (Table S1) to determine the effect of pH on
the kinetic resolution of ( )-1-phenylethyl acetate for
the preparation of (R)-1-phenylethanol. As shown in
Figure S7, with the increase of pH, the conversion
increased, but the e.e.p increased first and then
decreased. When the pH was set at 6.5 (phosphate
buffer), the e.e.p reached the maximal value (95.9%).
Therefore, the optimal pH for the enzymatic resolution
of ( )-1-phenylethyl acetate using immobilized pro-
teases was confirmed to be 6.5, with the conversion
being 23.4%.
Effect of reaction time on the kinetic resolution of
( )-1-phenylethyl acetate
The standard reactions were carried out for different
times (1–12h) to investigate the optimal reaction time
for the kinetic resolution of ( )-1-phenylethyl acetate
(Figure S11). The results showed that the highest e.e.p
reached 95.9%, with the conversion being 18.1%, when
the enzymatic reaction was carried out at 2 h. With the
extension of reaction time, the e.e.p decreased. Thus,
2 h was determined to be the optimal reaction time for
the enzymatic resolution of ( )-1-phenylethyl acetate.
Effect of temperature on the kinetic resolution of
( )-1-phenylethyl acetate
Enzymatic reactions were set at different temperatures
ranging from 20 to 50 ꢀC at pH 6.5 to evaluate the
effect of temperature on the kinetic resolution of
( )-1-phenylethyl acetate for the preparation of (R)-1-

BIOCATALYSIS AND BIOTRANSFORMATION
7
Table 1. Effect of metallic ions and surfactants on the kinetic resolution of ( )-1-phenylethyl acetate catalyzed by immobilized
extracellular proteases of Bacillus sp. DL-2 for the preparation of (R)-1-phenylethanol.
Metallic ions (2 mM)
e.e.p (%)
C (%)
Metallic ions (2 mM)
e.e.p (%)
C (%)
Control
Liþ
95.8 0.1
95.7 0.1
95.8 0.1
96.0 0.1
95.6 0.2
95.9 0.2
96.0 0.1
22.3 0.7
26.5 0.5
22.9 0.8
23.5 0.6
29.7 0.3
25.2 0.7
20.2 0.3
Cu2þ
94.9 0.3
95.7 0.2
96.8 0.1
96.0 0.1
95.7 0.2
96.1 0.1
96.0 0.1
9.6 1.8
16.5 1.2
17.4 1.3
14.9 0.7
18.2 0.7
19.9 0.4
19.1 1.1
Mg2þ
Mn2þ
Ni2þ
Naþ
Kþ
Ba2þ
Ca2þ
Co2þ
Zn2þ
Al3þ
Fe3þ
Surfactants 0.05 % (w/v)
e.e.p (%)
C (%)
Surfactants 0.05 %(w/v)
e.e.p (%)
C (%)
Control
95.8 0.2
95.6 0.3
95.7 0.2
94.9 0.1
95.8 0.2
26.5 0.4
21.6 0.5
21.9 0.4
26.6 0.5
22.1 0.9
STPP
CTAB
SDBS
CMC-Na
–
96.1 0.2
95.2 0.2
67.8 0.1
95.7 0.2
–
18.6 0.4
34.2 0.4
1.1 0.5
22.9 0.6
–
Tween-20
Tween-80
TritonX-100
Span 65
with the e.e.p, conversion and yield being 97, 18, and
41%, respectively (Figure 1).
Effect of metallic ions and surfactants on the kin-
etic resolution of ( )-1-phenylethyl acetate
Different metallic ions and surfactants, at final concen-
trations of 2 mM and 0.05% (w/v), respectively, were
added into standard reaction systems to determine
their effect on the kinetic resolution of ( )-1-phenyl-
ethyl acetate. As shown in Table 1, the presence of
Kþ, Ca2þ, Co2þ, Mn2þ, Al3þ, Fe3þ, and surfactant
sodium tripolyphosphate (STPP) exhibited slightly
positive effect on the e.e.p of the enzymatic resolu-
tions. Especially, the existence of Mn2þ increased the
e.e.p to 97%. While Cu2þ and surfactant Sodium dode-
cylbenzene sulphonate (SDBS) showed significantly
inhibitory effect on the kinetic resolution of ( )-1-phe-
nylethyl acetate.
Process optimization for the preparation of (S)-1-
phenylethyl acetate through the kinetic resolution
of ( )-1-phenylethyl acetate by immobilized
extracellular proteases
Enzyme concentration and substrate concentration
can distinctly affect the enantioselectivity and conver-
sion rate in enzymatic kinetic resolution. The immobi-
lized extracellular proteases of Bacillus sp. DL-2
hydrolysed ( )-1-phenylethyl acetate and preferentially
generated (R)-1-phenylethanol with high optical purity.
With the increase of enzyme concentrations or
decrease of substrate concentrations, (R)-1-phenylethyl
acetate was completely hydrolysed and (S)-1-phenyl-
ethyl acetate began to be hydrolysed. Therefore, in
order to produce (S)-1-phenylethyl acetate with high
optical purity, enhancing the concentration of immobi-
lized enzyme or reducing the concentration of ( )-1-
phenylethyl acetate in hydrolytic reaction system
might be helpful. Finally, (S)-1-phenylethyl acetate
with high enantiomeric excess was obtained by opti-
mizing the catalytic reaction conditions as follows.
Effect of organic solutions on the kinetic resolution
of ( )-1-phenylethyl acetate
Nineteen organic solvents were added into standard
reaction systems to study the effect of organic sol-
vents on the kinetic resolution of ( )-1-phenylethyl
acetate catalysed by immobilized extracellular pro-
teases (Table 2). Most organic solvents used in this
reaction systems exhibited negative effect on the e.e.
of (R)-1-phenylethanol and conversion rate. While,
compared with control experiment, the presence of
N,N-dimethylformamide (DMF) and n-propanol slightly
enhanced the e.e.p.
To conclude, after process optimization of the kin-
etic resolution of ( )-1-phenylethyl acetate using
immobilized extracellular proteases, the optimal reso-
lution reaction condition for generating (R)-1-phenyle-
thanol was found to be 5-mM ( )-1-phenylethyl
acetate, 360 mg/mL immobilized extracellular pro-
teases, pH 6.5 (PB), 2-mM Mn2þ, and 20 ꢀC for 2 h,
Effect of pH on the kinetic resolution of ( )-1-phe-
nylethyl acetate
Under the standard hydrolytic reaction system of pro-
ducing (S)-1-phenylethyl acetate with different pH val-
ues (from 5.0 to 10.0), the highest e.e.s of (S)-1-
phenylethyl acetate was found to be 98.0% at pH 7.5
(PB), with a conversion rate of being 62.1% (Figure
S12). The e.e.s quickly decreased when the reaction pH
was below 7.5 or above 8.5, indicating that pH could
greatly affect the optical purity of (S)-1-phenyl-
ethyl acetate.

8
L. DONG ET AL.
Table 2. Effect of organic solvents on the kinetic resolution of ( )-1-phenylethyl acetate catalyzed by immobilized extracellular
proteases of Bacillus sp. DL-2 for the preparation of (R)-1-phenylethanol.
Organic solvents 5% (v/v)
e.e.p (%)
C (%)
Organic solvents 5% (v/v)
e.e.p (%)
C (%)
Control
Acetone
95.4 0.3
95.8 0.1
95.2 0.3
91.8 0.2
95.5 0.2
66.4 0.3
94.3 0.4
75.0 0.3
95.4 0.2
95.7 0.2
17.9 0.5
15.8 0.9
12.9 1.3
3.5 1.2
12.7 0.3
0.7 0.4
16.4 0.8
1.1 0.4
7.3 0.6
14.4 0.4
1,4-Dioxane
DMF
DMSO
Tetrahydrofuran
n-Propanol
ttert-Butanol
sec-Butanol
n-Butanol
Isopropanol
Isobutanol
94.5 0.3
95.9 0.1
95.3 0.5
94.9 0.4
96.0 0.1
94.0 0.2
94.5 0.5
93.4 0.3
95.4 0.3
93.2 0.2
19.3 0.6
22.7 0.4
30.6 0.7
8.7 1.2
11.3 0.6
18.3 0.4
5.4 0.7
4.7 0.5
14.9 0.8
3.7 0.5
Acetonitrile
Dichloromethane
n-Hexane
Cyclohexanone
Cyclohexane
Chloroform
Ethanol
Methanol
Figure 1. (R)-1-phenylethanol and (S)-1-phenylethyl acetate prepared through asymmetric hydrolysis of ( )-1-phenylethyl acetate by
immobilized extracellular proteases after process optimization (1: standards of ( )-1-phenylethyl acetate and ( )-1-phenylethanol; 2: (R)-
1-phenylethanol prepared after enzymatic kinetic resolution; 3: (S)-1-phenylethyl acetate prepared after enzymatic kinetic resolution).

BIOCATALYSIS AND BIOTRANSFORMATION
9
Table 3. Effect of metallic ions and surfactants on the kinetic resolution of ( )-1-phenylethyl acetate catalyzed by immobilized
extracellular proteases of Bacillus sp. DL-2 for the preparation of (S)-1-phenylethyl acetate.
Metallic ions (2 mM)
e.e.s (%)
C (%)
Metallic ions (2 mM)
e.e.s (%)
C (%)
Control
Liþ
98.1 0.1
97.9 0.2
96.8 0.3
98.0 0.3
98.4 0.1
99.3 0.1
93.7 0.2
63.5 0.3
56.5 0.5
60.7 0.7
56.8 0.5
59.1 0.6
59.9 0.3
54.8 1.1
Cu2þ
27.5 1.2
99.1 0.1
95.6 0.4
76.8 0.3
98.2 0.3
98.8 0.2
98.8 0.1
24.4 3.7
60.7 0.5
57.1 0.6
47.8 1.1
55.7 0.8
58.7 0.6
61.1 0.4
Mg2þ
Mn2þ
Ni2þ
Naþ
Kþ
Ba2þ
Ca2þ
Co2þ
Zn2þ
Al3þ
Fe3þ
Surfactants 0.05% (w/v)
e.e.s (%)
C (%)
Surfactants 0.05% (w/v)
e.e.s (%)
C (%)
Control
99.1 0.1
95.8 0.1
98.1 0.4
98.8 0.2
96.9 0.5
62.7 0.3
56.5 0.5
55.6 0.6
62.8 0.3
61.0 0.7
STPP
CTAB
SDBS
CMC-Na
–
98.9 0.1
97.3 0.5
3.7 0.3
98.7 0.2
–
60.8 1.1
58.0 2.5
4.3 1.1
61.2 0.8
–
Tween-20
Tween-80
TritonX-100
Span 65
Table 4. Effect of organic solvents on the kinetic resolution of ( )-1-phenylethyl acetate catalyzed by immobilized extracellular
proteases of Bacillus sp. DL-2 for the preparation of (S)-1-phenylethyl acetate.
Organic solvents 5% (v/v)
e.e.s (%)
C (%)
Organic solvents 5% (v/v)
e.e.s (%)
C (%)
control
Acetone
99.2 0.2
96.4 0.5
16.0 0.5
4.6 0.3
78.3 0.2
1.0 0.1
74.4 0.5
4.3 0.4
89.6 0.3
98.5 0.2
64.3 0.3
54.4 0.6
14.7 1.3
4.8 1.9
47.2 0.9
1.4 0.3
45.8 2.5
4.5 2.5
50.8 1.2
56.9 0.6
1,4-Dioxane
DMF
DMSO
Tetrahydrofuran
n-Propanol
tert-Butanol
sec-Butanol
n-Butanol
Isopropanol
Isobutanol
74.9 0.2
96.5 0.7
99.0 0.1
47.8 0.4
88.9 0.2
95.7 0.3
29.6 0.4
5.9 0.6
45.9 1.8
54.6 2.9
61.1 0.6
34.0 2.1
50.4 0.9
54.4 0.8
23.9 1.9
6.1 1.0
Acetonitrile
Dichloromethane
n-Hexane
Cyclohexanone
Cyclohexane
Chloroform
Ethanol
91.2 0.3
29.8 0.5
52.5 1.5
24.0 1.7
Methanol
phenylethyl acetate. As shown in Figure S14, 440 mg/
mL was the optimal enzyme concentration for the kin-
etic resolution of ( )-1-phenylethyl acetate by immobi-
lized extracellular proteases, with the e.e.s being 98.3%
and the conversion rate being 64.0%.
Effect of temperature on the kinetic resolution of
( )-1-phenylethyl acetate
The kinetic resolution of ( )-1-phenylethyl acetate cat-
alysed by immobilized extracellular proteases was ana-
lysed after incubating enzymatic reactions at
temperatures ranging from 20 to 50 ꢀC. The e.e. and
conversion rate of (S)-1-phenylethyl acetate generated
from enzymatic reactions under different temperatures
are shown in Figure S13. The optimal temperature to
obtain the highest e.e.s was found to be 35 ꢀC, with
the e.e. and conversion rate of (S)-1-phenylethyl acet-
ate being 98.8 and 64.3%, respectively. The e.e.s
quickly decreased when the reaction temperature was
below 35 ꢀC or above 40 ꢀC, indicating that reaction
temperature could also greatly affect the optical purity
of (S)-1-phenylethyl acetate.
Effect of substrate concentration on the kinetic
resolution of ( )-1-phenylethyl acetate
Under the standard hydrolytic reaction system, differ-
ent concentrations of substrate were tested to deter-
mine the ideal concentration of ( )-1-phenylethyl
acetate for the kinetic resolution reaction. The highest
e.e.s (98.0%) of (S)-1-phenylethyl acetate was obtained
when substrate ( )-1-phenylethyl acetate was added
at a concentration of 2.5 mM (Figure S15). With the
increasing of ( )-1-phenylethyl acetate concentration,
the e.e.s and the conversion of substrate gradually
decreased. Thus, 2.5-mM was the optimal substrate
concentration for producing (S)-1-phenylethyl acetate.
Effect of enzyme concentration on the kinetic reso-
lution of ( )-1-phenylethyl acetate
Under the optimal reaction temperature (35 ꢀC) and
the optimal pH (7.5), different concentrations of immo-
bilized extracellular proteases were added to the
enzymatic reactions to determine the effect of enzyme
concentrations on the kinetic resolution of ( )-1-
Effect of reaction time on the kinetic resolution of
( )-1-phenylethyl acetate
Under the optimized conditions (440 mg/mL enzyme
concentration, 2.5-mM substrate concentration, pH 7.5,

10
L. DONG ET AL.
and 35 ꢀC), different reaction times (from 1 to 12 h)
were set to investigate the optimal reaction time for
the resolution of ( )-1-phenylethyl acetate (Figure
S16). The optimal reaction time was found to be 10 h,
with the highest e.e.s being 98.8% and the conversion
rate being 61.0%, respectively. With the elongation of
reaction time, the e.e.s increased gradually. However,
when reaction time was over 10 h, the e.e.s was
unchanged basically but conversion rate was
unsatisfactory.
repeated usage, probably because the proteases were
immobilized on kieselguhr through simple physical
adsorption and were easily desorbed from the carrier
without further cross-linking. The conversion rate and
the e.e. of the kinetic resolution of ( )-1-phenylethyl
acetate during repeated usage were also analysed. As
shown in Figure S18, both the C and the e.e.
decreased after repeated usage in the kinetic reso-
lution, especially during the process for the generation
of (R)-1-phenylethanol. Similarly, the decreases of both
the conversion and the e.e. were due to the reasons
of the leakage of enzyme from the support as well as
possible catalyst deactivation. Some previous literature
also reported some problems encountered about the
reusability of immobilized enzyme adsorbed on celite
(Wehtje et al. 1990; Triantafyllou et al. 1995; Ertan
et al. 2006; Ozcan and Sagiroglu 2009). Hence, cross-
linking after adsorption of enzyme on the support
might provide great help in keeping the stability and
reusability of immobilized enzyme, and will be investi-
gated in our following work.
Effect of metallic ions and surfactants on the kin-
etic resolution of ( )-1-phenylethyl acetate
Under the optimal reaction conditions, thirteen metal-
lic ions (2 mM) and eight different surfactants (0.05%)
were added to the enzymatic reactions to investigate
the effect of metallic ions and surfactants on the kin-
etic resolution of ( )-1-phenylethyl acetate. Reactions
without the addition of metal ions or surfactants were
carried out as a control. As shown in Table 3, the pres-
ence of Ca2þ (e.e.s >99%), Mg2þ (e.e.s >99%), Al3þ
,
and Fe3þ promoted the stereo-selectivity. A majority
of the tested metallic ions and surfactants, especially
Cu2þ, Ni2þ and SDBS, decreased the e.e.s.
Comparison of immobilized extracellular
proteases from Bacillus sp. DL-2 with other
proteases/esterases/lipases in the resolution of
( )-1-phenylethyl acetate through
Effect of organic solutions on the kinetic resolution
of ( )-1-phenylethyl acetate
asymmetric hydrolysis
Previously, there were some reports about the kinetic
resolution of ( )-1-phenylethyl acetate through asym-
metric hydrolysis using esterases/lipases (Ou et al.
2008; Fan et al. 2011; Liang et al. 2016; Huang et al.
2017; Melais et al. 2016). However, to the best of our
knowledge, there was no report about the kinetic
resolution of ( )-1-phenylethyl acetate through asym-
metric hydrolysis using proteases, although a great
variety of proteases have been demonstrated to be
useful industrial enzymes and biocatalysts.
In the cases of asymmetric hydrolysis of ( )-1-phe-
nylethyl acetate by esterases/lipases, generally (S)-1-
phenylethyl acetate and (R)-1-phenylethanol were pro-
duced after enzymatic kinetic resolution. In some
cases, (R)-1-phenylethanol was generated with the e.e.
being 99%. But in almost all cases, (S)-1-phenylethyl
acetate was generated with the e.e. being less than
97%, which was not too satisfying. In only one case,
the biocatalyst commercial lipase Novozym 435 exhib-
ited opposite enantio-preference and gave (S)-1-phe-
The effect of organic solvents on the kinetic resolution
of racemic 1-phenylethyl acetate by immobilized
extracellular proteases was shown in Table 4. Almost
all the tested organic solvents decreased the e.e.s. As a
consequence, no organic solvent was added to the
enzymatic reactions for the kinetic resolution of ( )-1-
phenylethyl acetate by immobilized extracellu-
lar proteases.
Ultimately, the optimal reaction conditions for the
kinetic resolution of ( )-1-phenylethyl acetate for the
preparation of (S)-1-phenylethyl acetate by immobi-
lized extracellular proteases were found to be 2.5-mM
( )-1-phenylethyl acetate, 440 mg/mL immobilized
extracellular proteases, pH 7.5 (PB), 2-mM Ca2þ/Mg2þ
,
and 35 ꢀC for 10 h. Under the optimal resolution condi-
tions, (S)-1-phenylethyl acetate was obtained with the
e.e.s, conversion, yield being 99, 64, and 71%, respect-
ively (Figure 1).
Operational stability of immobilized proteases
nylethanol
after
asymmetric
hydrolysis
(Fan
The operation stability of the immobilized extracellular
proteases was shown in Figure S17. The hydrolytic
activity of the immobilized proteases declined after
et al. 2011).
In our work, the extracellular proteases from deep-
sea Bacillus sp. DL-2 were developed into useful

BIOCATALYSIS AND BIOTRANSFORMATION
11
biocatalyst in the asymmetric hydrolysis of ( )-1-phe-
nylethyl acetate. After process optimization, (S)-1-phe-
nylethyl acetate was prepared with the e.e. being as
high as 99%, which was the highest report so far. Also
after process optimization, (R)-1-phenylethanol was
generated, with the e.e. being 97% (Table 5).
Additionally, neither the fermentation broth
from Bacillus sp. DL-2 nor the immobilsed extracellular
proteases exhibited the activity of lipase/esterase to
hydrolyse some other tested esters such as racemic
methyl lactate (an linear aliphatic ester), racemic
ethyl 2-methyl butyrate (an linear aliphatic ester) and
racemic methyl mandelate (an ester contain-
ing phenyl).
Conclusions
One microorganism Bacillus sp. DL-2 was isolated from
the deep sea of the Western Pacific. The immobilized
extracellular proteases of Bacillus sp. DL-2 was further
developed into a green biocatalyst for the kinetic reso-
lution of ( )-1-phenylethyl acetate. After process opti-
mization of the asymmetric hydrolysis of ( )-1-
phenylethyl acetate using immobilized extracellular
proteases, (R)-1-phenylethanol was generated with an
optical purity of 97% and a yield of 41%, and (S)-1-
phenylethyl acetate was prepared with an optical pur-
ity of more than 99% and a yield of 71% under differ-
ent enzymatic reaction conditions. Generally, chiral
alcohol and esters are obtained through kinetic reso-
lution catalysed by lipases or esterases. Herein, we
developed the extracellular protease of Bacillus sp. DL-
2 isolated from the deep sea into useful biocatalysts
and generated (R)-1-phenylethanol and (S)-1-phenyl-
ethyl acetate with high optical purities by asymmetric
hydrolysis of the ester bonds of ( )-1-phenylethyl acet-
ate. Our report is the first report about the resolution
of ( )-1-phenylethyl acetate using proteases and the
enantio-preference of the proteases was found to be
the same as those of most other esterases/lipases.
Also notably, the optical purity of (S)-1-phenylethyl
acetate generated through kinetic resolution using the
proteases of Bacillus sp. DL-2 (e.e. > 99%) was the
highest report so far, possibly because previous
reported esterases/lipases could not well discriminate
the two enantiomers of ( )-1-phenylethyl acetate.
Novel microbial extracellular proteases are new contri-
butions to the biocatalysts library for the preparation
of valuable chiral alcohols and chiral esters in the
chemical and pharmaceutical industries.

12
L. DONG ET AL.
Huang XL, Catignani GL, Swaisgood HE. 1997. Comparison of
the properties of trypsin immobilized on 2 Celite deriva-
tives. J Biotechnol. 53:21–27.
Huang JL, Zhang Y, Sun AJ, Hu YF. 2017. Enantioselective
production of (R)-1-phenylethanol by a novel marine
microbial carboxylesterase. J Trop Oceanogr. 36:55–60.
Husain Q. 2018. Nanocarriers immobilized proteases and
Acknowledgements
We are grateful for the Research Vessel KEXUE of the Chinese
Academy of Sciences for collecting deep-sea samples.
Disclosure statement
their industrial applications: an overview.
Nanotechnol. 18:486–499.
J Nanosci
The authors declare that they have no conflict of interest.
€
Jakubke HD, Konnecke A. 1987. [18] Peptide synthesis using
immobilized proteases. Method Enzymol. 163:178–188.
Kennedy J, Marchesi JR, Dobson AD. 2008. Marine metage-
nomics: strategies for the discovery of novel enzymes
with biotechnological applications from marine environ-
ments. Microb Cell Fact. 7:1–8.
Kroutil W, Mang H, Edegger K, Faber K. 2004. Recent advan-
ces in the biocatalytic reduction of ketones and oxidation
of sec-alcohols. Curr Opin Chem Biol. 8:120–126.
Li HY, Li ZY, Ruan GH, Yu YK, Liu XM. 2016. Asymmetric
reduction of acetophenone into R-(þ)-1-phenylethanol by
endophytic fungus Neofusicoccum parvum BYEF07 isolated
from Illicium verum. Biochem Bioph Res Co. 473:874–878.
Liang J, Zhang Y, Sun A, Deng D, Hu Y. 2016.
Enantioselective resolution of ( )-1-phenylethanol and
( )-1-phenylethyl acetate by a novel esterase from Bacillus
sp. SCSIO 15121. Appl Biochem Biotechnol. 178:558–575.
Mansour EH, Dawoud FM. 2003. Immobilization of invertase
on Celite and on polyacrylamide by an adsorption proced-
ure. J Sci Food Agric. 83:446–450.
Funding
This work was financially supported by the Scientific and
Technological Project of Ocean and Fishery from Guangdong
Province [A201701C12], the Senior User Project of RV KEXUE
[KEXUE2018G05], the Priority Research Program of the Chinese
Academy of Sciences [XDA11030404] and Natural Science
Foundation of Guangdong Province [2018A030313151].
References
Anderson WA, Bay P, Legge RL, Moo-Young M. 1990.
Adsorption of Streptococcus faecalis on diatomite carriers
for use in biotransformations. J Chem Technol Biotechnol.
47:93–100.
Burg B. 2003. Extremophiles as a source for novel enzymes.
Cur Opin Microbiol. 6:213–218.
Cabana H, Alexandre C, Agathos SN, Jones JP. 2009.
Immobilization of laccase from the white rot fungus
Coriolopsis polyzona and use of the immobilized biocata-
lyst for the continuous elimination of endocrine disrupt-
ing chemicals. Bioresource Technol. 100:3447–3458.
Chen CS, Fujimoto Y, Girdaukas G, Sih CJ. 1982. Quantitative
analyses of biochemical kinetic resolutions of enantiomers.
J Am Chem Soc. 104:7294–7299.
Melais N, Aribi-Zouioueche L, Riant O. 2016. The effect of
the migrating group structure on enantioselectivity in lip-
ase-catalyzed kinetic resolution of 1-phenylethanol. CR
Chim. 19:971–977.
Olivier R, Fascetti E, Angelini L, Degen L. 1979. Enzymatic
conversion of N carbamoyl-d-amino acids to d-amino
acids. Enzyme MicrobTech. 1:201–204.
Cho SH, Shim J, Yun SH, Moon SH. 2008. Enzyme-catalyzed
conversion of phenol by using immobilized horseradish
peroxidase (HRP) in a membrane less electrochemical
reactor. Appl Catal A General. 337:66–72.
Contesini FJ, Ibarguren C, Grosso CRF, Carvalho PDO, Sato
HH. 2012. Immobilization of glucosyltransferase from
Erwinia sp. using two different techniques. J Biotechnol.
158:137–143.
Ertan F, Yagar H, Balkan B. 2006. Some properties of free
and immobilized a-amylase from penicillium griseofulvum
by solid state fermentation. Prep Biochem Biotech. 36:
81–91.
Fan Y, Xie Z, Zhang H, Qian J. 2011. Kinetic resolution of
both 1-phenylethanol enantiomers produced by hydroly-
sis of 1-phenylethyl acetate with Candida antarctica lipase
B in different solvent systems. Kinet Catal. 52:686–690.
Fǜlling G, Sih CJ. 1987. Enzymatic second-order asymmetric
hydrolysis of ketorolac esters: in situ racemization. J Am
Chem Soc. 109:2845–2846.
Ou L, Xu Y, Ludwig D, Pan J, Xu JH. 2008. Chemoenzymatic
deracemization of chiral secondary alcohols: process opti-
mization for production of (R)-1-indanol and (R)-1-phenyl-
ethanol. Org Process Res Dev. 12:192–195.
Ozcan HM, Sagiroglu A. 2009. Production of ricinoleic acid
from castor oil by immobilised lipases. Prep Biochem
Biotechnol. 39:170–182.
Rao MB, Tanksale AM, Ghatge MS, Deshpande VV. 1998.
Molecular and biotechnological aspects of microbial pro-
teases. Microbiol Mol Biol Rev. 62:597–635.
Reetz MT. 2002. Lipases as practical biocatalysts. Curr Opin
Chem Biol. 6:145–150.
Sabotic J, Kos J. 2012. Microbial and fungal protease inhibi-
tors-current and potential applications. Appl Microbiol
Biotechnol. 93:1351–1375.
Sagiroglu A, Telefoncu A. 2004. Immobilization of lipases on
different carriers and their use in synthesis of pentyl iso-
valerates. Prep Biochem Biotechnol. 34:169–178.
Satar R, Husain Q. 2009. Adsorption of peroxidase on Celite
545 directly from ammonium sulfate fractionated white
radish (Raphanus sativus) proteins. Biotechnol J. 4:
408–416.
Gupta R, Beg QK, Lorenz P. 2002. Bacterial alkaline proteases:
molecular approaches and industrial applications. Appl
Microbiol Biot. 59:15–32.
Habulin M, Knez Z. 2009. Optimization of (R,S)-1-phenyletha-
nol kinetic resolution over Candida antarctica lipase B in
ionic liquids. J Mol Catal B-Enzym. 58:24–28.
Sharma S, Kanwar SS, Dogra P, Chauhan GS. 2015. Gallic
acid-based alkyl esters synthesis in a water-free system by
celite-bound lipase of Bacillus licheniformis SCD11501.
Biotechnol Progress. 31:715–723.

BIOCATALYSIS AND BIOTRANSFORMATION
13
Souza PMD, Bittencourt MLDA, Caprara CC, Freitas MD,
Almeida RPCD, Silveira D, Fonseca YM, Ferreira Filho EX,
Bacillus cereus MTCC 8372. Acta Microbiol Imm H. 56:
229–242.
Wehtje E, Adlercreutz P, Mattiasson B. 1990. Formation of C-
C bonds by mandelonitrile lyase in organic solvents.
Biotechnol Bioeng. 36:39–46.
Wu HY, Xu JH, Tsang SF. 2004. Efficient resolution of a chiral
alcohol (R,S)-HMPC by enzymatic transesterification with
vinyl acetate using surfactant-modified lipase. Enzyme
Microb Tech. 34:523–528.
Yamaguchi S, Komeda H, Asano Y. 2007. New
enzymatic method of chiral amino acid synthesis by
dynamic kinetic resolution of amino acid amides: use of
stereoselective amino acid amidases in the presence of
a-amino-e-caprolactam racemase. Appl Environ Microb.
73:5370–5373.
~
Pessoa Junior A, Magalhaes PO. 2015. A biotechnology
perspective of fungal proteases. Braz J Microbiol. 46:
337–346.
Tang XL, Lu XF, Wu ZM, Zheng RC, Zheng YG.
2018. Biocatalytic production of (S)-2-aminobutanamide
by
a novel daminopeptidase from Brucella sp. with
high activity and enantioselectivity. J Biotechnol. 266:
20–26.
Triantafyllou AO, Wehtje E, Adlercreutz P, Mattiasson B.
1995. Effects of sorbitol addition on the action of free and
immobilized hydrolytic enzymes in organic media.
Biotechnol Bioeng. 45:406–414.
Verma ML, Azmi W, Kanwar SS. 2009. Synthesis of ethyl
acetate
employing
celite-immobilized
lipase
of