Characterization of a new thermophilic and acid tolerant esterase from Thermotoga maritima capable of hydrolytic resolution of racemic ketoprofen ethyl ester

Article abstract of DOI:10.1016/j.molcatb.2012.08.006

A gene coding for a putative thermostable esterase (Tm1160) from the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The purified enzyme displayed optimal activity at 70 °C and had a half-life of 60 min at 90 °C. It was stable over a range of pHs from 5.0 to 7.5 with an optimum around 5.0-5.5. The enzyme was found to have high acid tolerance and maintained about 50% of its activity even after 60 min of treatment at pH 4.5 and 70 °C. Furthermore, the enzyme exhibited the highest specific activity with p-nitrophenyl butyrate (318 ± 7 s ^-1 mM^-1). Under native conditions, Tm1160 forms a ～74 kDa dimer in solution. In addition, the esterase Tm1160 could enantioselectively hydrolyze the racemic ketoprofen ethyl ester and with an enantiomeric excess (ee_p) of 91.4% at a conversion of 41.1%, which makes it as a promising biocatalyst for the chiral resolution of (S)-ketoprofen.

Full text of DOI:10.1016/j.molcatb.2012.08.006

Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 23–30
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Characterization of a new thermophilic and acid tolerant esterase from
Thermotoga maritima capable of hydrolytic resolution of racemic ketoprofen
ethyl ester
∗
Wei Tao , Feng Shengxue, Mao Duobin, Yu Xuan, Du Congcong, Wang Xihua
School of Food and Biological Engineering, Zhengzhou University of Light Industry, 5 Dongfeng Rd, Zhengzhou 450002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 May 2012
Received in revised form 29 July 2012
Accepted 8 August 2012
Available online 16 August 2012
A gene coding for a putative thermostable esterase (Tm1160) from the hyperthermophilic bacterium
Thermotoga maritima was cloned and expressed in Escherichia coli. The purified enzyme displayed opti-
◦
◦
mal activity at 70 C and had a half-life of 60 min at 90 C. It was stable over a range of pHs from 5.0
to 7.5 with an optimum around 5.0–5.5. The enzyme was found to have high acid tolerance and main-
◦
tained about 50% of its activity even after 60 min of treatment at pH 4.5 and 70 C. Furthermore, the
−
1
−1
enzyme exhibited the highest specific activity with p-nitrophenyl butyrate (318 ± 7 s mM ). Under
native conditions, Tm1160 forms a ∼74 kDa dimer in solution. In addition, the esterase Tm1160 could
enantioselectively hydrolyze the racemic ketoprofen ethyl ester and with an enantiomeric excess (eep)
of 91.4% at a conversion of 41.1%, which makes it as a promising biocatalyst for the chiral resolution of
(S)-ketoprofen.
Keywords:
Esterase
Thermoacidophilic
Thermophile
Thermotoga maritima
Enantioselective hydrolysis
© 2012 Elsevier B.V. All rights reserved.
1
. Introduction
Esterases/lipases from mesophilic organisms have been used
in food detergent production, chemicals synthesis and biodiesel
Esterases (EC3.1.1.1, carboxylester hydrolases) and lipase
EC3.1.1.3, triacylglycerol hydrolases) catalyze the cleavage and
production. However, mesophilic enzymes are not suitable under
harsh reaction conditions. Thus, esterases from thermophiles have
attracted attention due to their high thermal and chemical stability
to organic solvents, detergents, low and high pH and serve as a value
enzyme source of biocatalysts for industrial applications [8,9].
Several thermophilic esterases have been identified and character-
ized from Archaea and thermophilic bacteria including Sulfolobus
[10–13], Pyrococcus [14,15], Archaeoglobus fulgidus [16], Aeropyrum
pernix [17], Picrophilus torridus [18], Pyrobaculum calidifontis [19],
Thermoanaerobacter tengcongensis [20], Thermus thermophilus [21],
Thermotoga maritima [22] and from metagenomic libraries [23–26].
T. maritima is a hyperthermophilic bacterium that grows opti-
mally at 80 C and thrives at temperatures of up to 95 C, which
makes it a potential source for thermostable enzymes [27]. From
analysis of the genome of T. maritima, it is evident that at least ten
ORFs encoding esterases or lipases are present in this organism.
However, only three esterases (TM0033, TM0053 and TM0336)
(
formation of ester bonds and are found in various organisms
including animals, plants, and microorganisms [1,2]. These
enzymes have many important biotechnological applications
because of their unique features, such as remarkable stability in
organic solvents, broad substrate specificity, and high regio- and
stereo-selectivity [3,4]. Esterases/lipases belong to the family of
␣
/␤ hydrolases which share a highly conserved catalytic triad: a
serine, histidine, and an aspartic acid or glutamic acid. A consensus
signature sequence containing the active site serine is usually
characterized by a conserved pentapeptide GXSXG motif [5,6]. In
general, esterases have the ability to hydrolyze ester substrates
with short-chain fatty acids (≤C10), whereas lipases hydrolyze
long-chain fatty acids (≥C10) [7].
◦
◦
[
22,28,29] and one acetyl esterase (TM0077) [30,31] have been
identified and characterized. T. maritima is not known to possess
a thermophilic esterase which has the ability to resolve mixtures
of chiral esters. Herein, we report the cloning, expression and
biochemical characterization of a new putative esterase (Tm1160)
from T. maritima. Furthermore, Tm1160 is identified as the first
thermoacidophilic esterase from a hyperthermophilic bacteria
and also displays high hydrolytic resolution of racemic ketoprofen
ethyl ester.
Abbreviations: IPTG, isopropyl-␤-d-thiogalactopyranoside; SDS-PAGE, SDS-
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; pNP-C2,
p-nitrophenyl acetate; pNP-C4, p-nitrophenyl butyrate; pNP-C8, p-nitrophenyl
octanoate; pNP-C10, p-nitrophenyl decanoate; pNP-C12, p-nitrophenyl laurate;
pNP-C16, p-nitrophenyl palmitate; PBS, phosphate buffered saline; EDTA, ethylene
diamine tetraacetic acid.
∗ Corresponding author. Tel.: +86 371 63556627; fax: +86 371 63556627.
E-mail address: weitao8008@yahoo.com.cn (W. Tao).
1
381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.08.006

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W. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 23–30
2
. Materials and methods
or 60 min. The reaction was stopped by adding glycine at a final
concentration of 0.15 M. The reaction products were separated by
SDS-PAGE with 8% separating gel on a vertical slab mini gel appa-
ratus (Model SE 250; Amersham Bioscience) at 30 mA for 2 h. The
samples were diluted with electrophoresis sample buffer contain-
ing 5% (w/v) SDS and boiled for 5 min. After migration, gels were
stained for protein detection with Coomassie Brilliant Blue R-250
following standard procedures.
2
.1. Chemicals and strains
The genomic DNA of T. maritima was obtained from Professor
Weilan Shao at Nanjing Normal University. Restriction enzymes,
Pyrobest DNA polymerase, and DNA ligase were purchased from
TaKaRa Biotechnology (Dalian, China). p-Nitrophenyl esters and
(
S)-ketoprofen were obtained from Sigma (St. Louis, USA). Racemic
ketoprofen was purchased from Xinan Synthetic Pharmaceutical
Factory (Chongqing, China). The ethyl ester of racemic ketoprofen
was prepared as described previously [32]. Another two profens
2
.5. Enzymatic activity assay
Esterase activity was determined at 405 nm by a spectrophoto-
(
R,S)-naproxen methyl ester and (R,S)-ibuprofen methyl ester were
metric method using p-nitrophenyl esters of various chain lengths
as described previously [33]. The standard reaction mixture (1 ml),
containing 2 mM p-nitrophenyl butyrate (pNP-C4), 50 mM sodium
acetate buffer (pH 5.5) and 1% (v/v) isopropanol, was preincu-
bated for 2 min and then reaction was started by adding 18 ␮g of
purified enzyme. The esterase activity was measured at 70 C for
2
enzyme releasing 1 ␮mol of p-nitrophenyl per min. The background
hydrolysis of the substrate was determined by using a reference
sample of identical composition to the incubation mixture in the
absence of enzyme. The extinction coefficient (ε405) for pNP-C4 was
purchased from J&K Scientific Ltd. (Beijing, China). All the other
chemicals were of the highest reagent grade and obtained from
Sangon (Shanghai, China). Nickel columns and Superdex 200 gel
filtration columns were from GE Healthcare (Buckinghamshire,
UK). The plasmid pET15b (Novagen, Madison, USA) was modified
with NdeI changed to NcoI in order to facilitate the purification.
Escherichia coli DH5␣ and BL21-CodonPlus (DE3)-RIL (Stratagene,
La Jolla, CA) were used as the cloning and expression hosts, respec-
tively.
◦
0 min. One unit of esterase activity was defined as the amount of
2.2. Cloning of the putative esterase Tm1160 gene
−1 −1
1
6540 M cm . All enzyme activities were carried out at least
three times and the data were averaged.
The gene coding for a putative esterase Tm1160 from T. mar-
itima was amplified using PCR with the following primers: the
ꢀ
ꢀ
2.6. Substrate specificity
forward primer: 5 -GCC CATATG GTT AAG CTG ATA ATC AAG-3 and
ꢀ
the reverse primer: 5 -GAT GTCGAC TCACCTCTTCAAAAAGGAAAC-
3
ꢀ
Substrate specificity toward pNP-esters including pNP-acetate
(the underlined indicates the NdeI and SalI sites, respectively).
(C2), pNP-C4, pNP-octanoate (C8), pNP-decanoate (C10), pNP-
The PCR product was digested with NdeI and SalI and cloned into
pET15b. The resulting plasmid was designated pET15b–Tm1160
and sequenced to ensure that no unintended mutation had
occurred.
laurate (C12), and pNP-palmitate (C16), were performed as
described previously. Enzymatic assays to determine the Km
and Vmax values were done at different substrate concentrations
(0.02–1.0 mM) in three independent trials. Corresponding Km and
Vmax values were computed using Hanes–Wolff plots and the
Michaelis–Menten equation.
2.3. Expression and purification of the recombinant enzymes
About 1% of overnight culture of E. coli BL21(DE3)-CodonPlus-
RIL harboring the recombinant plasmid pET15b–Tm1160 was
inoculated into 500 ml of LB broth containing 100 mg/l ampicillin
and 34 mg/l chloramphenicol. The cells were cultured at 37 C
2.7. Effect of temperatures and pH on enzyme activity and
stability
◦
with shaking. When the OD600 reached 0.5, IPTG was added to a
final concentration of 0.2 mM. After culture for 4 h at 37 C, cells
The pH and temperature profiles for the esterase activity were
established using pNP-C4 as the substrate. The effect of tempera-
ture on the esterase activity was examined at temperatures ranging
◦
were harvested by centrifugation, resuspended in buffer A (50 mM
Tris–HCl, pH 8.0, 50 mM NaCl) and then disrupted by sonication.
◦
◦
from 30 C to 100 C in 50 mM of sodium acetate buffer (pH 5.5).
The buffer was adjusted to pH 5.5 at each assayed temperature.
The thermostability of the purified esterase was evaluated by incu-
bating the enzyme (18 ␮g/ml) in 50 mM of sodium acetate buffer
◦
The disrupted cells were incubated at 70 C for 30 min to remove
the cell debris and denatured proteins and then centrifuged at
1
2,000 × g for 15 min. The supernatant was loaded on a nickel
◦
◦
◦
column (GE Healthcare, Buckinghamshire, UK). The recombinant
enzymes were eluted with the elution buffer (20 mM Tris–HCl,
pH 8.0, 500 mM NaCl, 300 mM imidazole) and were dialyzed
overnight in buffer A to remove imidazole. For further purifica-
tion, the enzymes were dialyzed in buffer B (50 mM Tris–HCl,
pH 8.0, 200 mM NaCl), concentrated using a microfilter (Micro-
con YM-10, Millipore Corp, Beddord, MA) and loaded onto a Hiload
Superdex-200 (16/60) column (GE Healthcare, Buckinghamshire,
UK), which was pre-equilibrated with buffer B (50 mM Tris–HCl,
pH 8.0, 200 mM NaCl). The fraction size was 0.5 ml and the flow
rate was 0.5 ml/min. The peak fractions were collected and concen-
trated. The purified protein was analyzed by SDS-PAGE and protein
concentration was determined according to the Bradford method.
(pH 5.5) at three different temperatures (70 C, 80 C, and 90 C).
Each sample (40 ␮l) was obtained at 30, 60, 90 and 120 min inter-
vals during incubation, quickly cooled on ice and centrifuged at
12,000 × g for 15 s. The obtained supernatants were kept frozen at
◦
−20 C until the residual activity was determined under the stan-
dard conditions.
◦
The effect of pH on the esterase activity was examined at 70 C
from pH 3.0 to 9.0. The buffers used were 50 mM of sodium citrate
(pH 3.0–4.0), sodium acetate (pH 4.5–6.0), sodium phosphate (pH
6.5–8.0), and Tris–HCl (pH 8.5–9.0). The pHs of the different buffers
◦
were adjusted at 70 C. Under each pH condition, the pH stabil-
ity was determined by incubating the purified enzyme (18 ␮g/ml)
◦
in the reaction mixture at 70 C for 30 min, and then the residual
enzyme activity was measured by the standard method. The acid
tolerance of the esterase Tm1160 was examined by incubating the
enzyme (18 ␮g/ml) in 50 mM of sodium acetate buffer at three dif-
2
.4. Chemical cross-linking
◦
Cross-linking was carried out by incubating 20 ␮g of the enzyme
ferent pHs (4.5, 5.0, and 5.5) and at 70 C. At regular times intervals
in PBS containing 2% formaldehyde at room temperature for 0, 30
(0, 30, 60, 90 and 120 min), aliquots (40 ␮l) were withdrawn and

W. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 23–30
25
Fig. 1. Multiple sequence alignment for Tm1160 and its homologs. An alignment was carried out using Clustal W program with manual adjusting. Numbering of the amino
acids is indicated on the right. Conserved, conservative, and semi-conservative sites are indicated by asterisks, double, and single dots, respectively. Gaps introduced during the
alignment are indicated as dashes. The residues forming the oxyanion hole are indicated by the closed squares. Triangles represent the consensus active sites. Abbreviation:
Tm1160 from Thermotoga maritima (NP 228966); THA 1490 from Thermosipho africanus TCF52B (YP 002335272); Tlet 0739 from Thermotoga lettingae TMO (YP 001470369);
BCA 3321 from Bacillus cereus 03BB102 (YP 002750590); SACE 0950 from Saccharopolyspora erythraea NRRL 2338 (YP 001103209).
quickly cooled on ice. The residual enzyme activity was determined
using the standard method to analyze the acid tolerance of Tm1160.
2.9. Effect of metal ions, EDTA and surfactants on enzyme activity
The effect of metal ions, EDTA and surfactants (Tween 20, Tween
8
0, Triton X-100 and SDS) on the esterase activity was investigated
by adding each metal salt (5 mM), EDTA (5 mM) and each surfac-
tants (1% and 5%, v/v) to the assay solution containing the purified
enzyme (18 ␮g/ml) and 50 mM sodium acetate buffer (pH 5.5). The
reaction mixture was incubated at room temperature for 60 min.
The residual enzyme activity was measured under the standard
assay conditions and was initiated by addition of the substrate pNP-
C4. The esterase activity of enzyme without additives was taken as
the control and defined as 100% activity.
2
.8. Organic solvent stability of Tm1160
The stability of the esterase against organic solvents was deter-
mined by incubating the enzyme in the presence of organic solvents
50% or 90%, v/v) in 50 mM sodium acetate buffer (pH 5.5). The
(
purified enzyme was diluted to 18 ␮g/ml with buffer containing
each organic solvent of interest, and incubated at 30 C with con-
◦
stant shaking at 160 rpm for 12 h in screw-capped tubes. A sample
was taken periodically from the aqueous phase, and then the resid-
ual enzyme activity was determined using pNP-C4 as the substrate
under standard assay conditions. Blank samples were prepared
with the reaction buffer instead of the enzyme. Residual activity
of enzyme without organic solvents was taken as the control and
defined as 100% activity.
2
.10. Enantioselective hydrolysis of the chiral compounds
◦
Enzymatic reactions were carried out at 70 C in 1 ml of a
reaction mixture containing the purified enzyme (0.235 mg) and
5 mg of racemic ketoprofen ethyl ester dissolved in 50 mM
sodium acetate buffer (pH 5.5). The reaction mixture was stirred
2

2
6
W. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 23–30
at 200 rpm. The resulting solution was analyzed by HPLC using the
chiral column (25 cm × 4.6 cm, Daical Chemical Industries, Tokyo,
Japan). Samples were eluted with n-hexane:2-propanol:acetic acid
(
2
90:10:0.5, v/v/v) at a flow rate of 1.0 mL/min and detected at
54 nm. The retention times of racemic ketoprofen ethyl ester,
R-ketoprofen, and S-ketoprofen were detected at 5.4, 14.8 and
8.2 min, respectively. The enantioselectivity of the enzyme was
1
calculated with E = ln[1 − c(1 + eep)]/ln[1 − c(1 − eep)], where c and
eep represent the degree of conversion and the enantiomeric excess
of product, respectively [34].
3
. Results and discussion
3.1. Amino acid sequence comparison
The Tm1160 gene encodes a 306 amino acid protein with a
calculated molecular mass of 35.2 kDa. The deduced amino acid
sequence of Tm1160 showed 54, 45, 30, and 28% sequence identity
with the esterase from Thermosipho africanus TCF52B, Thermotoga
lettingae TMO, Saccharopolyspora erythraea NRRL 2338, and Bacillus
cereus 03BB102, respectively. The functions of Tm1160 have not
been determined. Ser160, Asp250 and His282 formed a catalytic
triad in Tm1160 (Fig. 1), which is typical of those commonly found
in esterases and lipases. Multiple sequence alignment indicated
that Tm1160 has the highest similarity to the family of mammalian
HSLs, which is characterized by a conserved GXSAGG hexapeptide
containing the active site residue and a oxyanion hole, which is
composed of Gly88 and Gly89 within the conserved motif HGGG
Fig. 2. SDS-PAGE of the recombinant esterase Tm1160. Lanes 1, crude cell extract; 2,
supernatant after heat treatment; 3, Tm1160 after nickel affinity chromatography;
4, purified Tm1160 after gel filtration chromatography.
(
amino acid residues 87–90) and Ala161 [3,5].
3
.2. Overexpression and purification of Tm1160
molecular mass (Fig. 3B). These results indicated that Tn1160
appeared to form a dimer in solution, which is probably con-
sistent with the proposed mechanism that oligomerization is a
major factor contributing to the thermostability of thermophilic
enzymes including the esterases [35]. The thermophilic esterases
PestE, EstE1, and AFEst form tetramers and octamers while the
esterases EstE5, SspEst, and EfaHyd from mesophilic organisms are
monomers [24,36–38].
To evaluate the biochemical properties of Tm1160, the plasmid
pET15b–Tm1160 for the wild type enzyme was constructed. The
recombinant Tm1160 were expressed in E. coli as the His6–Tm1160
fusion (calculated to be a 36.9 kDa polypeptide) and purified
from the soluble extracts by heat treatment, Ni-NTA affinity and
Superdex-200 gel filtration. The relative molecular mass of the His6
fusion enzyme Tm1160 was ∼37 kDa on SDS-PAGE (Fig. 2). The
purification procedure is shown in Table 1. The specific activity
of the purified Tm1160 reached 2458 U/mg using pNP-C4 as sub-
◦
3.4. Substrate specificity and kinetics
strates in 50 mM of sodium acetate buffer at pH 5.5 and 70 C. The
enzyme was purified to 9.2-fold with a yield of 59.5%. To determine
the molecular mass of the native enzyme, the purified enzyme were
pooled, lyophilized and resuspended in 50 mM sodium acetate
buffer, 200 mM NaCl, pH 5.5. Three micrograms of the pure enzyme
was loaded on a Superdex 200 gel filtration column. The enzyme
was eluted at 12.47 ml corresponding to ∼74 kDa molecular mass
The substrate specificity of Tm1160 was determined
using pNP-esters with acyl chains of different lengths as the
substrates. The kcat/Km value for Tm1160 toward different
−
1
−1
pNP-esters followed the order pNP-C4 (318 ± 7 s mM ) > pNP-
−
−1
1
−1
−1
−1
−1
−1
C2 (158 ± 4 s mM ) > pNP-C8 (85 ± 2 s mM ) > pNP-C10
−
1
(
(
68 ± 2 s mM ) > pNP-C12
(14 ± 0.5 s mM ) > pNP-C16
(
Fig. 3B).
−
1
−1
3 ± 0.6 s mM
)
(Table 2). This result demonstrated that
Tm1160 exhibited the highest activity for pNP-C4 among the sub-
strates tested. Compared with Tm1160, the known esterases from
T. maritima exhibited different substrate preferences. Tm0033,
Tm0053 and Tm0336 preferred pNP-C8, pNP-C10, and pNP-C5,
respectively, while the acetyl esterase Tm0077 showed the high-
est activity with pNP-C2 [22,28–30]. On the basis of substrate
specificity, Tm1160 could be classified as a true esterase [7].
3.3. Dimer formation of Tm1160
In chemical cross-linking analysis, Tm1160 was found to be
covalently linked by formaldehyde and the bands corresponding
to ∼74 kDa in addition to 37 kDa were detected on the SDS-PAGE
(
Fig. 3A). This result was confirmed by gel filtration, which showed
that Tm1160 was eluted in a peak corresponding to ∼74 kDa
Table 1
Summary of purification procedures of Tm1160 expressed in E. coli.
Step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification fold
Yield (%)
Supernatant of cell lysate
Thermal treatment
Ni-NTA agarose affinity
Superdex-200 gel filtration
29.5
20.3
2.5
7845
6256
5385
4670
266
308
2154
2458
1
100
1.2
8.1
9.2
79.7
68.6
59.5
1.9

W. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 23–30
27
Fig. 4. Temperature optima (A) and thermostability (B) of the esterase Tm1160.
(
a) Temperature optima of Tm1160 was determined with pNP-C4 as substrates
◦
in 50 mM sodium acetate buffer (pH 5.5) at temperatures ranging from 30 C to
1
◦
◦
00 C. Activity at 70 C (optimum temperature) was taken to be 100% (2458 U/mg).
(
b) Thermostability of Tm1160. The residual enzyme activity was measured after
◦
◦
◦
incubation of the purified enzyme at 70 C (diamonds), 80 C (boxes), and 90 C (tri-
angles), respectively. Relative activity was calculated by defining original activity
(
◦
◦
◦
2576 U/mg at 70 C, 1972 U/mg at 80 C and 914 U/mg at 90 C, respectively) as
00%. The values are means of three independent experiments.
1
Fig. 3. (A) Chemical cross-linking of Tm1160. Tm1160 proteins (20 ␮g) were cross-
linked using 2% formaldehyde in PBS at room temperature for 0, 30 and 60 min
(
lanes 1, 2 and 3). (B) Gel filtration of Tm1160. The arrows indicated the eluted
positions of Tm1160 and protein markers (from left to the right): alcohol dehy-
drogenase (150 kDa, 11.85 ml), Tm1160 (12.45 ml), albumin (66 kDa, 12.66 ml) and
carbonic anhydrase (29 kDa, 13.52 ml).
Most of the enzyme activity was maintained after incubation at
70 C for at least 120 min, whereas incubation at 90 C for 60 min
reduced the maximum activity by approximately 50% (Fig. 4B).
◦
◦
Table 2
Kinetic parameters for hydrolysis of various pNP-esters.a
kcat (s−1
−1
−1
)
3.6. Effects of pH on enzyme activity
Substrate
Km (␮M)
)
kcat/Km (s mM
pNP-acetate (C2)
152 ± 10
239 ± 18
47 ± 7
24 ± 5
76 ± 6
158 ± 4
318 ± 7
85 ± 2
The effect of pH on the esterase activity between pH 3.0 and
.0 was measured using various buffers. Contrary to the reported
pNP-butyrate (C4)
pNP-caprylate (C8)
pNP-butyrate (C10)
pNP-laurate (C12)
pNP-myristate (C16)
9
4.0 ± 0.3
1.5 ± 0.2
0.2 ± 0.2
0.055 ± 0.2
22 ± 3
68 ± 2
esterases from T. maritima, which display an optimal activity in the
range 7.0–9.0, Tm1160 show a maximum at pH 5.0–5.5. In addi-
tion, Tm1160 maintained over 90% of its maximal activity in the pH
14 ± 2
14 ± 0.5
3 ± 0.6
16 ± 2
a
◦
The specificity was determined by using pNP-acetate (C2), pNP-butyrate (C4),
pNP-octanoate (C8), pNP-decanoate (C10), pNP-laurate (C12) and pNP-palmitate
C16) as substrates at different substrate concentrations (0.02–1.0 mM). The values
are means of three independent experiments.
range of 5.0–7.5, even after incubation at 70 C for 30 min (Fig. 5A).
Furthermore, the acid tolerance of the esterase was determined
(
◦
by incubating the enzyme at 70 C at different pHs (4.5, 5.0 and
5
.5) and then measuring the residual activity. No significant loss of
esterase activity was found after treatment at pH 5.0 and 5.5 for
120 min, but 50% of the initial activity was lost after incubation at
pH 4.5 for 60 min (Fig. 5B). These results indicated that Tm1160
possesses a high stability to higher temperatures and under acidic
conditions and is a thermoacidophilic esterase. The optimal pHs of
thermophilic esterases from hyperthermophilic archaea and bacte-
ria are observed at around neutrality (pH 6.0–8.0) or alkalinity (pH
8.0–11.0) [13,39,40]. To our knowledge, this is the first example of a
thermophilic and acid tolerant esterase from a hyperthermophilic
bacteria.
3
.5. Effects of temperature on enzyme activity
The effects of temperature on Tm1160 activity were studied
using pNP-C4 as a substrate. The esterase activity of Tm1160 was
examined over the range of 30–100 C. The esterase Tm1160 had
maximal activity at around 70 C and showed more than half
of maximal activity at 50–85 C (Fig. 4A). The thermostability of
Tm1160 was evaluated at three different temperatures (70 C,
8
◦
◦
◦
◦
◦ ◦
0 C, and 90 C) with increasing incubation times up to 120 min.

2
8
W. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 23–30
Table 3
a
Effect of organic solvents on the esterase activity of Tm1160.
Organic solvents
None
Concentration (%)
Relative activity (%)
–
100
Methanol
50(v/v)
90 ± 4
83 ± 1
90(v/v)
Ethanol
50(v/v)
87 ± 3
77 ± 2
90(v/v)
DMF
50(v/v)
72 ± 2
46±
90(v/v)
Acetone
50(v/v)
83 ± 5
53 ± 4
90(v/v)
Isopropanol
Isoamyl alcohol
Chloroform
DMSO
50(v/v)
95 ± 3
68 ± 2
90(v/v)
50(v/v)
98 ± 5
49 ± 7
90(v/v)
50(v/v)
70 ± 5
54 ± 3
90(v/v)
50(v/v)
88 ± 7
52 ± 3
90(v/v)
n-Hexane
Heptane
Isooctane
Formaldehyde
Toluene
90(v/v)
90(v/v)
90(v/v)
90(v/v)
90(v/v)
18 ± 4
22 ± 2
6 ± 2
8 ± 5
12 ± 4
a
The purified Tm1160 was incubated in the presence of organic solvents (50%
or 90%, v/v) at room temperature for 12 h. Residual activity was determined by the
standard method and the values are means of three independent experiments.
Fig. 5. pH optima, pH stability (A) and acid tolerance (B) of the esterase Tm1160.
Table 4
(
a) pH optima (closed boxes) and pH stability (open diamonds) of the enzyme at pHs
a
Effect of various reagents on the esterase activity of Tm1160.
◦
ranging from 3 to 9 was measured using pNP-C4 as substrates for 30 min at 70 C.
The buffers used were 50 mM of sodium citrate (pH 3.0–4.0), sodium acetate (pH
Compound
None
Concentration
Relative activity (%)
4
5
.5–6.0), sodium phosphate (pH 6.5–8.0), and Tris–HCl (pH 8.5–9.0). Activity at pH
.0 was taken to be 100% (2498 U/mg). (b) Acid tolerance of Tm1160. The residual
–
100
109 ± 2
98 ± 3
90 ± 5
73 ± 6
82 ± 3
65 ± 5
102 ± 3
98 ± 1
117 ± 3
99 ± 2
103 ± 6
93 ± 3
97 ± 2
80 ± 4
83 ± 2
55 ± 3
78 ± 4
52 ± 6
23 ± 6
12 ± 2
102 ± 2
98 ± 6
95 ± 2
88 ± 3
2
+
Ca
Al
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
1%(w/v)
5%(w/v)
1%(w/v)
enzyme activity was measured with pNP-C4 as substrates after incubation of the
purified enzyme at pH 4.5 (diamonds), 5.0 (boxes), and 5.5 (triangles). Relative activity
was calculated by defining original activity (1533 U/mg at pH 4.5, 2359 U/mg at pH
3
+
2
+
Co
Cu
2
+
5
.0 and 2503 U/mg at pH 5.5, respectively) as 100%. The values are means of three
2
+
Ni
Fe
independent experiments.
2
+
2
+
Mg
Zn
Mn
2
+
3.7. Stability of Tm1160 in organic solvents
2
+
+
Na
Organic solvents are used in many industrial biocatalysts
and therefore the effect of organic solvents on the activity of
this esterase is of special interest. The effects of polar solvents
+
K
EDTA
Tween20
(
methanol, ethanol, DMF, acetone, isopropanol, isoamyl alcohol
and chloroform) on the enzyme activity were not significant at 50%
v/v) organic solvent and approximately half of the enzyme activity
Tween80
Trtion-X100
SDS
5
%(w/v)
1%(w/v)
%(w/v)
1%(w/v)
%(w/v)
0.5 mM
mM
(
was retained at 90% (v/v) solvent (Table 3). Non-polar solvents had
a remarkably inhibitory effect on the enzyme activity. The relative
activity of Tm1160 was reduced below 20% at 90% (v/v) concentra-
tions of n-hexane, heptane, isooctane, formaldehyde and toluene.
This result demonstrated that the esterase Tm1160 is stable in the
presence of various organic solvents.
5
5
DTT
5
Urea
4 M
8 M
a
Various reagents were added to reaction mixture containing the purified
Tm1160 in 50 mM sodium acetate buffer (pH 5.5) and then incubated at room tem-
perature for 60 min. The reaction was initiated by addition of 0.2 mM pNP-butyrate
and residual activity was measured under the standard conditions. The values are
means of three independent experiments.
3
.8. Effect of metal ions, EDTA and surfactants on the enzymatic
activity
The effect of 5 mM of various metal ions, Ca +, Al3+, Co2+, Cu2+
2
,
Fe2+, Na , K , Ni , Mg , Mn , and Zn , on the enzymatic activ-
+
+
2+
2+
2+
2+
ity was investigated by the standard esterase assay. The activity
The effect of surfactants (Tween 20, Tween 80, Triton-X100, and
SDS), urea, and DTT on the esterase activity were also investigated
by the standard assay. Incubation with Tween 20, Tween 80, or
Triton-X100 at 1% and 5% reduced the esterase activity by less than
50% (Table 4). The enzyme activity was remarkably decreased after
incubation for 60 min with SDS at both concentrations (1% and 5%).
3+
2+
2+
2+
of Tm1160 was not significantly affected by Al , Mg , Zn , Ca
,
2+
+
+
Mn , K , Na and the chelating reagent EDTA (0.5 mM and 5 mM),
2
+
indicating the enzyme was not a metalloprotein. However, Cu
,
2+
2+
Ni and Fe had a slight inhibitory effect on the enzyme activity
Table 4).
(

W. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 23–30
29
Fig. 6. Tm1160-catalyzed enantioselective hydrolysis of racemic ketoprofen ethyl ester. (A) Reaction scheme. (B) Conversion of substrates and enantiomeric excess of racemic
◦
ketoprofen ethyl ester. Reactions were carried out using 0.235 mg of Tm1160 and 25 mg of racemic ketoprofen ethyl ester in 50 mM sodium acetate buffer (pH 5.5) at 70 C.
(
C) E profile of racemic ketoprofen ethyl ester.
Furthermore, the enzyme was not inactivated by 4 M and 8 M urea,
which indicated that the purified Tm1160 showed high stability
toward various compounds known to affect protein folding. The
enzyme activity was not reduced by the addition of DTT (0.5 mM
and 5 mM).
and E value of 10.5 at 20% conversion in 32 h. Two other pro-
fens, (R,S)-naproxen methyl ester and (R,S)-ibuprofen methyl ester,
were used as racemic substrates to test the enantioselectivity
of Tm1160. Tm1160 showed low selectivity in the resolution of
(R,S)-naproxen and (R,S)-ibuprofen (E < 10) (data not shown). These
results demonstrated that Tm1160 has high enantioselectivity to
hydrolyze racemic ketoprofen ethyl ester under high temperatures
conditions and the kinetic resolution is well-suited for industrial
applications.
3.9. Enantioselective hydrolysis of racemic ketoprofen ethyl ester
Several thermophilic esterases have been found to have the
ability to resolve mixtures of chiral esters. The ability of Tm1160
for enantioselective hydrolysis of a chiral compound was tested
with the racemic ketoprofen ethyl ester. Conversion of substrates
4. Conclusions
(
c), enantiomeric ratio (E) and enantiomeric ratio (eep) profiles
In this study, the putative esterase Tm1160 from T. mar-
for the hydrolysis of racemic ketoprofen ethyl ester are shown
in Fig. 6B and C. About 41.1% conversion of (S)-ketoprofen was
obtained after a reaction time of 23 h and c increased very little (c
was 45.2% for 60 h) after 23 h. The highest E value of (S)-ketoprofen
was 91.4 and the eep was 95.7% after 23 h. Keroprofen [(R,S)-2-
itima was cloned, purified and characterized for the first time.
The enzyme behaved as a dimer of ∼74 kDa under native condi-
tions. The purified recombinant Tm1160 possesses high activity
and extreme stability at high temperatures and low pHs, and in
the presence of organic solvents and surfactants. Its biochemical
properties demonstrated that Tm1160 is a thermophilic and acid
tolerant esterase. To our knowledge, Tm1160 is the first character-
ized thermoacidophilic esterase from hyperthermophilic bacteria.
Strikingly, Tm1160 could hydrolyze the racemic ketoprofen ethyl
ester with an eep of 95.7% and a conversion of 41.1% in 23 h. Taken
together, these results indicate sufficient advantages for the devel-
opment of Tm1160 as a potential biocatalyst for chiral resolution
and organic synthesis under thermoacidophilic conditions.
(
3-benzoyl-phenyl) propionic acid] is one of the most prevalent
anti-inflammatory drugs (NSAIDs) and only (S)-ketoprofen is phar-
macologically active to act to reduce inflammation and alleviate
pain [41]. Esterases from the mesophilic organisms are known
to have a high specificity for the hydrolysis of racemic ketopro-
fen ester [42–44]. However, so far only one thermophilic esterase
Est3 from S. solfataricus with a strict stereoselectivity toward (S)-
ketoprofen was identified and characterized [5]. The E value of
Tm1160 was higher than that of Est3, which showed eep of 80%

3
0
W. Tao et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 23–30
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