Cloning, expression and characterization of a new enantioselective esterase from a marine bacterium Pelagibacterium halotolerans B2T

Article abstract of DOI:10.1016/j.molcatb.2013.09.002

An esterase, designated as PE8 (219 aa, 23.19 kDa), was cloned from a marine bacterium Pelagibacterium halotolerans B2^T and overexpressed in Escherichia coli Rosetta, resulting an active, soluble protein which constituted 23.1% of the total cell protein content. Phylogenetic analysis of the protein showed it was a new member of family VI lipolytic enzymes. Biochemical characterization analysis showed that PE8 preferred short chain p-nitrophenyl esters (C2-C6), exhibited maximum activity toward p-nitrophenyl acetate, and was not a metalloenzyme. PE8 was an alkaline esterase with an optimal pH of 9.5 and an optimal temperature of 45 C toward p-nitrophenyl acetate. Furthermore, it was found that PE8 exhibited activity and enantioselectivity in the synthesis of methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG) from the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG). (R)-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction under optimized conditions (0.6 M phosphate buffer (pH 8.0) containing 17.5% 1,4-dioxane under 30 C). In addition, PE8 was tolerant to extremely strong basic and high ionic strength solutions as it exhibited high activity even at pH 11.0 in 1 M phosphate buffer. Given its highly soluble expression, alkalitolerance, halotolerance and enantioselectivity, PE8 could be a promising candidate for the production of (R)-3-MFG in industry. The results also demonstrate the potential of the marine environment as a source of useful biocatalysts.

Full text of DOI:10.1016/j.molcatb.2013.09.002

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270–277
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Cloning, expression and characterization of a new enantioselective
esterase from a marine bacterium Pelagibacterium halotolerans B2T
Xiaolian Weia,1, Xiawei Jiangb,1, Lidan Yea, Shenfeng Yuana, Zhirong Chena,
Min Wuc,∗∗, Hongwei Yua,∗
a Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
b State Key Laboratory for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou
310003, China
c College of Life Sciences, Zhejiang University, Room 209, 866 Yuhangtang Road, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 June 2013
Received in revised form 26 August 2013
Accepted 2 September 2013
Available online 10 September 2013
An esterase, designated as PE8 (219 aa, 23.19 kDa), was cloned from a marine bacterium Pelagibacterium
halotolerans B2T and overexpressed in Escherichia coli Rosetta, resulting an active, soluble protein which
constituted 23.1% of the total cell protein content. Phylogenetic analysis of the protein showed it was
a new member of family VI lipolytic enzymes. Biochemical characterization analysis showed that PE8
preferred short chain p-nitrophenyl esters (C2–C6), exhibited maximum activity toward p-nitrophenyl
acetate, and was not a metalloenzyme. PE8 was an alkaline esterase with an optimal pH of 9.5 and an opti-
mal temperature of 45 ◦C toward p-nitrophenyl acetate. Furthermore, it was found that PE8 exhibited
activity and enantioselectivity in the synthesis of methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG)
from the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG). (R)-3-MFG was obtained in 71.6% ee
and 73.2% yield after 36 h reaction under optimized conditions (0.6 M phosphate buffer (pH 8.0) con-
taining 17.5% 1,4-dioxane under 30 ◦C). In addition, PE8 was tolerant to extremely strong basic and high
ionic strength solutions as it exhibited high activity even at pH 11.0 in 1 M phosphate buffer. Given its
highly soluble expression, alkalitolerance, halotolerance and enantioselectivity, PE8 could be a promising
candidate for the production of (R)-3-MFG in industry. The results also demonstrate the potential of the
marine environment as a source of useful biocatalysts.
Keywords:
Esterase
Marine
Enzyme biocatalysis
Methyl (R)-3-(4-fluorophenyl)glutarate
Pelagibacterium halotolerans B2T
© 2013 Published by Elsevier B.V.
1. Introduction
bacteria may be expected to have unusual habitat-related prop-
erties such as salt tolerance, hyperthermostability, barophilicity,
Esterases (EC 3.1.1.1) are a class of versatile biocatalysts belong-
ing to the family of hydrolase and are often applied in chemical
synthesis processes due to their activity and stereospecificity
toward chemicals and high stability in organic solvents [1]. They
catalyze the cleavage and formation of ester bonds and are widely
distributed in microorganisms from various environments [2].
Oceans constitute about 71% of the earth’s surface. Such a huge
habitat provides a vast pool of microorganisms, which become
great donors of novel esterases. In addition, enzymes from marine
cold adaptivity, and often excellent region- and stereospecificity,
which make them attractive for industrial application [3,4]. Fur-
thermore, marine enzymes usually possess novel chemical and
stereochemical characteristics, which increase their applicability
in pharmaceutical and chemical fields [4].
Optically pure methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-
MFG) is a pharmaceutically important precursor in the synthesis
of the widely used antidepressant – (-)-paroxetine hydrochloride
[5–8]. (R)-3-MFG can be synthesized from the prochiral dimethyl
3-(4-fluorophenyl)glutarate (3-DFG) by either enzymatic or non-
enzymatic methods. The enzymatic desymmetrization of prochiral
diester has been demonstrated to be an effective method for
the synthesis of 3-substituted glutaric acid monoesters [5,8–10]
(Table 1). The enantioselective aminolysis and ammonolysis of 3-
DFG in organic solvent has been investigated in previous reports [9]
with unsatisfactory yield, though high ee values were obtained. Yu
et al. [5] employed porcine liver esterase (PLE) to conduct enantios-
elective hydrolysis of 3-DFG, which gave the undesired (S)-3-MFG
in 95% ee and 86% yield. Recently, lipase B from Candida antarctica
Abbreviations:
methyl (R)-3-(4-fluorophenyl)glutarate;
3-DFG, dimethyl 3-(4-fluorophenyl)glutarate; (R)-3-MFG,
(S)-3-MFG, methyl (S)-3-(4-
fluorophenyl)glutarate; 3-FGA, 3-(4-fluorophenyl)glutaric acid; IPE, isopropyl
ether; MTBE, methyl tert-butyl ether; IPA, isopropanol; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide.
∗
Corresponding author. Fax: +86 571 8795 1873.
Corresponding author. Fax: +86 571 8820 6261.
∗∗
E-mail addresses: wumin@zju.edu.cn (M. Wu), yuhongwei@zju.edu.cn (H. Yu).
These authors contributed equally to this work.
1
1381-1177/$ – see front matter © 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.molcatb.2013.09.002

X. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270–277
271
Table 1
Enantioselective hydrolysis of 3-DFG using different enzymes.
Enzyme
Yield (%)
86
ee (%)
Configuration
Major advantages
Major drawbacks
References
Porcine liver
95
(S)
Relatively high ee value
Undesired
[5]
esterase (PLE)
configuration(S);high cost of
PLE
Lipase B from
Candida antarctica
(Novozym 435)
92.6
72.3
95.6
71.6
(R)0
(R)
Relatively high ee value and
conversion
Long reaction time (64 h); high
cost of the commercial
enzyme; required large
amount of the enzyme (20 g/l)
Moderate ee value and yield;
should be further engineered
to make it more suitable for
industrial applications
[8]
Esterase PE8 from
Pelagibacterium
halotolerans B2T
Highly soluble expression, easy
preparation and low cost of the
enzyme; short reaction time
(36 h)
Current
study
(Novozym 435) was used in the preparation of (R)-3-MFG with high
ee value and yield [8], but was not suitable for industrial application
due to its high cost. The increasing demand of the pharmaceu-
tical industry for the production of (-)-paroxetine hydrochloride
requires new and efficient hydrolases for the preparation of
(R)-3-MFG.
In the present study, an esterase (PE8) from the marine bacteria
Pelagibacterium halotolerans B2T isolated from East China Sea [11]
was cloned, heterologously expressed in Escherichia coli Rosetta in
soluble forms and biochemically characterized. We also attempted
to apply PE8 in the enantioselective hydrolysis of 3-DFG in an
aqueous-organic phase (Scheme 1). Moreover, effects of various
reaction conditions on the activity and enantioselectivity of PE8
were investigated and optimized.
AB, Sweden. The p-nitrophenyl hexanoate was purchased from
TCI, Japan, while other p-nitrophenyl esters and isopropyl-␤-d-
thiogalactoside (IPTG) were purchased from Sigma. Isopropanol,
n-hexane and trifluoroacetic acid were of HPLC grade. All other
chemicals were also commercially available, with purity of analyt-
ical grade. 3-DFG (HPLC purity >99%) was synthesized as described
previously [12].
2.3. Sequence analysis
A gene coding a putative esterase (PE8) was identified from the
genome sequence of Pelagibacterium halotolerans B2T [13]. The sim-
ilarity of deduced protein sequence was analyzed using the BLASTP
program (http://blast.ncbi.nlm.nih.gov/). Phylogenetic and molec-
ular evolutionary analysis were conducted using MEGA software
version 5 [14].
2. Materials and methods
2.1. Bacterial strains, plasmids and media
2.4. Cloning, expression and purification of recombinant esterase
Pelagibacterium halotolerans B2T was previously isolated by our
lab [11], and cultivated in marine 2216 broth (BD, USA) at 30 ◦C. The
E. coli strains DH5␣ and plasmid pET28b (+) (Novagen, Germany)
were used as the host and vector, respectively, for cloning the
esterase, and the plasmid pET28b (+) was used for gene cloning
and sequencing. E. coli Rosetta (DE3) (Novagen, Germany) and
pET28b (+) were used as the host and vector, respectively, for het-
erologous expression of the esterase. E. coli cells were grown in
Luria-Bertani (LB) medium containing 5 g/L yeast extract, 10 g/L
tryptone, and 10 g/L NaCl at pH 7.2. Chloramphenicol (34 ␮g/ml)
and/or kanamycin (20 ␮g/ml) was added to the medium when
needed.
Genomic DNA was extracted from Pelagibacterium halotoler-
ans B2T using genomic DNA Purification Kit described above. The
gene encoding PE8 was amplified by PCR using PrimeSTARTM
HS DNA Polymerase with the following primers: PE8F-Nde I:
5ꢀ-AGGACATATGACCGAACCCGTAAAG-3ꢀ and PE8R-Hind III: 5ꢀ-
CGATAAGCTTCTAGAGGATCTCGCG-3ꢀ. The digested PCR fragment
was inserted between the Nde I and Hind III sites of the pET-
28b (+) vector, creating pET-28a-PE8, which was then transformed
into chemically competent E. coli Rosetta (DE3) following standard
protocols. Chloramphenicol (34 ␮g/ml) and kanamycin (20 ␮g/ml)
were used as the selective pressure. Protein expression was induced
in E. coli Rosetta cells carrying the pET-28a-PE8 vector by addi-
tion of 0.5 mM isopropyl-␤-d-thiogalactoside (IPTG) to the culture
when OD600 reached 0.6 followed by overnight cultivation at 25 ◦C.
The cells were harvested by centrifugation at 4000 × g, 4 ◦C and
washed twice in phosphate-buffered saline (PBS) buffer (137 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, pH 7.4). The cell pellet was then
resuspended in Tris–HCl buffer (pH 8.0) and ruptured by ultrason-
ication. Cell debris was removed by centrifugation at 12,000 × g
and 4 ◦C for 20 min, and the crude extract containing PE8 was
purified by Ni-NTA affinity chromatography column following the
2.2. Reagents
Restriction endonucleases, PrimeSTARTM HS DNA Polymerase
and T4 DNA ligase were purchased from Takara, China. Genomic
DNA Purification Kit was obtained from Dongsheng, China. Gel
extraction kit and Plasmid Miniprep kit were obtained from
Axygen, China. Primers were synthesized by Invitrogen, China.
Ni–NTA resins were purchased from GE Healthcare Bio-Science
Scheme 1. Enzymatic enantioselective hydrolysis of 3-DFG in an aqueous-organic phase using PE8 as catalyst.

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X. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270–277
manufacturer’s instructions. The elute obtained was dialyzed twice
against 0.1 M Tris–HCl buffer (pH 8.0) before the determination of
purity and apparent molecular mass by 12% SDS-PAGE. The rela-
tive quantities of the protein bands on the gels were analyzed by
gel documentation unit assay (BioRad, USA).
of 3-DFG before and after reaction. The ee values and yield were
calculated according to methods previously reported [12].
2.8. Nucleotide sequence accession number
The nucleotide sequence of PE8 from Pelagibacterium halotol-
erans B2T was deposited in GenBank under accession number of
KF544956.
2.5. Enzyme assays
Activity was measured using p-nitrophenyl acetate (C2) or other
p-nitrophenyl esters (pNPEs) as substrates. The standard reac-
tion mixture contained 1 mM substrate (dissolved in acetonitrile),
100 mM Tris–HCl buffer and the appropriate amount of enzyme
in a total volume of 1 ml [15]. Unless otherwise specified, the
enzyme reaction was performed for 3 min at 30 ◦C and the release
of p-nitrophenol was measured at 405 nm using a UV–Visible
spectrophotometer (WFZ-UV2800H, China). All samples were mea-
sured in triplicate and corrected for the autohydrolysis of the
substrate. One unit of enzyme activity was defined as the amount
of enzyme required to release 1 ␮mol of p-nitrophenol from
p-nitrophenyl ester per minute. The substrate specificity was ana-
lyzed with the following p-nitrophenyl derivatives: acetate (C2),
butyrate (C4), hexanoate (C6), octanoate (C8), decanoate (C10),
laurate (C12), myristate (C14), and palmitate (C16). The effects of
3. Results and discussion
3.1. Sequence analysis
A putative ORF of 660 bp, encoding an esterase of 219 amino
acids (PE8) with a theoretical molecular mass of 23.19 kDa and a
deduced pI of 4.73 (http://web.expasy.org/protparam/) was iden-
tified from the genome sequence of Pelagibacterium halotolerans
B2T. BLASTP of the translated protein sequence showed maximum
identity (46%) with the esterase from Parvibaculum lavamentivorans
DS-1.
According to the phylogenetic analysis, PE8 and its closest rela-
tive protein could be classified into family VI esterases (Fig. 1). This
family of esterases showed the smallest molecular mass among all
esterases, in the range of 23 26 kDa [2]. Due to the presence of the
major conserved sequence motifs GFSQG including catalytic serine,
PE8 was identified as a new member of family VI esterases.
metal ions (Co2+, Cu2+, Ca2+, Mg2+, Zn2+, Sr2+, Mn2+, Ni2+, Ba2+
)
and EDTA on the activity of PE8 were examined at a final con-
centration of 10 mM using p-nitrophenol acetate as substrate at
30 ◦C and pH 7.5. The optimal temperature for esterase activity was
determined by measuring enzyme activity at temperatures rang-
ing from 0 to 70 ◦C using p-NP acetate as substrate following the
same protocol. The optimal pH was measured at 45 ◦C using p-NP
octanoate as substrate over a pH range of 5.0–12.0. In this experi-
ment, p-NP octanoate was used due to its high stability at alkaline
pHs, at which pHs p-NP acetate was rapidly auto-hydrolyzed. The
Michaelis-Menten constant (Km), maximum velocity (Vmax) and
the turnover number (kcat) were calculated by Lineweaver–Burk
plot. Enzyme assay was carried out under optimal conditions with
varying p-NP acetate concentrations (0.1–3 mM).
3.2. Purification and characterization of PE8
To eliminate the possible influence of the remaining imidazole
for the enzymatic assay, the enzyme was dialyzed after elution
from the Ni-NTA affinity chromatography column. PE8 was 2.8-
fold purified, with a specific activity of 48.4 U/mg and a yield of
93.8% (Table 2). The Km, Vmax and kcat values for p-NP acetate were
0.83 mM, 288 ␮M/min, and 9.21 s−1, respectively.
The purified PE8 was tested for lipase/esterase activity toward
p-nitrophenyl esters with various acyl chain lengths (C2–C16).
As shown in Fig. 2a, PE8 preferred short-chain p-nitrophenyl
esters and the highest activity was obtained in the hydrolysis
of p-nitrophenyl acetate (C2). However, the enzyme exhibited
poor activities toward middle-chain and long-chain p-nitrophenyl
esters. More specifically, no significant enzyme activity was
observed for the substrates with a chain length ≥ C10. Consequently,
the enzyme can be classified as an esterase rather than a lipase.
Many hydrolases are known to require metal ions [16,17]. As
shown in Fig. 2b, PE8 was stable in the presence of many metal ions,
and it could retain more than 50% of activity with most of the ions
except for Ni2+, Zn2+ and Cu2+. Moreover, the enzyme activity was
totally inhibited by 10 mM Zn2+and Cu2+. The chelating agent EDTA
had no obvious inhibition on enzyme activity, which indicated this
esterase was not a metalloenzyme [17].
2.6. Enantioselective hydrolysis of 3-DFG using PE8
Enzymatic hydrolysis was performed in a 1.5 ml reaction ves-
sel by suspending 3-DFG (40 mM) in phosphate buffer containing
different amounts of organic co-solvents (v/v). The reaction mix-
ture (0.5 ml) containing 5 mg of lyophilized crude PE8 (crPE8) were
orbitally shaken at 20–40 ◦C, 200 rpm for 24 h. After that, the reac-
tion was terminated by adjusting the pH to 2.0 with 5 M HCl
and extracted with ethyl acetate (2 ml × 0.5 ml). Ethyl acetate was
removed through vacuum drying and isopropanol (300 ␮l) was
added to dissolve the residues, then the extracts were analyzed
by high performance liquid chromatography (HPLC). All measure-
ments were conducted in triplicate.
The activity of PE8 was measured over a temperature range
of 0–70 ◦C and a pH range of 5.0–12.0 with p-nitrophenyl acetate
or p-nitrophenyl octanoate as substrates. As shown in Fig. 2c, the
optimal temperature for the PE8 activity was 45 ◦C, similar to that
of another esterase (PE10) isolated from Pelagibacterium halotoler-
ans B2T [18]. PE8 showed activity also at low temperatures. It can
retain 27% relative activity at 4 ◦C, which might be related to the
low temperature of the sea water, where Pelagibacterium halotoler-
ans B2T was isolated [11]. Moreover, 53.6% of its maximum activity
remained at 65 ◦C, making it feasible for a broad range of indus-
trial applications. As shown in Fig. 2d, PE8 is an alkaline esterase
with maximum activity toward p-nitrophenyl octanoate at pH 9.5,
and still exhibited esterase activity at pH 11, which makes PE8 an
attractive catalyst for future applications in industry.
2.7. Product analysis
The determination of conversion, ee, and yield was performed
by chiral HPLC analysis carried out on a HPLC system (Agilent
1100 Series) equipped with a UV detector and a system controller.
Samples were run using a mobile phase of n-hexane (containing
0.1% trifluoroacetic acid) and isopropanol (95:5, v/v) at a flow rate
of 0.5 ml/min on a Chiralpak AD-H column (250 mm × 4.6 mm) at
30 ◦C. The peaks were monitored at 266 nm, and the samples were
analyzed in triplicate. The retention time of 3-DFG, (R)-3-MFG, (S)-
3-MFG and 3-FGA were 19.4 min, 37.9 min, 43.9 min and 51.6 min,
respectively. The conversion was calculated as follows: conver-
sion = (A1 − A2)/A1, where A1 and A2 were the HPLC peak areas

X. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270–277
273
Fig. 1. Phylogenetic tree of PE8 from Pelagibacterium halotolerans B2T (bold type) and other members of family VI lipolytic enzymes. The neighbor-joining tree was constructed
using MAGE software. Bootstrap values were based on 1000 replications. Scale bar, 0.1 substitutions per amino acid position.
Table 2
Purification of the recombinant esterase PE8 after expression in E. coli Rosseta (DE3).
Purification step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purification (fold)
PE8 (crude extract)
PE8 (Ni-NTA)
9.23
3.13
161.36
151.38
17.48
48.43
100.00
93.81
1.00
2.77
3.3. Enantioselective hydrolysis of 3-DFG using PE8
in 3-DFG hydrolysis, yielding products with a moderate ee value.
To obtain a satisfactory ee value and conversion rate for industry
applications, the reaction conditions were optimized.
Methyl (R)-3-MFG is a pharmaceutically important precur-
sor, which has applications in the synthesis of the widely used
antidepressant – (-)-paroxetine hydrochloride. Due to the excellent
properties of PE8, the enzyme was tested for its activity in enantios-
elective hydrolysis of 3-DFG for the synthesis of methyl (R)-3-MFG.
It turned out the enzyme had activity and enantioselectivity
3.3.1. Screening of organic co-solvents
Due to the poor solubility of 3-DFG in aqueous medium, a
suitable organic solvent was required in which the substrate
would be properly dissolved, meanwhile, the required activity and
Fig. 2. Biochemical properties of PE8. (a) Substrate specificity of PE8. The esterase activity of the purified recombinant enzyme PE8 toward various chain lengths of p-NP
esters was assayed at 30 ◦C with a pH 7.5. The highest level of activity with the C2 substrate was set as 100%. (b) Effect of metal ions (10 mM) on PE8 activity. Enzymatic assay
was performed at 30 ◦C in 100 mM Tris–HCl buffer (pH7.5) with p-nitrophenol acetate as a substrate. The value obtained with no additives in the reaction mixture was taken
as 100%. (c) Effect of temperature on PE8 activity. The activity was measured in the temperature range of 0–70 ◦C in 100 mM Tris–HCl buffer (pH 7.5) with p-nitrophenol
acetate as substrate. The value obtained at 45 ◦C was taken as 100%. (d) Effect of pH on PE8 activity. The activity was determined at 45 ◦C over a pH range from 5.0 to 12.0
using p-nitrophenol octanoate as substrate. Buffers used were: 100 mM Citrate buffer (pH 5.0–6.0); 100 mM sodium phosphate buffer (pH 6.0–8.0); 100 mM Tris–HCl buffer
(pH 7.0–9.5), and 100 mM glycine–NaOH buffer (pH 9.5–12). The values obtained using Tris–HCl at pH 9.5 was taken as 100%.

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X. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270–277
Fig. 3. Effect of co-solvents (10%, v/v) on enantioselective hydrolysis of 3-DFG. Reaction conditions: 5 mg crude PE8 (crPE8), 0.02 mmol 3-DFG in 0.5 ml phosphate buffer
(0.1 M, pH 8.0), containing 10% (v/v) various co-solvents, 200 rpm, 30 ◦C, 24 h.
enantioselectivity of the enzyme would not be negatively affected.
The effect of different organic solvents on the esterase-catalyzed
synthesis of (R)-3-MFG was investigated (Fig. 3). Hydrolysis was
performed in various co-solvents at a concentration of 10% (v/v).
The highest conversion (74%) and ee values (63%) were obtained
after 24 h at 30 ◦C when 1,4-dioxane was used as co-solvent. In
the absence of any organic co-solvent, the conversion and ee
values were only 39% and 40%, respectively. The results demon-
strate that the modification of a reaction medium by adding an
appropriate organic co-solvent could improve the catalytic activity
and enantioselectivity of an enzyme. The same phenomena have
been reported for other esterases and lipases in previous studies
[19–24]. The lowest conversion (25.6%) was determined in iso-
propyl ether. The results also demonstrate a correlation between
the activity of PE8 and polarity of organic solvents. A good mea-
sure of polarity of an organic solvent is its log P value, defined
as the logarithm of its partition coefficient in the standard n-
octane/water two-phase system [25]. Furthermore, log P has been
the most commonly used to describe the solvent’s effect on the
activity and/or stability of enzymes [26]. The activity of PE8 was
profoundly enhanced in highly polar, water-miscible solvents such
as DMSO (log P = −1.3), 1,4-dioxane (log P = −1.1) and DMF (log
P = −1.0), as compared to the phosphate buffer without co-solvent.
In contrast, the addition of water-immiscible solvents with low
polarity, such as n-hexane (log P = 3.1), isopropyl ether (log P = 2.2)
and toluene (log P = 2.0) greatly reduced the activity. This might
be attributed to the fact that water plays an indispensable part in
the hydrolysis of 3-DFG. The use of water-miscible solvents could
increase the accessibility of water, thus enhancing the conversion
of 3-DFG. These results indicate that the property of the solvent
could significantly affect the catalytic efficiency of an enzyme.
However, there is still no general understanding between the
physicochemical properties of the solvent and the catalytic effi-
ciency of the enzyme due to the diversity of substrates and enzymes
used [26,27]. Therefore, taking both the ee value and conversion
into consideration, in the subsequent experiments, 1,4-dioxane
was selected as co-solvent for the enantioselective hydrolysis
of 3-DFG.
will depend on the activity, enantioselectivity (to produce mainly
one enantiomer of the monoester) and the enantiospecificity (to
hydrolyze the undesired (S)-3-MFG) of PE8. In our enzymatic reac-
tion, three products were detected, namely, (R)-3-MFG, (S)-3-MFG,
and 3-FGA. Therefore, in the following optimization, we will discuss
the effects of various reaction parameters on the activity (conver-
sion), enantioselectivity (ee) and enantiospecificity (yield of 3-FGA)
during the hydrolysis of 3-DFG. Considering the fact that enzyme
activity and enantioselectivity are profoundly influenced by the
concentration of co-solvent [28,29], the influence of 1,4-dioxane
concentration on the ee value of (R)-3-MFG and conversion of 3-DFG
was investigated (Fig. 4a). When the concentration of 1,4-dioxane
was relatively low (≤17.5%, v/v), both the ee value and conver-
sion increased with the addition of 1,4-dioxane, probably due to
the increased solubility of 3-DFG caused by the addition of co-
solvent. However, an excess of 1,4-dioxane resulted in the decrease
of ee value as well as conversion. This may be attributed to the
fact that high concentration of organic solvent molecules may alter
the enzyme conformation by penetrating into the active center of
the enzyme and consequently change the enzyme performance
[30]. Taking both the ee value and conversion into considera-
tion, although using 17.5% 1,4-dioxane, the ee value (66.8%) was
by 0.26% lower than that using 15% 1,4-dioxane (67.1%), its con-
version (98.9%) was much higher than that of 15% 1,4-dioxane
(82.9%). Therefore, 17.5% was chosen as the optimal co-solvent
concentration.
3.3.3. Effect of buffer pH
The efficiency of an enzyme is greatly influenced by the pH
value of its surroundings [31,32]. The variation of pH could alter
the ionic state of the substrate and the stereochemical configura-
tion of enzyme in the neighborhood of active sites, which in turn
influences the enzymatic activity and enantioselectivity [33,34].
The effect of buffer pH on the activity and enantioselectivity of PE8
toward 3-DFG was investigated using different buffered solutions
with pH ranging from 4.0 to 12.0. As shown in Fig. 4b, the con-
version was extremely low in acidic medium, with less than 20%
conversion at pH 4.0, 5.0, and 6.0. The optimal pH for 3-DFG hydrol-
ysis was 10, which is a little higher than that for p-nitrophenyl
octanoate hydrolysis (pH 9.5). Nevertheless, the ee value at pH 8
(66.8%, sodium phosphate buffer) was much higher than that of pH
10 (59.2%, glycine–NaOH buffer). Considering the similar conver-
sion (both ∼98%) at the two pH values and the higher ee value at
3.3.2. Selection of co-solvent concentration
It is well known that an ideal biocatalyst needs to be fully enan-
tioselective, producing the desired monoester without hydrolyzing
the first product of the reaction. The final yield of (R)-3-MFG

X. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270–277
275
Fig. 4. Effect of 1,4-dioxane concentration, buffer pH, buffer concentration, and temperature on enantioselective hydrolysis of 3-DFG. (a) Effect of 1,4-dioxane concentration.
Reaction conditions: 5 mg crPE8, 0.02 mmol 3-DFG in 0.5 ml phosphate buffer (0.1 M, pH 8.0) containing different amount of 1,4-dioxane, 200 rpm, 30 ◦C, 24 h. (b) Effect of
buffer pH. Reaction conditions: 5 mg crPE8, 0.02 mmol 3-DFG in 0.5 ml buffer (0.1 M) containing 17.5% (v/v) 1,4-dioxane with various pH values, 200 rpm, 30 ◦C, 24 h. Buffers
used were: 100 mM Citrate buffer (pH 4.0–6.0); 100 mM sodium phosphate buffer (pH 6.0–8.0); 100 mM Tris–HCl buffer (pH 7.0–9.5), and 100 mM glycine–NaOH buffer
(pH 9.5–12). (c) Effect of buffer concentration. Reaction conditions: 5 mg crPE8, 0.02 mmol 3-DFG in 0.5 ml phosphate buffer (pH 8.0) containing 17.5% (v/v) 1,4-dioxane
with different ionic strength, 200 rpm, 30 ◦C, 24 h. (d) Effect of temperature. Reaction conditions: 5 mg crPE8, 0.02 mmol 3-DFG in 0.5 ml phosphate buffer (pH 8.0, 0.6 M)
containing 17.5% (v/v) 1,4-dioxane, 200 rpm, 20 ◦C-40 ◦C, 24 h. Conversion (solid circle); ee values (empty triangle); yield of 3-FGA (star). Conversion (left Y axis); ee and yield
of 3-FGA (right Y axis), indicated by the arrows beside the curves.
pH 8, pH 8 (sodium phosphate buffer) was selected as the optimal
pH condition for this hydrolytic reaction.
increased from 0 to 0.1 M. With further increase of buffer con-
centration, the ee value was first leveled off and then decreased a
little when the concentration exceeded 0.6 M. The highest ee value
(67.4%) was achieved at 0.6 M. Conversion showed the same trend
as ee value, while the yield of the undesired 3-FGA exhibited a
different trend with the highest value of 13.2% observed at 0.1 M
and the decreasing of the value with the further increasing of the
buffer concentration. The results demonstrate that ionic strength
plays a crucial role in activity and enantioselectivity of PE8. Similar
3.3.4. Effect of ionic strength
Ionic strength is another parameter that might affect the activity
and enantioselectivity of enzymes [35,36]. Therefore, the effect of
buffer concentration (0–1 M) was tested at pH 8.0 (Fig. 4c). Both
ee value and conversion increased sharply from 50.4% to 66.8%
and 16.6% to 98.9%, respectively as the buffer concentration was
Fig. 5. Reaction time courses in the enantioselective hydrolysis of 3-DFG. Reaction conditions: 5 mg crPE8, 0.02 mmol 3-DFG in 0.5 ml phosphate buffer (pH 8.0, 0.6 M)
containing 17.5% (v/v) 1,4-dioxane, 200 rpm, 30 ◦C. (a) Developing of the yield of 3-DFG; (R)-3-MFG; (S)-3-MFG; and 3-FGA during the hydrolysis of 3-DFG. (b) Developing
of ee value and conversion during the hydrolysis of 3-DFG.

276
X. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270–277
activity and enantioselectivity of PE8 for 3-DFG, five different tem-
peratures were employed, ranging from 20 ◦C to 40 ◦C (Fig. 4d).
It was observed that the conversion first increased with temper-
ature, plateaued of 100% at 30 ◦C after 24 h, and then decreased
rapidly with further elevation of temperature. Similar results were
also found in the production of (S)-1-phenyl-1,2-ethanediol using
a Bacillus subtilis esterase as catalyst [33]. The highest ee value was
also achieved at 30 ◦C. It is noteworthy that the optimal temper-
ature of PE8 for 3-DFG hydrolysis (30 ◦C) was much lower than
that for p-NP acetate hydrolysis (45 ◦C). PE8 exhibited poor activity
toward 3-DFG at 40 ◦C. It is well known that the temperature opti-
mum of an enzyme differs for different substrates. Since 3-DFG has
much larger steric hindrance than p-NP acetate, the poor activity
for 3-DFG hydrolysis at elevated temperatures might be caused by
the decreased size of the binding pocket of PE8 at relatively high
temperatures. Furthermore, the enantioselectivity was lower at rel-
atively high temperature. This phenomenon has been reported in a
number of cases and a rational understanding of this phenomenon
has also been proposed [8,40,41]. Taking above into considera-
tion, 30 ◦C was chosen as the optimal temperature for further
reactions.
3.3.6. Time course of yield, ee and conversion in the hydrolysis of
3-DFG
A time course of the reaction was monitored under the
optimized conditions. Fig. 5a showed the evolution of yield of
all products during the hydrolytic reaction. At beginning, the
enzyme mainly hydrolyzed 3-DFG, producing (R)-3-MFG much
more rapidly than (S)-3-MFG. However, after depletion of the
diester, the enzyme tended to utilize the monoester, hydrolyzing
the minority (S)-3-MFG. As shown in Fig. 5, ee value increased from
56.8% (16 h) to 71.6% (36 h) while the yield of (R)-3-MFG decreased
from 74.5% to 73.2%. Although extending reaction time lowered the
yield of (R)-3-MFG, it enabled the enzyme to generate a product
with higher ee.
Fig. 6. SDS-PAGE of Rosseta (DE3), crPE8 and purified PE8. Lane 1, Rosseta (DE3)
grown overnight at 37 ◦C; lane 2, marker (beta-galactosidase (116 kDa), bovine
serum albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa),
REase Bsp98I (25.0 kDa), beta-lactoglobulin (18.4 kDa)); lane 3, 5 mg crPE8 enzyme
powder dissolved in 1 ml phosphate buffer (pH 8.0); lane 4, 2 mg purified PE8 pow-
der dissolved in phosphate buffer (pH 8.0).
to most halophilic enzymes from halophilic archaea, which are
inactive at low ionic concentrations [37], PE8 also exhibited low
activity under low ionic strength. Furthermore, PE8 was stable in
high ionic strength buffers. Since PE8 was cloned from Pelagibac-
terium haoltolerants B2T, which was isolated from sea water, it is
capable of growing in the presence of 0–13% (0–2.22 M) NaCl [11],
so it is reasonable that its enzymes have significant halotolerance.
Halophilic archaea established an osmotic balance with their high
salt environment by maintaining internal levels of salt that are iso-
tonic with the exterior [38]. Therefore it could be understood that
relatively high level of ionic strength should be more favorable for
the enzymes from the halophilic archaea.
As shown in Fig. 5, it is apparent that both the yield and ee
were time-dependent. The 24 h time point was selected to cal-
culate the conversion yield and ee by taking into consideration
the optimal conditions for the organic co-solvent, co-solvent con-
centration, buffer pH, buffer concentration, and the temperature.
The activity of the enzyme was selected as the first metric for
optimizing the reaction conditions. Therefore, after 24 h of reac-
tion, those conditions with low conversion were excluded. When
the conversion rates were approximately the same (∼90%), reac-
tion conditions were optimized based on the more favorable ee
value.
3.3.7. Effect of protein purification
3.3.5. Effect of reaction temperature
To determine whether purification could affect the enantios-
elective hydrolysis of 3-DFG, the crude PE8 (crPE8) was further
purified by Ni-NTA affinity chromatography column (Fig. 6), and
both the crPE8 and the purified PE8 were used in the hydrolysis
of 3-DFG. As shown in Table 3, the ee value and the conversion
In enzyme-catalyzed reactions, temperature also has a signif-
icant impact on the activity, enantioselectivity and stability of a
biocatalyst and the thermodynamic equilibrium of the catalyzed
reaction [39]. To investigate the effect of temperature on the
Table 3
Effect of purification on enantioselective hydrolysis of 3-DFG.
Yield (%)
ee (%)
Conversion (%)
3-DFG
(R)-3-MFG
(S)-3-MFG
3-FGA
Before purificationa
After purificationb
1.1 0.2
1.2 0.1
71.4 0.4
74.4 0.9
14.2 0.3
14.9 0.1
13.2 0.3
9.4 0.7
66.8% 0.7
66.6% 0.6
98.9 0.2
98.8 0.1
Results are presented as means standard deviation (n = 3).
a
Reaction conditions: 5 mg crPE8 powder (containing 0.487 mg PE8), 0.02 mmol 3-DFG in 0.5 ml phosphate buffer (pH 8.0, 0.1 M) containing 17.5% (v/v) 1,4-dioxane,
200 rpm, 30 ◦C, 24 h.
b
Reaction conditions: 1.1 mg purified PE8 (containing 0.487 mg PE8), 0.02 mmol 3-DFG in 0.5 ml phosphate buffer (pH 8.0, 0.1 M) containing 17.5% (v/v) 1,4-dioxane,
200 rpm, 30 ◦C, 24 h.

X. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 270–277
277
showed no significant difference between the two. Therefore, crPE8
could be directly used for biocatalytic reaction without further
purification.
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4. Conclusion
In this study, an esterase gene (pe8) from the marine bac-
terium Pelagibacterium halotolerans B2T was successfully cloned
and expressed in E. coli Rosseta (DE3). PE8 was assigned into fam-
ily VI of lipolytic enzymes based on the phylogenetic analysis. A
substrate specificity study showed that PE8 preferred short chain
p-nitrophenyl esters, and exhibited maximum activity toward p-
nitrophenyl acetate. PE8 was stable under a variety of metal ions,
and was found not to be a metalloenzyme. Biochemical charac-
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optimal pH of 9.5 and an optimal temperature of 45 ◦C toward
p-nitrophenyl acetate. Also it exhibited high activity and stabil-
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(R)-3-MFG was obtained in 71.6% ee and 73.2% yield after a 36 h
reaction under optimized conditions (0.6 M phosphate buffer (pH
8.0) containing 17.5% 1, 4-dioxane under 30 ◦C). To the best of our
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potential biocatalyst for the production of (-)-paroxetine. Never-
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the future, PE8 will be further engineered to make it more suitable
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This work is financially supported by National High-tech R&D
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