Enantioselective resolution of (±)-1-phenylethyl acetate using the immobilized extracellular proteases from deep-sea Bacillus sp. DL-1

Article abstract of DOI:10.1080/10242422.2021.1897579

Bacillus sp. DL-1 was isolated from the deep sea of the Western Pacific Ocean and behaved very good resistance to NaCl. The extracellular proteases of Bacillus sp. DL-1 were found to exhibit excellent enantioselectivity for the kinetic resolution of (±)-1-phenylethyl acetate. To improve the stability of the enzyme, the immobilized extracellular proteases were preparated by using 60 g kieselguhr and 1-L crude fermentation broth containing extracellular proteases of Bacillus sp. DL-1, shaking at 25 °C, 200 r/min for 10 h. Every gram of kieselguhr adsorbed 25.7 mg extracellular protease and the enzymatic activity recovery was 79.86%. The immobilized proteases preserved about 35.8% of its activity after 6 repeated uses, which were also used as biocatalyst to asymmetrically hydrolyse (±)-1-phenylethyl acetate for the preparation of (R)-1-phenylethanol and (S)-1-phenylethyl acetate with high optical purities. The effects of pH, temperature, enzyme concentration, substrate concentration, reaction time and additives (metal ions/surfactants) on the resolution were investigated by single factor experiments. Under the optimal reaction conditions (10 mM (±)-1-phenylethyl acetate, 40 mg/mL immobilized extracellular proteases, pH 7.5 (Tris-HCl), 5% (v/v) methanol and 45 °C for 2 h), (R)-1-phenylethanol was generated with the e.e._p being > 97%, and the yield being 53%, respectively. Analogously, under the optimal reaction conditions (10-mM (±)-1-phenylethyl acetate, 360 mg/mL immobilized extracellular proteases, pH 6.0 (PB), 5% (v/v) DMSO and 35 °C for 1.5 h), (S)-1-phenylethyl acetate was generated with the e.e._s being over 99% and the yield being 79%, respectively. Compared with the extracellular proteases from Bacillus sp. DL-2, the immobilized extracellular proteases from Bacillus sp. DL-1 exhibited higher hydrolytic activity and could asymmetrically hydrolyse (±)-1-phenylethyl acetate by using higher substrate concentrations, shorter reaction times to obtain higher yields. Notably, the extracellular proteases of Bacillus sp. DL-1 were demonstrated to behave the same enantio-preference as those of most other reported esterases/lipases. Proteases from deep-sea Bacillus sp. DL-1 are promising biocatalysts for the synthesis of valuable chiral chemicals.

Full text of DOI:10.1080/10242422.2021.1897579

Biocatalysis and Biotransformation
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Enantioselective resolution of (±)-1-phenylethyl
acetate using the immobilized extracellular
proteases from deep-sea Bacillus sp. DL-1
Lu Dong, Shujuan Qi, Jianwei Jia, Yun Zhang & Yunfeng Hu
To cite this article: Lu Dong, Shujuan Qi, Jianwei Jia, Yun Zhang & Yunfeng Hu (2021):
Enantioselective resolution of (±)-1-phenylethyl acetate using the immobilized extracellular
proteases from deep-sea Bacillus sp. DL-1, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2021.1897579
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2021.1897579
RESEARCH ARTICLE
Enantioselective resolution of (± ±)-)ꢀpenꢁletpꢁl acetate using tpe
immobilized extracellular ꢀroteases from deeꢀ)sea Bacillus sꢀ. DL)-
a,b
c
d
a,b,e
a,b
Lu Dong , Shujuan Qi , Jianwei Jia , Yun Zhang
and Yunfeng Hu
a
Guangdong Key Laboratory of Marine Materia Medical, South China Sea Institute of Oceanology, Chinese Academy of Sciences, CAS
b
Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangzhou, PR China; Southern Marine Science and Engineering
Guangdong Laboratory (Guangzhou), Guangzhou, PR China; The Affiliated Traditional Chinese Medicine Hospital of Guangzhou
c
d
Medical University, Guangzhou, PR China; International College, Guangzhou University of Chinese Medicine, Guangzhou, PR China;
e
Equipment Public Service Center, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, PR China
ABSTRACT
ARTICLE HISTORY
Bacillus sp. DL-1 was isolated from the deep sea of the Western Pacific Ocean and behaved very
good resistance to NaCl. The extracellular proteases of Bacillus sp. DL-1 were found to exhibit
excellent enantioselectivity for the kinetic resolution of (±)-1-phenylethyl acetate. To improve
the stability of the enzyme, the immobilized extracellular proteases were preparated by using
Received 19 November 2020
Revised 24 February 2021
Accepted 25 February 2021
KEYWORDS
6
0 g kieselguhr and 1-L crude fermentation broth containing extracellular proteases of Bacillus
Deep-sea microorganisms;
extracellular proteases;
immobilization; asymmetric
hydrolysis; (R)-1-
phenylethanol; (S)-1-
phenylethyl acetate
ꢀ
sp. DL-1, shaking at 25 C, 200 r/min for 10 h. Every gram of kieselguhr adsorbed 25.7mg extra-
cellular protease and the enzymatic activity recovery was 79.86%. The immobilized proteases
preserved about 35.8% of its activity after 6 repeated uses, which were also used as biocatalyst
to asymmetrically hydrolyse (±)-1-phenylethyl acetate for the preparation of (R)-1-phenylethanol
and (S)-1-phenylethyl acetate with high optical purities. The effects of pH, temperature, enzyme
concentration, substrate concentration, reaction time and additives (metal ions/surfactants) on
the resolution were investigated by single factor experiments. Under the optimal reaction condi-
tions (10 mM (±)-1-phenylethyl acetate, 40 mg/mL immobilized extracellular proteases, pH 7.5
ꢀ
(
Tris-HCl), 5% (v/v) methanol and 45 C for 2 h), (R)-1-phenylethanol was generated with the e.e.
p
being > 97%, and the yield being 53%, respectively. Analogously, under the optimal reaction
conditions (10-mM (±)-1-phenylethyl acetate, 360mg/mL immobilized extracellular proteases, pH
ꢀ
6
.0 (PB), 5% (v/v) DMSO and 35 C for 1.5 h), (S)-1-phenylethyl acetate was generated with the
e.e. being over 99% and the yield being 79%, respectively. Compared with the extracellular pro-
s
teases from Bacillus sp. DL-2, the immobilized extracellular proteases from Bacillus sp. DL-1
exhibited higher hydrolytic activity and could asymmetrically hydrolyse (±)-1-phenylethyl acetate
by using higher substrate concentrations, shorter reaction times to obtain higher yields. Notably,
the extracellular proteases of Bacillus sp. DL-1 were demonstrated to behave the same enantio-
preference as those of most other reported esterases/lipases. Proteases from deep-sea Bacillus
sp. DL-1 are promising biocatalysts for the synthesis of valuable chiral chemicals.
Introduction
range of 6 to 8 that do not require stoichiometric
cofactors, being highly stereo- and regioselective
Currently, the great demands of enzymes as biocata-
lysts in diverse industries are increasing rapidly, due to
some attracting advantages such as mild working con-
ditions, outstanding selectivity, high efficiency and
benign to the environment (Hudlicky and Reed 2009).
Protease, one important industrial hydrolase, has
received special attention in some industries such as
pharmacy, chemistry and food, and has accounted for
nearly 60% of the total enzyme sale globally (Jemli
et al. 2016). Proteases are robust and stable enzymes,
active at mild conditions, with optimal pH in the
(
Bordusa 2002). These properties of proteases are
important to their use as catalysts in organic synthesis,
which may be better alternatives for chemical cata-
lysts. Protease derived from microorganisms may be
located intracellular, periplasmic, or extracellular (Ray
et al. 2012). Considering availability and costs, extra-
cellular proteases have advantages over intracellular
one. A multitude of microorganism produce extracellu-
lar proteases, and most of the protease producing
candidates belongs to Bacillus species (Banerjee and
CONTACT Yunfeng Hu
yunfeng.hu@scsio.ac.cn
CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, Guangdong Key Laboratory of
Marine Materia Medical, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, PR China
Supplemental data for this article can be accessed here.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
L. DONG ET AL.
Ray 2017). At present, using enzymes as promising
biocatalysts to synthesize optically pure compounds is
an important method. The conventional methods for
purification of enzyme are ultrafiltration, precipitation
and affinity chromatography. However, some of these
methods are complicated, laborious, time-consuming
and expensive. As a consequence, the crude enzymes
with high stereoselectivity behave a better application
prospect in industry.
With the development of the research on enan-
tiomers, it has been recognized that due to the stereo-
selective interaction between drugs and their
receptors, different enantiomers of chiral compounds
have different or even opposite pharmacological and
toxicological activities. Therefore, the synthesis of
enantiomers with optical purities is particularly import-
ant. Biocatalytic reactions have become an integral
part of modern technological processes and many
chiral drug intermediates have been synthesized by
biocatalysis (Zhao et al. 2006; Ghosh et al. 2015;
Pinheiro et al. 2018, 2019). For instance, protease 6SD
was covalently immobilized on multi-walled carbon
nanotubes (MWCNT), and the as-prepared immobili-
synthesis of many chiral compounds in pharmaceut-
ical, fine chemical, and agrochemical industries (Tao
and Xu 2009). (R)-1-phenylethanol is needed as a key
precursor for the generation of pharmaceuticals and is
also used as ophthalmic preservative, cholesterol intes-
tinal adsorption inhibitor, and solvatochromic dye
(Chua and Sarmidi 2004; Patel 2008). Chiral 1-phenyl-
ethyl acetate, 1-phenylethanol ester derivative, is
widely used as additive in cosmetics, foods, and phar-
maceuticals, owing to its mild floral odour
(Gassenmeier et al. 2017; Boraty nꢀ ski et al. 2018).
Immobilization is one of the most useful strategies
to improve various enzyme performance such as activ-
ity, selectivity, specificity, resistance to inhibitors, etc.
(Garcia-Galan et al. 2011). There are several
approaches of enzymatic immobilization, such as cova-
lent attachment, multipoint covalent attachment, mul-
tipoint covalent attachment of an enzyme to a
functionalized support, immobilization of enzymes by
cross-linking, matrix entrapment, encapsulation, immo-
bilization by adsorption, magnetic nanoparticles
hybrids formation, enzyme immobilization via forma-
tion mechanism of hybrid nanoflowers (Qian et al.
2019; Reis et al. 2019; Xu et al. 2019; Lin et al. 2020;
Zhu et al. 2020). The extracellular proteases contains
many other enzymes in microbial fermentation broth,
sometimes with activities against the same substrate,
or with opposite (or just different) properties, such as
different stereoselectivity, which may reduce the
apparent performance, including catalytic activity and
stereoselectivity, of the prepared biocatalyst. However,
even in a best-case scenario, the contaminant proteins
will reduce the volumetric activity of the biocatalyst
by competitive inhibition. In some cases, a fine control
of the support and immobilization conditions may
realize the one-step immobilization and purification.
The choice of solid activated support is significantly
important in the immobilization of enzymes. The car-
riers need to have the following properties: enough
mechanical strength, resistance of chemical attack and
microbial decomposition. In addition, the surface of
carriers should have abundant reactive groups and
hydrophilic chain. A proper design of a support may
permit to shift equilibrium of very weak protein com-
plexes permitting their accumulation on the support,
or improve the stability of an enzyme by several-thou-
sand-fold, or its activity, or their selectivity (dos Santos
et al. 2015a). Pinheiro and his colleagues (Pinheiro
et al. 2019) used chitosan activated by divinyl sul-
phone as a novel support to immobilize Candida ant-
arctica Lipase B (CALB). The result show that the
thermal stability of immobilized enzyme was better
zate P-6SD@NH -MWCNT was applied in the enantio-
2
selective resolution of (R,S)-2-chlorophenylglycine
methyl ester to yield (S)-2-chlorophenylglycine methyl
ester, a key chiral building block of clopidogrel (anti-
aggregatory and antithrombotic drug), with the e.e.
being over 99%, and the conversion being 71% (Weng
et al. 2021). (S)-pindolol, a non-selective b-blocker, act-
ing effectively as antianginal, antihypertensive, and
antiarrhythmic, 200 times more active than (R)-
Pindolol, was synthesized by enzymatic kinetic reso-
lution of rac-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloro-
propane with lipase from Pseudomonas fluorescens to
obtain (2 R)-2-acetoxy-1-(1H-indol-4-yloxy)-3-chloropro-
pane (97% ee) which was subjected to a hydrolysis
reaction catalysed by Candida rugosa leading to (2 R)-
1-(1H-indol-4-yloxy)-3-chloro-2-propanol (97% ee), fol-
lowed by a reaction with isopropylamine, with the e.e.
being 97% (Lima et al. 2017). Lipase from
Pseudomonas fluorescens was immobilized on the
superparamagnetic nanoparticles coupled with APTES
and then activated with glutaraldehyde for the kinetic
resolution of secondary alcohols as rac-indanol, rac-1-
phenylethanol, leading to (S)-indanol with high select-
ꢀ
ivity (e.e. > 99%, E > 200) in 1.75 h at 50 C, (R)-1-phe-
nylethyl acetate with e.e. > 99% and to the remaining
(
S)-phenylethanol with e.e. of 94%, conversion of 49%
ꢀ
and E > 200, after 48 h of reaction at 40 C (Galv ~a o
et al. 2018). Chiral secondary alcohols, represented by
1-phenylethanol, are important intermediates for the

BIOCATALYSIS AND BIOTRANSFORMATION
3
than that obtained when using glutaraldehyde as the
support-activating reagent. Using a heterofunctional
support, such as the heterofunctional glyoxyl-octyl
agarose can overcome the problem that the enzyme
easily released from the octyl-agarose support (phys-
ical absorption), which couples the numerous advan-
tages of the octyl-agarose support to covalent
immobilization and creates the possibility of using the
biocatalyst under any experimental conditions without
the risk of enzyme desorption and leaching (Rueda
et al. 2016). Fernandez-Lopez improved immobilization
and stabilization of lipase from Rhizomucor miehei
carried out by lipases/esterases (Musidlowska-Persson
and Bornscheuer 2003; Cross et al. 2004; Wang et al.
2011; de los R ꢀı os et al. 2012; Merabet-Khelassi et al.
2012; Jia et al. 2013; Xia et al. 2014; Zaïdi et al. 2015;
Cao et al. 2016; Huang et al. 2016; Liang et al. 2016a;
Deng et al. 2018; Gong et al. 2018; Wang et al. 2019),
a few studies utilized proteases for kinetic resolution
(Huang et al. 2018; Dong et al. 2019, 2020). In this
work, one deep-sea microorganism Bacillus sp. DL-1
could produce extracellular proteases, which can
resolve (±)-1-phenylethyl acetate to (R)-1-phenyletha-
nol and (S)-1-phenylethyl acetate. To improve the sta-
bility and enantioselectivity, the extracellular proteases
were immobilized by adsorption on kieselguhr. And
the results indicated that extracellular proteases of
Bacillus sp. DL-1 were effectively immobilized on the
natural diatomite. But due to steric resistance, the
amount of enzymes adsorbed by diatomite is limited.
We will activate the surface of diatomite and then use
it for the immobilization of the extracellular protease
to further improve the activity, rigidity and stability of
the immobilized enzyme. In addition, the prepared
immobilized proteases, further utilized to asymmetric-
ally hydrolyse (±)-1-phenylethyl acetate, showed good
stability, resolution activity and stereoselectivity and
generated (R)-1-phenylethanol and (S)-1-phenylethyl
acetate with high optical purities (Scheme 1).
(RML) on octyl-glyoxyl (OCGLX) agarose beads by
using CaCl , and the final preparation is 2.7 fold more
2
active than the free enzyme and a 50% more active
than the standard OCGLX-RML and more stable than
thestandard glyoxyl (Fernandez-Lopez et al. 2016).
Adsorption using inorganic matrices such as kiesel-
guhr, combined with enzyme by the force of hydro-
gen bonds, van der Waals forces and hydrophobic
bonds, is an attractive option, due to its procedural
simplicity, cost-effective, retention of the enzyme
activities, high efficiency and ease of industrial applica-
tion (Satar and Husain 2009; Contesini 2012; Lin et al.
2019). In the case of adsorption immobilization, exces-
sive enzyme loading may cause intermolecular steric
hindrance, resulting in a decline in enzyme activity. A
long spacer arm will increase the possibilities of multi-
interaction to absorb more proteins, but the selectivity
of the adsorption will decrease (Armis ꢀe n et al. 1999).
In the case of covalent immobilization, a long spacer
arm may reduce steric hindrances for the enzyme-sup-
port reaction, and involve more percentage of the
protein surface in the immobilization, increasing the
number of covalent attachment groups but reducing
the rigidity conferred through multipoint covalent
attachment (dos Santos et al. 2015a). If the arm is
hydrophobic, this may have some negative effects on
enzyme thermal stability. Kieselguhr (celite) is white to
off-white natural sedimentary rock powder, similar to
pumice powder and very light, and has high porosity,
which is desirable/recommended for the immobiliza-
tion of enzyme so that proper amounts of enzymes
can be adsorbed without conformational changes
Materials and methods
Microorganisms and reagents
Bacillus sp. DL-1 was isolated from the deep-sea sedi-
ment samples of the Western Pacific Ocean by the
Research Vessel “KEXUE”. High protein level skimmed
milk powder was purchased from Inner Mongolia Yili
Industrial Group Co., Ltd. (Inner Mongolia, China).
Yeast extract and tryptone were bought from
Guangdong
Huankai
Microbial
Science
and
Technology Co., Ltd. (Guangdong, China). PCR
reagents were obtained from Shanghai Generay
Biotech Co., Ltd. (Shanghai, China). Kieselguhr was
purchased from Tianjin Damao Chemical Reagent Co.,
Ltd. (Tianjin, China). (±)-1-phenylethyl acetate and cor-
responding chiral enantiomers were gained from
Adamas Reagent Co., Ltd. (Shanghai, China). Dodecane
used as an internal standard was obtained from
Aladdin Industrial Corporation (Shanghai, China). Other
reagents were of analytical grade.
(Verma et al. 2009). Enzymes immobilized on celite via
adsorption have been used in many reactions, such as
effluent treatment, ester synthesis, dye decolorization,
and biodiesel production (Prieto et al. 2002; Sagiroglu
2008; Andrade et al. 2013; Saun et al. 2014; Kanmani
et al. 2015; Sharma et al. 2015; Zhou et al. 2017).
Notably, most of the kinetic resolution of
racemic mixtures by asymmetric hydrolysis were

4
L. DONG ET AL.
1)
4
0 mg/mL
O
O
immobilized extracellaluar
proteases of Bacillus sp. DL-1
(R) OH
(S) O
O
+
pH 7.5 (Tris-HCl), 45 °C,
5
% (v/v) methanol, 2 h
1
0 mM
(
R)-1-Phenethyl alcohol
(S)-1-phenylethyl acetate
(
± )-1-phenylethyl acetate
e.e. > 97%
p
Yield = 53%
3
60 mg/mL
2)
O
O
O
immobilized extracellaluar
proteases of Bacillus sp. DL-1
(R) OH
(S) O
+
pH 6.0 (PB), 35 °C,
% (v/v) DMSO, 1.5 h
5
1
0 mM
(
± )-1-phenylethyl acetate
(R)-1-Phenethyl alcohol
(S)-1-phenylethyl acetate
e.e. > 99%
s
Yield = 79%
Scheme 1. Kinetic resolution of (±)-1-phenylethyl acetate using immobilized extracellular proteases from Bacillus sp. DL-1 for the
generation of (R)-1-phenylethanol (1) and (S)-1-phenylethyl acetate (2).
PCR amplification and sequencing of 16S
rDNA gene
bacteria liquid at the wavelength of 600 nm (OD600)
were tested every 2 h during the culture process by
using a Tecan NanoQuant Infinite M200 Pro plate
reader. Furthermore, the growth curve of Bacillus sp.
DL-1 under different concentrations of NaCl (0% ꢂ
The 16S rDNA of Bacillus sp. DL-1 was obtained by
0
PCR with the following primers: 5 -AGAGTTTATC
0
0
0
CTGGCTCAG-3 and 5 -GGTTACCTTGTTACGACTT-3 . The
PCR system included 1 lL of incubating medium as
template directly, 2 lL of both 10 lM forward and
reverse primers, 25 lL of 2ꢁTaq Master Mix containing
1
5%) at four culture time (4 h, 14 h, 24 h, and 36 h)
were also analysed by measuring OD600. Subsequently,
% bacteria liquid was inoculated to milk powder
1
liquid medium (1% high protein level skimmed milk
4
mM MgCl and 0.4 mM dNTP mix and 20 lL aseptic
2
ꢀ
powder) for fermentation cultivation at 37 C and 200
ddH O in 50 lL reaction volume. The PCR program
2
ꢀ
r/min. According to the SB/T 10317-1999 standard, the
enzymic activities of extracellular proteases were
measured at culture times of 14 h, 24 h, 36 h and 48 h.
Meanwhile, the extracellular proteases at the same cul-
ture times were used to hydrolyse (±)-1-phenylethyl
acetate and the reaction products were analysed by
chiral gas chromatograph (GC) to evaluate the enan-
tioselectivity of the kinetic resolution.
comprised a pre-denaturation step at 94 C for 10 min
ꢀ
and 30 cycles of 30 s denaturation at 94 C, 30 s
ꢀ
ꢀ
annealing at 56 C, and 150 s extension at 72 C. A
ꢀ
final extension of 10 min was given at 72 C. The PCR
products were purified by using 0.8% agarose gel
electrophoresis.
16s rDNA gene sequence analysis for molecular
identification of strains
Effect of pH on the activity and stability of the
extracellular protease
The sequencing of the 16S rDNA of Bacillus sp. DL-1
was carried out by Shanghai Majorbio Bio-Pharm
Technology Co., Ltd. The BLASTN program was used
to blast against the NCBI nucleotide database (http://
www.ncbi.nlm.nih.gov/blast/).
In order to determine the optimal pH, the crude fer-
mentation broth cultivated for 14 h was freeze-dried
by freeze drier to obtain extracellular proteases pow-
der which were dissolved by various pH values buffer:
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).
Subsequently, the activities of extracellular proteases
with different pH were measured according to the SB/
T 10317-1999 standard. The extracellular proteases in
the different pH buffers were incubated for 2 h at
Biochemical characterization of bacillus sp. DL-1
In order to measure the growth curve of Bacillus sp.
DL-1, Lysogeny broth (LB) liquid medium with 1%
inoculation was cultured in a rocking incubator at
ꢀ
3
7 C and 200 r/min and the optical densities of

BIOCATALYSIS AND BIOTRANSFORMATION
5
room temperature, and then the residual activities
were analysed at 45 C to confirm the pH stability of
extracellular proteases.
A series of pH (5.0 ꢃ 10.0) were carried out in stand-
ard enzymatic reaction system to optimize the pH for
the kinetic resolution of (±)-1-phenylethyl acetate. The
optimal reaction temperature for the kinetic resolution
was determined by incubating standard enzymatic
ꢀ
Effect of temperature on the activity and stability
of the extracellular protease
ꢀ
ꢀ
reactions at temperatures ranging from 20 C to 50 C
at pH 7.5 (Tris-HCl). In order to confirm the optimal
immobilized extracellular proteases concentrations,
20 mg/mL to 160 mg/mL immobilized enzymes were
The effect of temperature on the activities of extracel-
lular proteases were evaluated by incubating the
ꢀ
extracellular proteases at temperatures ranging from
loaded in standard reaction systems reacted at 45 C
ꢀ
2
5 to 70 C by using 2% casein as the substrate. The
for 2 h. Various concentrations of (±)-1-phenylethyl
acetate ranging from 5 mM to 50 mM were employed
to confirm the effect of substrate concentration on
the kinetic resolution of (±)-1-phenylethyl acetate. The
effect of reaction time was studied by investigating
the reaction time ranging from 1 h to 12 h. The effects
of metallic ions and surfactants on the kinetic reso-
lution of (±)-1-phenylethyl acetate were researched by
adding different metallic ions and surfactants into
standard reaction systems at final concentrations of
thermal stability of the extracellular proteases was
determined by incubating the enzymes at different
ꢀ
temperatures ranging from 25 to 70 C for 2 h, and
ꢀ
then analysing the residual activities at 45 C.
Immobilization of extracellular proteases and
measurement of enzyme activities
Bacillus sp. DL-1 was cultured in milk powder liquid
medium for 14 h. Subsequently, the cultured filtrates,
crude extracellular proteases of Bacillus sp. DL-1, were
gained by centrifugation and filtration. Sixty-gram kie-
selguhr was put into 1-L crude fermentation broth
containing extracellular proteases. The adsorption
2
mM and 0.05% (w/v), respectively. Similarly, a variety
of organic solvents at final concentrations of 5% (v/v)
were also put into standard enzymatic reactions to
measure the effect of organic solvents on the kinetic
resolution of (±)-1-phenylethyl acetate.
reaction was performed in shaking table at pH 8.0,
ꢀ
2
5 C, 200 r/min for 10 h. Finally, the kieselguhr in the
Kinetic resolution of (± ±-1-phenylethyl acetate for
the preparation of (S±-1-phenylethyl acetate by
immobilized extracellular proteases
reaction mixture was obtained by vacuum suction fil-
tration, which was dried at 38 C so as to gain the
ꢀ
immobilized extracellular proteases, whose activity
was measured according to the SB/T 10317-1999
standard. Some parameters of immobilization process
can fully understand the effect of immobilization on
enzyme activity (Boudrant et al. 2020). But the activ-
ities of mixed proteases did not represent the activ-
ities of resolution reactions, so we did not optimize
the immobilization process, but only optimized the
resolution reaction conditions.
Standard reactions were executed by adding 360 mg/mL
immobilized extracellular proteases to a 500 mL reaction
mixture containing 50 mM phosphate buffer (pH ¼ 6.0)
and 10 mM (±)-1-phenylethyl acetate followed by incu-
ꢀ
bation at 35 C, 200 r/min for 1.5 h. An equal volume of
ethyl acetate was used to terminate the reaction and
extract both substrates and products. Afterwards, the
ethyl acetate phase was analysed by a chiral GC to
determine the efficiency of kinetic resolution reaction.
The effect of pH on the kinetic resolution of (±)-1-
phenylethyl acetate was ascertained by assaying the
enantiomeric excess and conversion rate of the substrate
at different pH in the range of 5.0–6.5. The optimal tem-
Kinetic resolution of (± ±-1-phenylethyl acetate for
the preparation of (R±-1-phenylethanol using
immobilized extracellular proteases
ꢀ
ꢀ
Standard 500-lL pH 7.5 (50 mM Tris-HCl) reaction sys-
tem containing 40 mg/mL immobilized extracellular
proteases and 10 mM (±)-1-phenylethyl acetate was
perature was determined from 20 C to 50 C at pH 6.0
(50 mM phosphate buffer). 120 mg/mL to 400 mg/mL
immobilized extracellular proteases were added to the
standard reaction to investigate the effect of enzyme
concentration on the kinetic resolution of (±)-1-phenyl-
ethyl acetate. The optimal substrate concentration for
the kinetic resolution of (±)-1-phenylethyl acetate was
tested by adding different concentrations of (±)-1-phe-
nylethyl acetate ranging from 5 mM to 60 mM.
ꢀ
implemented at 45 C for 2 h. After the enzyme-cata-
lysed reaction was completed, 500-lL ethyl acetate
was used to extract substrates and reaction products
and the ethyl acetate phase was tested by chiral GC
to evaluate the enzymatic resolution of (±)-1-phenyl-
ethyl acetate.

6
L. DONG ET AL.
Subsequently, the hydrolytic reaction system containing
YR ¼ A=A0; YS ¼ B=B0
3
60 mg/mL immobilized enzyme and 10 mM (±)-1-phe-
C stands for the conversion ratio of the substrate,
and e.e. and e.e. represent the enantiomeric excess
ꢀ
nylethyl acetate were carried out at 35 C and pH 6.0 for
different times (from 0.5 to 12 h) to investigate the opti-
mal reaction time. Under the optimal reaction conditions
for the preparation of (S)-1-phenylethyl acetate, 2 mM
various metallic ions, 0.05% (w/v) surfactants, and 5% (v/
v) organic solvents were added to the kinetic resolution
reactions catalysed by immobilized extracellular pro-
teases of Bacillus sp. DL-1, to test the effect of additives
on the kinetic resolution of (±)-1-phenylethyl acetate by
the biocatalyst.
p
s
of the product and substrate, respectively. S and R ,
0
0
respectively, stand for the concentrations of the (S)-
and (R)-enantiomers of 1-phenylethyl acetate before
reaction, while S and R represent the concentrations
of the (S)-enantiomer and (R)-enantiomer of 1-phenyl-
ethyl acetate after reaction, respectively. YR and YS
stand for the yields of (R)-1-phenylethanol and (S)-1-
phenylethyl acetate, respectively. A represents the
concentration of (R)-1-phenylethanol after the reaction.
A0 represent the concentration of (R)-1-phenylethanol
when (R)-1-phenylethyl acetate completely transform
Operational stability of immobilized
extracellular proteases
into (R)-1-phenylethanol. B and B represent the con-
0
The operational stability of immobilized extracellular
proteases was studied by repeating the hydrolysis pro-
cess, and the residual enzyme activities were meas-
ured after each hydrolysis process. The enzyme
activities of each reaction were calculated as relative
activity by taking the activity of the first cycle as
centration of (S)-1-phenylethyl acetate after and before
the reaction, respectively.
Results and discussion
Isolation and characterization of protease-
producing strain which can resolve (± ±-1-
phenylethyl acetate
1
00%. Meanwhile, the celite-bound extracellular pro-
teases were repetitively used to perform asymmetric
hydrolysis of (±)-1-phenylethyl acetate for five times
under the optimized conditions.
One bacterium strain isolated and purified from deep-
sea sediments was found to produce extra-cellular
proteases which could asymmetric hydrolyse (±)-1-
phenylethyl acetate. The 16S rRNA nucleotide
sequence of this deep-sea bacterium was used to blast
against the NCBI nucleotide sequence database, which
revealed 99% identity with those of some Bacillus spe-
cies, such as Bacillus cereus strain RTR, Bacillus sp.
F221, Bacillus cereus strain L1.3, Bacillus anthracis strain
ES-9, and Bacillus subtilis strain 263AY4. Consequently,
the deep-sea bacterium was named as Bacillus sp. DL-
1. The growth curve of Bacillus sp. DL-1 was deter-
mined by measuring the OD600 during the time of cul-
ture, which showed that the logarithmic phase was
Analytical methods
The reaction products were analysed by gas chromato-
graph (FULI GC-9700II) equipped with H flame ioniza-
2
tion detector and 1112-6632 CYCLOSIL-B chiral
capillary column (30 m ꢁ 0.25 mm ꢁ 0.25 lm). Nitrogen
served as the carrier gas at a flow rate of 1.20 mL/min.
The column temperature was ranging from 80 to
ꢀ
2
10 C, and the injector and detector temperature
ꢀ
were 210 C. The enantiomeric excess (e.e.), conversion
rate (C), and yield (Y) were calculated by the equation
as follows (Chen et al. 1982).
1
–10 h, the stationary phase was 10–20 h, and the
decline phase was after 20 h (Supplementary Figure
S1). The growth curve of Bacillus sp. DL-1 under differ-
ent concentrations of NaCl revealed that Bacillus sp.
S þ R ꢂðS þ RÞ
0
0
C ¼
S þ R
0
0
½
ðRÞꢂ1ꢂphenyletha nolꢄ–½ðSÞꢂ1ꢂphenyletha nolꢄ
e:e:p ¼
½
ðRÞ ꢂ 1 ꢂ phenyletha nolꢄ þ ½ðSÞꢂ1 ꢂ phenyletha nolꢄ
½
ðSÞꢂ1ꢂphenylethyl acetateꢄꢂ½ðRÞꢂ1ꢂphenylethyl acetateꢄ
e:e:s ¼
½
ðSÞ ꢂ 1 ꢂ phenylethyl acetateꢄ þ ½ðRÞ ꢂ 1 ꢂ phenylethyl acetateꢄ

BIOCATALYSIS AND BIOTRANSFORMATION
7
DL-1 exhibited very good resistance to NaCl at con-
centrations of 0%ꢃ6% and Bacillus sp. DL-1 could
grow quite well even in the presence of 5% NaCl
proteases was in Tris–HCl buffer of pH 8.0. The pH sta-
bility of the proteases was determined after incubating
the enzymes in different buffers (pH 5.0–9.0) for 2 h.
The extracellular proteases were relatively stable when
pH was over 6.0, and exhibited the highest activity
and stability in Tris–HCl buffer of pH 8.5
(Supplementary Figure S4).
(Supplementary Figure S2). Thus, Bacillus sp. DL-1, one
bacterium isolated from the deep sea of the Western
Pacific Ocean was characterized to be a strain with
very good resistance to NaCl.
Comparation of the protease activity and
resolution ability of bacillus sp. DL-1
Effect of temperature on the activity and stability
of the extracellular protease
The highest enzyme activity (208.8 U/mL) of the extra-
cellular proteases from Bacillus sp. DL-1 was observed
when cultured for 36 h, while the extra-cellular proteases
from 14-h cultures were proved to be most appropriate
The hydrolytic activities of the extracellular proteases at
ꢀ
ꢀ
different temperatures (25 C–70 C) were shown in
Supplementary Figure S5, which demonstrated that the
hydrolytic activity of the extracellular proteases was the
ꢀ
ꢀ
to resolve (±)-1-phenylethyl acetate, with the e.e. being
highest at 45 C. When the temperature was over 55 C,
the activities of the proteases decreased sharply. After
incubating the extracellular proteases at different tem-
p
9
1
1%, but the enzyme activity was only 99.9 U/mL (Figure
). The reason why the highest enzymatic activity of the
ꢀ
ꢀ
extracellular proteases was inconsistent with the best
resolution ability is possibly due to the fact that Bacillus
sp. DL-1 could produce multiple extracellular proteases
and the kinetic resolution of (±)-1-phenylethyl acetate
might be carried out by one of them.
peratures (25 C–70 C) for 2 h, the residual hydrolytic
activities of the enzyme were measured to evaluate the
thermal stability of the proteases. As shown in
Supplementary Figure S6, the extracellular protease was
ꢀ
relatively stable below 45 C, but when the temperature
was over 50 C the activities of the proteases decreased
distinctly, and when the temperature was over 60 C the
ꢀ
ꢀ
Effect of pH on the activity and stability of the
extracellular protease
hydrolytic activities of the free enzyme were almost zero,
indicating that high temperature had great influence on
free extracellular protease of Bacillus sp. DL-1.
The activity of extracellular proteases under pH 5.0–9.0
(Supplementary Table S1) were measured to evaluate
the effect of pH on the enzyme activity. As shown in
Supplementary Figure S3, the hydrolytic activities of
extracellular proteases increased first and then
decreased with the increase of pH, showing that the
highest hydrolytic activity of the extracellular
The activities of extracellular proteases
Before immobilization, the enzymatic activity of the
free extracellular proteases cultivated for 14 h in the
fermentation medium was 129.9 U/mL. After immobil-
ization, every gram of kieselguhr adsorbed 25.7 mg
extracellular protease and the hydrolytic activity of
immobilized protease was 1729.0 U/g. The enzymatic
activity recovery was 79.86%.
Enzyme activities
250
225
200
175
150
125
100
100
90
80
70
60
50
40
30
20
10
0
e.e.p
C
Process optimization for the preparation of (R±-1-
phenylethanol through the kinetic resolution of
(± ±-1-phenylethyl acetate using immobilized
extracellular proteases
7
5
2
5
0
5
0
Effect of pH on the kinetic resolution of (± ±)-)phe)
nylethyl acetate
The influence of pH on the stereo-selectivity and
enzymatic activity of biocatalysts is one major concern
14
24
36
48
Time (h)
in enzymatic reactions. Both the C and the e.e. during
p
the enzymatic kinetic resolution of (±)-1-phenylethyl
acetate were investigated at different pH ranging from
Figure 1. The hydrolytic activities and the kinetic resolutions
of (±)-1-phenylethyl acetate by the extracellaluar proteases of
Bacillus sp. DL-1 at different culture times.
5
.0 to 10.0 (Supplementary Table S1) to determine the

8
L. DONG ET AL.
1
00
100
90
80
70
60
50
40
30
20
10
0
100
1
00
90
80
70
60
50
40
30
20
9
8
7
6
5
4
3
2
1
0
9
8
7
6
0
0
0
0
0
0
0
0
0
0
0
0
0
e.e.
C
p
50
4
3
2
0
0
0
5
6
7
8
9
10
PH
e.e.p (Sodium citrate/Phosphate buffer)
e.e.p (Tris-HCl buffer)
e.e.p (Gly-NaOH buffer)
C (Sodium citrate/Phosphate buffer)
C (Tris-HCl buffer)
C (Gly-NaOH buffer)
20
25
30
35
40
45
50
Figure 3. Effect of temperature on the kinetic resolution of
(±)-1-phenylethyl acetate catalysed by the immobilized extrac-
ellaluar proteases of Bacillus sp. DL-1 for the generation of (R)-
1-phenylethanol. The reaction condition: 50 mM pH 7.5 Tris-
HCl, 40 mg/mL immobilized extracellular proteases, 10 mM
(±)-1-phenylethyl acetate, total 500-lL, reaction for 2 h.
Figure 2. Effect of pH on the kinetic resolution of (±)-1-phe-
nylethyl acetate catalysed by the immobilized extracellaluar
proteases of Bacillus sp. DL-1 for the generation of (R)-1-phe-
nylethanol. The reaction condition: 40 mg/mL immobilized
extracellular proteases, 10 mM (±)-1-phenylethyl acetate, total
ꢀ
5
00-lL, reaction at 45 C for 2 h.
1
00
100
90
80
70
60
50
40
30
20
10
0
effect of pH on kinetic resolution. As shown in Figure
, the highest e.e. (96.6%) was observed at pH 7.5
9
0
0
2
p
8
(Tris-HCl), and significant decrease of e.e.p was
observed at pH above 9. Thus, the optimal pH for the
asymmetric hydrolysis of (±)-1-phenylethyl acetate
using immobilized proteases was determined to be
70
60
e.e.
C
p
50
40
30
20
10
0
7.5 (Tris-HCl), with the C being 30.8%.
Effect of temperature on the kinetic resolution of
± ±)-)phenylethyl acetate
(
The effect of temperature on the enzyme-catalysed
reaction was investigated at various temperatures
0
20
40
60
80
100
120
140
160
180
ꢀ
ranging from 20 to 50 C at pH 7.5 (Tris-HCl). The e.e.p
Enzyme concentration (mg/mL)
during the kinetic resolution of (±)-1-phenylethyl acet-
Figure 4. Effect of enzyme concentration on the kinetic reso-
lution of (±)-1-phenylethyl acetate catalysed by the immobi-
lized extracellaluar proteases of Bacillus sp. DL-1 for the
generation of (R)-1-phenylethanol. The reaction condition:
0 mM pH 7.5 Tris-HCl, 10 mM (±)-1-phenylethyl acetate, total
00-lL, reaction at 45 C for 2 h.
ꢀ
ate increased as the temperature increased from 25 C
ꢀ
to 45 C and decreased when the temperature was
ꢀ
ꢀ
over 45 C. Therefore, 45 C was determined to be the
optimal temperature for the enzymatic kinetic reso-
lution of (±)-1-phenylethyl acetate for the preparation
5
5
ꢀ
of (R)-1-phenylethanol, with the e.e. being 95.7% and
the conversion being 35.7%, respectively (Figure 3).
p
4
0 mg/mL immobilized extracellular proteases were
selected to carry out the kinetic resolution for the
preparation of (R)-1-phenylethanol.
Effect of enzyme concentration on the kinetic reso)
lution of (± ±)-)phenylethyl acetate
The influence of biocatalyst loading on the kinetic
resolution of (±)-1-phenylethyl acetate was investi-
gated. As shown in Figure 4, with the increase of
enzyme concentration, the C increased, but the e.e.p
increased first and then decreased. When the biocata-
Effect of substrate concentration on the kinetic
resolution of (± ±)-)phenylethyl acetate
Substrate concentration was also a key factor which
can greatly affect enzymatic reactions. The kinetic
resolution of (±)-1-phenylethyl acetate using the
immobilized extracellular proteases of Bacillus sp. DL-1
with different substrate concentrations were
lyst loading was 40 mg/mL, the e.e. was the highest
p
(96.5%), with the conversion being 21.4%. Thus,

BIOCATALYSIS AND BIOTRANSFORMATION
9
1
00
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
e.e.p
C
e.e.
C
p
0
10
20
30
40
50
0
2
4
6
8
10
12
substrate concentration (mM)
Time (h)
Figure 5. Effect of substrate concentration on the kinetic reso-
lution of (±)-1-phenylethyl acetate catalysed by the immobi-
lized extracellaluar proteases of Bacillus sp. DL-1 for the
generation of (R)-1-phenylethanol. The reaction condition:
Figure 6. Effect of reaction time on the kinetic resolution of
(±)-1-phenylethyl acetate catalysed by the immobilized extrac-
ellaluar proteases of Bacillus sp. DL-1 for the generation of (R)-
1-phenylethanol. The reaction condition: 50 mM pH 7.5 Tris-
HCl, 40 mg/mL immobilized extracellular proteases, 10 mM
5
0 mM pH 7.5 Tris-HCl, 40 mg/mL immobilized extracellular
ꢀ
ꢀ
proteases, total 500-lL, reaction at 45 C for 2 h.
(±)-1-phenylethyl acetate, total 500-lL, reaction at 45 C.
investigated. As shown in Figure 5, the e.e. reached
experiments without the addition of additives.
Notably, the presence of Co increased the e.e. to
p
97%. While surfactant SDBS (Sodium dodecylbenzene
sulfonate) showed obvious inhibitory activity on the
kinetic resolution of (±)-1-phenylethyl acetate.
p
2
þ
its maximum 95.4% with the C of 17.1% at a substrate
concentration of 10 mM. With the further increase of
substrate concentration, the C of (±)-1-phenylethyl
acetate decreased significantly. Thus, the optimal reso-
lution results were gained when the substrate concen-
tration was set at 10 mM.
Effect of organic co)solvents on the kinetic reso)
lution of (± ±)-)phenylethyl acetate
Different organic co-solvents at final concentrations of
Effect of reaction time on the kinetic resolution of
5
% (v/v) were added into standard reaction systems.
(
± ±)-)phenylethyl acetate
As shown in Table 2, the e.e. was enhanced in the
The effect of reaction time (1 h ꢂ 12 h) on both the C
and the enantio-selectivity of chiral products was
studied. When the reaction time was 2 h, the e.e.p
reached 96.6%, with the conversion being 23.7%, and
when the reaction time was longer than 2 h, the e.e.p
gradually decreased (Figure 6). Thus, 2 h was chosen
as the optimal reaction time to asymmetrically hydro-
lyse (±)-1-phenylethyl acetate for the preparation of
p
presence of n-hexane and methanol, with the e.e. in
p
the existence of methanol being the highest (97.1%).
Most of the rest organic solvents used in this reaction
systems exhibited negative effects on both the e.e.p
and the C.
To summarize, after careful process optimization for
the kinetic resolution of (±)-1-phenylethyl acetate
using immobilized extracellular proteases of Bacillus
sp. DL-1, the optimal resolution reaction condition was
(R)-1-phenylethanol.
ꢀ
carried out at 45 C for 2 h in pH 7.5 Tris-HCl buffer
Effect of metallic ions and surfactants on the kin)
etic resolution of (± ±)-)phenylethyl acetate
with 10 mM (±)-1-phenylethyl acetate, 40 mg/mL
immobilized proteases, 5% (v/v) methanol, which led
Metallic ions and surfactants could possibly greatly
affect both the hydrolytic activity and the resolution
ability of enzyme biocatalysts. In this study, thirteen
different metallic ions and eight surfactants were
added to investigate the effect of metallic ions and
surfactants on the enzymatic kinetic resolution of
to the e.e. > 97%, the C and the yield being 27% and
p
5
3%, respectively (Figure 7).
(
±)-1-phenylethyl acetate (Table 1). Of all the tested
þ
2þ
2þ
metallic ions and surfactants, only Na , Ba , Co
and CMC-Na increased the enantio-selectivity of the
immobilized proteases, compared with control

1
0
L. DONG ET AL.
Table 1. Effect of metallic ions and surfactants on the kinetic resolution of (±)-1-phenylethyl acetate cat-
alysed by immobilized extracellular proteases of Bacillus sp. DL-1 for the preparation of (R)-1-
phenylethanol.
Metallic ions (2mM)
e.e.
p
(%)
C (%)
Metallic ions (2mM)
e.e.
p
(%)
C (%)
2þ
Control
96.0 ± 0.3
95.7 ± 0.6
96.9 ± 0.1
95.8 ± 0.5
96.8 ± 0.1
96.1 ± 0.6
97.0 ± 0.1
33.3 ± 1.5
33.3 ± 1.5
34.1 ± 0.4
32.8 ± 1.6
23.7 ± 0.4
30.3 ± 1.0
30.1 ± 1.5
Cu
Mg
95.3 ± 0.3
95.9 ± 0.1
95.6 ± 0.4
93.9 ± 0.3
96.0 ± 0.3
92.6 ± 1.5
96.0 ± 1.6
23.1 ± 0.4
23.8 ± 2.2
14.2 ± 2.1
42.7 ± 1.9
17.2 ± 1.8
32.4 ± 1.7
28.4 ± 1.4
þ
2þ
2þ
Li
þ
þ
Na
Mn
2þ
K
Ni
2
þ
þ
þ
2þ
Ba
Ca
Co
Zn
2
3þ
3þ
Al
2
Fe
Surfactants
.05 % (w/v)
0
e.e.
p
(%)
C (%)
Surfactants 0.05 %(w/v)
e.e.
p
(%)
C (%)
Control
95.4 ± 0.3
94.6 ± 0.3
95.6 ± 0.4
94.6 ± 0.9
94.9 ± 1.0
40.3 ± 0.9
38.5 ± 2.4
38.6 ± 1.9
36.1 ± 2.0
40.9 ± 1.8
STPP
CTAB
SDBS
CMC-Na
94.4 ± 1.2
90.4 ± 1.4
60.4 ± 2.5
96.2 ± 0.7
38.6 ± 2.1
11.1 ± 1.7
0.9 ± 0.4
Tween-20
Tween-80
TritonX-100
Span 65
34.7 ± 1.1
Note: The reaction condition: 50 mM pH 7.5 Tris-HCl, 40 mg/mL immobilized extracellular proteases, 10 mM (±)-1-phenylethyl
ꢀ
acetate, total 500-lL, reaction at 45 C for 2 h.
Table 2. Effect of organic solvents on the kinetic resolution of (±)-1-phenylethyl acetate catalysed by immobilized extracellular
proteases of Bacillus sp. DL-1 for the preparation of (R)-1-phenylethanol.
Organic solvents
% (v/v)
Organic solvents
5 % (v/v)
5
e.e.
p
(%)
e.e.
p
(%)
C (%)
Control
Acetone
95.9 ± 0.8
95.6 ± 1.4
96.0 ± 0.5
91.7 ± 1.5
96.6 ± 0.5
94.0 ± 0.2
96.3 ± 0.5
95.5 ± 0.3
96.1 ± 0.2
97.1 ± 0.1
25.9 ± 0.6
34.8 ± 1.7
28.0 ± 1.8
38.3 ± 1.8
19.4 ± 0.5
17 ± 1.4
12.2 ± 0.4
12.4 ± 0.5
14.1 ± 1.6
27.4 ± 0.6
1,4-Dioxane
DMF
DMSO
Tetrahydrofuran
n-Propanol
tert-Butanol
sec-Butanol
n-Butanol
Isopropanol
Isobutanol
95.5 ± 0.5
96.1 ± 0.8
95.2 ± 1.5
95.1 ± 0.6
90.1 ± 0.7
94.1 ± 0.9
89.7 ± 1.0
89.3 ± 1.1
93.2 ± 0.3
84.1 ± 1.5
29.7 ± 2.1
34.8 ± 2.0
33.9 ± 2.3
36.2 ± 2.4
6.4 ± 1.0
7.3 ± 1.8
4.6 ± 1.2
7.5 ± 1.8
7.2 ± 1.6
2.3 ± 0.7
Acetonitrile
Dichloromethane
n-Hexane
Cyclohexanone
Cyclohexane
Chloroform
Ethanol
Methanol
Note: reaction conditions are the same as in Table 1.
Process optimization for the preparation of (S±-1-
phenylethyl acetate through the kinetic resolution
of (± ±-1-phenylethyl acetate using immobilized
extracellular proteases
Effect of pH on the kinetic resolution of (± ±)-)phe)
nylethyl acetate
pH may exhibit great effect on the enantio-selectivity
and conversion rate in enzymatic kinetic resolution.
Under standard hydrolytic reaction system of produc-
ing (S)-1-phenylethyl acetate, when pH was equal to
or greater than 7.0, (±)-1-phenylethyl acetate was
completely hydrolysed. Thus, for the generation of (S)-
The immobilized extracellular proteases of Bacillus sp.
DL-1 preferentially hydrolysed (R)-1-phenylethyl acet-
ate firstly to generate (R)-1-phenylethanol and thus
(S)-1-phenylethyl acetate was generated during the
1
-phenylethyl acetate, the kinetic resolution of (±)-1-
process of asymmetric hydrolysis of (±)-1-phenylethyl
acetate. Suitable increasing enzyme loading can
enhance the conversion rate in enzymatic kinetic reso-
lution. With the increase of conversion rate, (R)-1-phe-
nylethyl acetate was consumed completely, (S)-1-
phenylethyl acetate began to be transformed into (S)-
phenylethyl acetate catalysed by immobilized extracel-
lular proteases was analysed after incubating enzyme
reactions at pH ranging from 5.0 to 6.5. As shown in
Supplementary Figure S7, the highest e.e. of (S)-1-
s
phenylethyl acetate was found to be 98.1% at pH 6.0
1-phenylethanol. Therefore, in order to generate (S)-1-
(PB), with the C being 64.8%.
phenylethyl acetate with high optical purity, enhanc-
ing the concentration of immobilized proteases in
hydrolytic reaction system might be useful. (S)-1-phe-
Effect of temperature on the kinetic resolution of
(± ±)-)phenylethyl acetate
nylethyl acetate was gained with high e.e. through
optimizing the asymmetric hydrolysis conditions
as follows.
To evaluate the effects of different temperatures on
enzyme reaction, the temperature range was varied
from 20 C to 50 C under standard working
s
ꢀ
ꢀ

BIOCATALYSIS AND BIOTRANSFORMATION
11
Figure 7. (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-phenyletha-
nol; 2: (R)-1-phenylethanol prepared after enzymatic kinetic resolution; 3: (S)-1-phenylethyl acetate prepared after enzymatic kin-
etic resolution).
conditions. With the increase of temperature, e.e.s
decreased. Thus, the optimal working temperature
was characterized as 35 C.
ꢀ
increased, and the maximal e.e. was recorded at
s
ꢀ
3
5 C, with the e.e. and the C being 98.0% and 63.1%,
s
respectively (Supplementary Figure S8). When the
reaction temperature was further increased, the e.e.s

1
2
L. DONG ET AL.
Table 3. Effect of metallic ions and surfactants on the kinetic resolution of (±)-1-phenylethyl acetate cat-
alysed by immobilized extracellular proteases of Bacillus sp. DL-1 for the preparation of (S)-1-phenyl-
ethyl acetate.
Metallic ions (2mM)
e.e.
s
(%)
C (%)
Metallic ions (2mM)
e.e.
s
(%)
C (%)
2þ
control
95.6 ± 1.1
93.7 ± 1.3
97.3 ± 0.2
96.8 ± 0.6
86.7 ± 0.4
92.5 ± 0.5
85.1 ± 2.2
52.9 ± 0.7
48.5 ± 1.4
55.1 ± 0.5
54.7 ± 0.6
54.2 ± 0.5
50.7 ± 1.7
47.8 ± 0.7
Cu
Mg
90.5 ± 0.6
96.9 ± 0.3
60.0 ± 1.4
49.2 ± 1.3
84.4 ± 0.9
47.4 ± 1.7
72.0 ± 1.2
49.8 ± 0.1
54.7 ± 0.4
38.7 ± 2.4
34.0 ± 1.4
47.7 ± 1.5
33.1 ± 1.9
43.2 ± 1.3
þ
2þ
2þ
Li
þ
þ
Na
Mn
2þ
K
Ni
2
þ
þ
þ
2þ
Ba
Ca
Co
Zn
2
3þ
3þ
Al
2
Fe
Surfactants
.05 % (w/v)
Surfactants
0.05 % (w/v)
0
e.e.
s
(%)
C (%)
e.e.
s
(%)
C (%)
control
95.8 ± 0.5
97.3 ± 0.2
97.0 ± 0.6
97.5 ± 0.2
97.2 ± 0.5
53.5 ± 0.4
71.4 ± 1.2
56.5 ± 2.5
68.5 ± 1.8
58.0 ± 1.8
STPP
CTAB
SDBS
CMC-Na
96.8 ± 0.4
93.7 ± 1.3
1.8 ± 0.9
55.0 ± 2.0
48.4 ± 1.1
2.1 ± 1.3
Tween-20
Tween-80
TritonX-100
Span 65
96.2 ± 0.4
54.3 ± 0.6
Note: The reaction condition: 50 mM pH 6.0 PB, 360 mg/mL immobilized extracellular proteases, 10 mM (±)-1-phenylethyl
ꢀ
acetate, total 500-lL, reaction at 35 C for 1.5 h.
Effect of enzyme concentration on the kinetic reso)
lution of (± ±)-)phenylethyl acetate
Effect of reaction time on the kinetic resolution of
(± ±)-)phenylethyl acetate
Under standard hydrolytic reaction system for the
preparation of (S)-1-phenylethyl acetate, different con-
centrations (120 mg/mL ꢃ 400 mg/mL) of immobilized
extracellular proteases were added to the enzymatic
reactions. As shown in Supplementary Figure S9, both
To investigate the influence of reaction time on the
resolution of (±)-1-phenylethyl acetate by the immobi-
lized extracellular proteases, the reaction time was var-
ied within the range of 0.5 h to 12 h. The results in
Supplementary Figure S11 indicated a marked ten-
the e.e. and the C slowly increased when the enzyme
concentration was less than 280 mg/mL, and a sharp
s
dency for the e.e. to increase as the reaction time
s
prolonged to 1.5 h. Then, the e.e. decreased with sub-
s
increase of both the e.e. and the C was observed
s
sequent increases in reaction time. Thus, the highest
from 280 mg/mL to 360 mg/mL. When the concentra-
e.e. (97.9%) was observed when the reaction time was
s
tions of proteases were higher, both the e.e. and the
s
set as 1.5 h, with the conversion rate being 58.5%
C decreased. Thus, the optimal enzyme concentration
was found to be 360 mg/mL, with the highest e.e.s
and the C being 96.5% and 59.2%, respectively.
(Supplementary Figure S11).
Effect of metallic ions and surfactants on the kin)
etic resolution of (± ±)-)phenylethyl acetate
Effect of substrate concentration on the kinetic
resolution of (± ±)-)phenylethyl acetate
High substrate loading in biocatalysis is of great
importance for its practical use. In order to enhance
the volumetric productivity of kinetic resolution of
Under optimal reaction conditions, various metallic
ions (2 mM) and surfactants (0.05%) were added to the
enzyme-catalysed reaction systems to determine the
effect of metallic ions and surfactants on the kinetic
resolution of (±)-1-phenylethyl acetate. As shown in
(±)-1-phenylethyl acetate, the substrate concentration
þ
þ
2þ
Table 3, the presence of Na , K , Mg and surfac-
tants Tween-20, Tween-80, TritonX-100, Span 65, STPP
promoted the stereoselectivity to some extent, com-
pared with control experiments. While most of the
was optimized. As illustrated in Supplementary Figure
S10, (S)-1-phenylethyl acetate was produced with the
highest e.e.s (96.8%) and a 46.5% conversion rate,
when the (±)-1-phenylethyl acetate concentration was
2þ
2þ
3þ
3þ
tested additives especially Mn , Ni , Al , Fe , and
SDBS inhibited the activity of the biocatalyst.
1
0 mM. With the further increasing of substrate con-
centration, the e.e. and the conversion of substrate
s
gradually decreased. Therefore, 10 mM was set as the
optimal substrate concentration for generating (S)-1-
phenylethyl acetate.
Effect of organic co)solvents on the kinetic reso)
lution of (± ±)-)phenylethyl acetate
We also tested the effects of different organic co-sol-
vents at final concentrations of 5% (v/v) on the bioca-
talytic process. From the results we found that the
presence of acetonitrile, ethanol, methanol, DMF,
DMSO, and THF significantly improved the

BIOCATALYSIS AND BIOTRANSFORMATION
13
Table 4. Effect of organic solvents on the kinetic resolution of (±)-1-phenylethyl acetate catalysed
by immobilized extracellular proteases of Bacillus sp. DL-1 for the preparation of (S)-1-phenyl-
ethyl acetate.
Organic solvents
% (v/v)
Organic solvents
5 % (v/v)
5
e.e.
s
(%)
C (%)
e.e.
s
(%)
C (%)
Control
Acetone
97.2 ± 0.2
97.8 ± 0.1
98.6 ± 0.2
69.8 ± 1.3
90.9 ± 0.7
88.9 ± 1.2
97.4 ± 0.3
59.7 ± 1.3
98.6 ± 0.3
99.1 ± 0.1
55.2 ± 0.4
81.6 ± 2.9
70.4 ± 0.1
44.2 ± 2.1
53.8 ± 0.5
50.6 ± 0.4
53.9 ± 0.3
39.9 ± 1.5
70.3 ± 1.1
67.6 ± 0.5
1,4-Dioxane
DMF
DMSO
Tetrahydrofuran
n-Propanol
tert-Butanol
sec-Butanol
n-Butanol
Isopropanol
Isobutanol
97.1 ± 0.4
99.1 ± 0.1
99.2 ± 0.1
99.3 ± 0.1
46.1 ± 1.2
59.0 ± 0.8
25.4 ± 1.6
54.9 ± 1.3
90.3 ± 0.4
14.5 ± 1.2
77.0 ± 0.2
80.9 ± 1.1
60.3 ± 2.2
77.4 ± 1.4
32.9 ± 1.9
38.6 ± 1.6
21.5 ± 0.8
38.7 ± 1.5
49.8 ± 1.7
13.7 ± 2.7
Acetonitrile
Dichloromethane
n-Hexane
Cyclohexanone
Cyclohexane
Chloroform
Ethanol
Methanol
Note: reaction conditions are the same as in Table 3.
stereoselectivity of the proteases compared with con-
trol experiments (Table 4). Particularly, DMSO, metha-
happen (Ertan et al. 2006; Nunes and Marty 2006; Silva
et al. 2018). Different immobilization protocols may
greatly alter enzyme features. Lipase B from Candida
antarctica (CALB) was covalently immobilized on
epoxy Immobead-350 (IB-350) for the circularly hydro-
lytic resolution of rac-indanyl acetate, which showed
that the reusability of the CALB-IB-350 was efficient in
five consecutive resolution cycles (Pinheiro et al.
2018). Lipase from Pseudomonas fluorescens was
immobilized on the superparamagnetic nanoparticles
coupled with APTES and then activated with glutaral-
nol, THF, and DMF could enhance the e.e. to 99%
s
with the C being 60.3%, 67.6%, 77.4%, and 80.9%,
respectively. As a consequence, considering the con-
version rate, DMSO was characterized to be the opti-
mal organic cosolvent for the kinetic resolution of
(±)-1-phenylethyl acetate to generate (S)-1-phenylethyl
acetate using immobilized extracellular proteases.
Finally, 10 mM (±)-1-phenylethyl acetate, 360 mg/mL
immobilized extracellular proteases, pH 6.0 (PB), 5%
ꢀ
(
v/v) DMSO, and 35 C for 1.5 h was determined as the
dehyde (Fe O @APTES-GLU-PF) for the circularly kin-
3 4
optimal reaction conditions for the kinetic resolution
of (±)-1-phenylethyl acetate to prepare (S)-1-phenyl-
ethyl acetate using immobilized extracellular proteases
of Bacillus sp. DL-1. Under the optimal resolution con-
ditions, the immobilized proteases could yield (S)-1-
etic resolution of rac-indanol, which showed that the
enzyme system remained with high activity and values
of selectivity unchanged during the first three cycles
of reaction, and in the fourth and fifth cycles, only a
slight decrease in selectivity and with conversion value
close to 50% (Galv ~a o et al. 2018). In consequence,
covalent immobilization may be more advantageous
in improving the reusability of the immobilized
enzyme. The conditions of immobilization, especially,
the pH, may have great influence on the performance
of immobilization enzyme. Changing the immobiliza-
tion pH can altere the orientation of the enzyme mol-
ecules on the support. For instance, using
nucleophiles (e.g. ethylenediamine) to block the sup-
port permitted to eliminate the chemical reactivity of
the support avoiding undesired enzyme-support inter-
action and being a useful reaction endpoint (dos
Santos et al. 2015b). Near to the isoelectric point, the
free enzyme may experience protein aggregation
caused by undesired enzyme-support interactions
where utilization of changing pH for inactivation that
can stabilize incorrect enzyme structures (Sanchez et al.
2016). Therefore, pH of immobilization will be investi-
gated carefully in our future research. In order to fur-
ther improve the thermal stability, storage stability and
reusability of immobilized enzyme, cross-linking after
phenylethyl acetate with high e.e. (99%), conversion
s
rate (60%) and yield (79%) (Figure 7).
Operational stability of immobilized proteases
As shown in Supplementary Figure S12, the immobi-
lized extracellular proteases were reused for ten times
to measure their enzyme activity. The bound enzyme
preserved about 35.8% of its activity after 6 repeated
uses. The operational stabilities of immobilized extra-
cellular proteases were also assayed by reusing the
immobilized enzyme five times for the kinetic reso-
lution of (±)-1-phenylethyl acetate under the optimal
conditions (Supplementary Figure S13). Both the con-
version rate and the enantiomeric excess decreased
after repeated usage in the asymmetric hydrolysis.
This unsatisfactory reusability of the immobilized
enzyme could be due to the weak physical adsorption
between the proteases and kieselguhr. In other words,
desorption or leakage of the enzyme (resulting from
changes in temperature, pH, and ionic strength) may

1
4
L. DONG ET AL.

BIOCATALYSIS AND BIOTRANSFORMATION
15
adsorption of the extracellular proteases on the sup-
port, various conditions of immobilization, even some
new activated supports and other immobilization meth-
ods, will be tried and optimized in our following work.
optimum reaction conditions for the preparation of
(R)-1-phenylethanol: 10 mM (±)-1-phenylethyl acetate,
40 mg/mL immobilized extracellular proteases, pH 7.5
ꢀ
(Tris-HCl), 5% (v/v) methanol and 45 C for 2 h, the (R)-
1
-phenylethanol was obtained with the e.e.p being
over 97%, conversion being 27%, and yield being
3%, respectively, and under the optimum reaction
conditions for the preparation of (S)-1-phenylethyl
acetate: 10-mM (±)-1-phenylethyl acetate, 360 mg/mL
Comparation of the immobilized extracellular
proteases from bacillus sp. DL-1 with other
proteases/eaterases/lipases in the resolution of
5
(± ±-1-phenylethyl acetate through
immobilized extracellular proteases, pH 6.0 (PB), 5%
asymmetric hydrolysis
ꢀ
(
v/v) DMSO and 35 C for 1.5 h, the (S)-1-phenylethyl
To the best of our knowledge, in the field of biocataly-
sis, most of the kinetic resolution of (±)-1-phenylethyl
acetate through asymmetric hydrolysis were carried
out by using esterases or lipases (Ou et al. 2008; Fan
et al. 2011; Melais et al. 2016; Liang et al. 2016b;
Huang et al. 2017). Protease, one of most important
industrial enzymes, was rarely used in the resolution
of racemic esters. The only example of asymmetrically
hydrolysing (±)-1-phenylethyl acetate by using pro-
teases was in our previous work (Dong et al. 2019).
Generally, (S)-1-phenylethyl acetate and (R)-1-phenyle-
thanol were generated in the asymmetric hydrolysis of
acetate was generated with the e.e. being over 99%,
s
conversion being 60%, and yield being 79%, respect-
ively. Notably, we noticed that some organic solvents,
such as methanol or DMSO, may improve the stereo-
selectivity during the process of enzymatic kinetic
resolution. In conclusion, the extracellular proteases of
Bacillus sp. DL-1 were efficient and cost-effective
deep-sea biocatalysts with high industrial potentials in
the preparation of optically pure (R)-1-phenylethanol
and (S)-1-phenylethyl acetate as well as of other
chiral chemicals.
(
±)-1-phenylethyl acetate, which was catalysed by
Disclosure statement
esterases/lipases/proteases. A commercial immobilized
Candida antarctica lipase B (Novozym 435) was found
to exhibited opposite stereoselectivity to generate (S)-
The authors all declare that they have no conflict of interest.
1
-phenylethanol with the e.e. being over 99%, and the
conversion being 41.2% in the kinetic resolution of
±)-1-phenylethyl acetate. Compared with esterases/
Funding
This work was supported by the Key Special Project for
Introduced Talents Team of Southern Marine Science and
Engineering Guangdong Laboratory (Guangzhou) under
Grant [GML2019ZD0406]; the Natural Science Foundation of
Guangdong Province under Grant [2018A030313151]; the
Scientific and Technological Project of the Ocean and
(
lipases, proteases showed its unique advantages of
producing (S)-1-phenylethyl acetate with high optical
pure in the asymmetric hydrolysis of (±)-1-phenylethyl
acetate (Table 5).
Compared with the extracellular proteases from
Bacillus sp. DL-2, the immobilized extracellular pro-
teases from Bacillus sp. DL-1 exhibited higher hydro-
lytic activity and could asymmetrically hydrolyse (±)-1-
phenylethyl acetate by using higher substrate concen-
tration, shorter reaction time to obtain higher yield,
which also generated (S)-1-phenylethyl acetate with
Fishery
from
Guangdong
Province
under
Grant
[A201701C12]; the Priority Research Program of the Chinese
Academy of Sciences under Grant [XDA11030404] and the
Senior User Project of the Research Vessel KEXUE under
Grant [KEXUE2018G05].
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