Cloning, expression, purification, and characterization of a thermostable esterase from the archaeon Sulfolobus solfataricus P1

Article abstract of DOI:10.1016/j.molcatb.2013.05.019

A genomic library of the thermoacidophilic archaeon Sulfolobus solfataricus P1 was constructed using 3.5 kb BamHI-fragments into pUC118 vector and the recombinant plasmids were hosted in Escherichia coli. One positive clone showing thermostable esterase a

Full text of DOI:10.1016/j.molcatb.2013.05.019

Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Cloning, expression, purification, and characterization of a
thermostable esterase from the archaeon Sulfolobus solfataricus P1
Jae-Kyung Nama,1, Young-Jun Parkb,1, Hee-Bong Leea,∗
a Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Republic of Korea
b Cell Therapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A genomic library of the thermoacidophilic archaeon Sulfolobus solfataricus P1 was constructed using
3–5 kb BamHI-fragments into pUC118 vector and the recombinant plasmids were hosted in Escherichia
coli. One positive clone showing thermostable esterase activity was directly selected on tributyrin-
emulsified agar plates. The open reading frame of the esterase gene isolated from this clone was composed
of 942 nucleotides encoding 314 amino acids. The recombinant enzyme stably expressed in E. coli was
purified to apparent homogeneity by two column chromatographies using butyl Sepharose followed by
Q-Sepharose. The molecular mass of the enzyme, estimated to be approximately 35 kDa by sodium dode-
cyl sulfate polyacrylamide gel electrophoresis and gel filtration, agreed with that deduced by sequence
analysis (34,260 Da). Maximal activity was observed at 80 ◦C and pH 8.0. The enzyme was extremely
stable without significant change in its activity up to 120 h at 50 ◦C, and even at 80 ◦C almost 30% of its
activity remained after 120 h. Among the p-nitrophenyl esters (C4–C16) tested, the best substrate was
p-nitrophenyl caprate (C10) with Km and kcat values of 24.0 mM and 2337 s−1, respectively. At 30 ◦C, the
enzyme displayed remarkable stability against up to 90% methanol, ethanol, and 2-propanol, and also
withstood, to a certain extent, 5% SDS and 8 M urea. Site-directed mutagenesis revealed that the enzyme
contains a catalytic triad composed of Ser144, Asp266, and His295 in the active site. The S. solfataricus P1
esterase is an archaeal esterase grouped into family V as well as B-type esterases.
Received 3 March 2013
Received in revised form 18 May 2013
Accepted 20 May 2013
Available online 27 May 2013
Keywords:
Sulfolobus solfataricus P1
Archaeal esterase
Thermostability
Family V
Catalytic triad
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
be grouped in only two families, IV and V. A number of bacterial
enzymes belonging to family IV show high amino acid similar-
Esterases (EC 3.1.1.x) are hydrolases catalyzing the cleavage and
formation of ester bonds. They are widespread enzymes found
in all domains of life: Bacteria, Archaea, and Eukarya [1]. These
ester hydrolases are generally classified into two major groups:
carboxylesterases (EC 3.1.1.1, carboxyl ester hydrolases), which
hydrolyze ester-containing molecules with short chains (<10 car-
bon atoms) that are at least partly soluble in water, and lipases (EC
3.1.1.3, triacylglycerol hydrolases), which catalyze the hydrolysis of
water-insoluble acylglycerols with long chains (≥10 carbon atoms)
[2].
Bacterial esterases, particularly carboxylesterases and lipases,
are classified into eight families (I–VIII) mainly based on the com-
parison of their amino acid sequences and some fundamental
biological properties [3]. Six more families (IX–XIV) were reported
later [4,5]. Among these families, archaeal esterases are known to
ity to the mammalian hormone-sensitive lipase (HSL) family [6].
Hence, esterases grouped in family IV are also called the HSL
family. Esterases from psychrophiles (e.g. Moraxella sp., X53868),
mesophiles (Escherichia coli, AE000153), and thermophiles (Alicy-
clobacillus acidocaldarius, YP 003186268; Archaeoglobus fulgidus,
NP 070544) belong to this family IV. Esterases grouped in fam-
ily V originate from mesophilic bacteria (Haemophilus influenza,
U32704; Acetobacter pasteurianus, AB013096), cold-adapted organ-
isms (Moraxella sp., X53869; Psychrobacter immobilis, X67712),
and heat-adapted organisms (Sulfolobus acidocaldarius DSM639,
YP 256792). These esterases also display significant homology to
non-lipolytic enzymes like epoxide hydrolase, dehalogenase, and
haloperoxidase, which possess the typical ␣/␤ hydrolase fold and a
catalytic triad [7,8]. Most bacterial and archaeal esterases classified
into the HSL family are well characterized. However, family V has
not been well characterized.
Structural studies of carboxylesterases and lipases have
increased remarkably in recent years through the analysis of their
gene sequences and crystal structures [9]. Most of them belong to
the ␣/␤ hydrolase fold enzymes, which usually contain a typical
∗
Corresponding author. Tel.: +82 33 250 8512; fax: +82 33 242 0459.
E-mail address: leehbong@kangwon.ac.kr (H.-B. Lee).
Both these authors contributed equally to this work.
1
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.05.019

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J.-K. Nam et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
catalytic triad composed of Ser, Asp, and His residues. The serine
residue usually appears in the conserved pentapeptide, Gly-x-Ser-
x-Gly (where x is any amino acid residue) [2,7–9].
X-gal
(5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside)
were purchased from MBI Fermentas. All other chemicals employed
in the study were purchased from Sigma Co. unless otherwise
stated.
Esterases currently attract enormous attention because of their
industrial applications such as in detergents, dairy, diagnostics,
and biotransformation [10–12]. Most esterases used in indus-
try are microbial enzymes, of both fungal and bacterial origin
[13,14]. However, their use in industrial processes has many restric-
tions at high temperatures or in the presence of organic solvents.
Hence, enzymes from hyperthermophilic archaea are good candi-
dates because these enzymes show generally high stability against
high temperatures and various chemicals such as sodium dode-
cyl sulfate (SDS), urea, and organic solvents known as denaturing
factors for the enzymes from mesophiles [15–18]. In recent years,
the identification of the complete genome sequences of hyper-
thermophiles makes the study on new esterases more active. S.
solfataricus P1 used in this study is a hyperthermophile belonging
to the thermoacidophilic archaeon, that inhabits sulfur rich vol-
canic hot springs in a high temperature (>75 ◦C) and low pH (<pH
4) environment.
To date, only a few esterases have been purified and character-
ized from archaea, in contrast to the large numbers from bacteria.
Most archaeal esterases are intracellular carboxylesterases as fol-
lows: Archaeoglobus fulgidus [19], Pyrobaculum calidifontis [17],
Pyrococcus furiosus [20], S. acidocaldarius [21–23], S. solfataricus
MT4 [24], S. solfataricus P1 (DSM1616) [15,25], S. solfataricus P1
(DSM5354) [26], and S. solfataricus P2 [16,27]. A few extracellular
archaeal esterases from halophiles like Haloarcula marismortui [28]
and from S. shibatae [29] have been characterized, but not purified.
In this study, we describe the cloning, subcloning, and overex-
pression of an esterase gene (accession number HF586419) from S.
solfataricus P1 (DSM1616) in E. coli cells, and the purification and
characterization of the expressed S. solfataricus P1 esterase from the
cell extract. The S. solfataricus P1 esterase was identified as a B-type
esterase/serine esterase and also belongs to family V esterases con-
taining a catalytic triad composed of Ser144, Asp266, and His295.
The enzyme showed high activity toward medium length acyl chain
(C8–C12) esters, and was found to possess remarkable stability
against high temperatures and various chemicals such as alcohols,
SDS, and urea. The enzyme is the second esterase/isozyme reported
from the same S. solfataricus P1.
2.2. Cloning and expression
Standard molecular cloning techniques were employed
throughout [30]. Chromosomal DNA from S. solfataricus P1 was
isolated from approximately 0.5 g of cells using the method of
Laderman et al. [31], and partially digested using the restriction
enzyme, BamHI. Digested DNA fragments in the size range of
approximately 3–5 kb isolated from agarose gel were ligated into
the Bam HI site in the polylinker region of the cloning vector
pUC118. These recombinant plasmid DNAs were transformed into
E. coli DH5␣ competent cells, and the transformed E. coli DH5␣
cells were screened on LB medium plates containing ampicillin
(100 ␮g/ml), X-gal, and IPTG. White colonies containing the
recombinant plasmid DNA were selected. To examine whether the
esterase gene of S. solfataricus stably existed and was expressed
in the transformed E. coli DH5␣ cells from the white colonies,
each colony was inoculated and cultured overnight at 37 ◦C in
LB medium with ampicillin (100 ␮g/ml). Cells were harvested
by centrifugation at 3000 × g for 15 min at 4 ◦C and washed with
10 mM Tris/HCl buffer (pH 8.0). Cells were resuspended in the same
buffer and then sonicated on ice three times every 30 s, with 30-s
intervals, with maximum power. The sonicated cell extract was
clarified by centrifugation (20,000 × g, 4 ◦C, 30 min). For denaturing
all proteins from E. coli DH5␣ cells except the expressed ther-
mostable proteins from S. solfataricus, the supernatant was heated
at 70 ◦C for 30 min. The denatured E. coli proteins were removed
by centrifugation (100,000 × g, 4 ◦C, 1 h). Subsequently, in order
to quickly identify the existence of active thermostable esterase
from S. solfataricus expressed in the transformed E. coli DH5␣ cells
according to the activity staining method as described below, an
aliquot of the supernatant was loaded on tributyrin-emulsified
agar plates. For further identification of the expression of the S.
solfataricus esterase gene, another aliquot was used for the detec-
tion of esterase activity on SDS-polyacrylamide gel electrophoresis
(PAGE) gels after electrophoretic separation according to another
activity staining method as described below.
2.3. Subcloning and sequencing
2. Materials and methods
Restriction and expression analyses of the recombinant plas-
mids isolated from transformed E. coli DH5␣ cells revealed the
insertion of the esterase gene from S. solfataricus and its expres-
sion. A restriction map of the insert DNA of the isolated plasmid for
subcloning was formulated by comparing the size of the fragments
produced by appropriate restriction enzymes, including the restric-
tion enzymes with recognition sites in the polylinker region, with
each other. Each fragment of the insert DNA digested by the identi-
fied restriction enzymes in the insert DNA was ligated into the same
restriction enzyme site in the polylinker region of the cloning vec-
tor pUC118 and transformed into E. coli DH5␣ competent cells. The
transformed E. coli DH5␣ cells, which formed white colonies, were
screened on LB medium plates containing ampicillin (100 ␮g/ml),
X-gal, and IPTG. Following this, transformants showing esterase
activity as well as containing the esterase gene from S. solfataricus in
the insert DNA were identified on tributyrin-emulsified agar plates
and on SDS-PAGE gels using activity staining methods as described
below. The nucleotide sequence of the insert DNA in the plasmid
obtained from transformed E. coli DH5␣ cells exhibiting esterase
activity was analyzed. DNA sequencing was performed with an ABI
PRISM kit and a model 310 capillary DNA sequencer (Perkin-Elmer
Applied Biosystems, Foster City, CA). The nucleotide sequence of
2.1. Strains, plasmids, growth conditions, and chemicals
S. solfataricus P1 (DSM1616) was purchased from the American
Type Culture Collection (ATCC). S. solfataricus P1 was aerobically
grown at 75 ◦C and pH 4.0 in a 5-L fermentor with moderate
stirring (120 rpm). Growth medium containing 0.2% (wt/vol) yeast
extract and inorganic salts was used according to the description of
ATCC media formulations. Cells were harvested by centrifugation
(4000 × g) for 45 min at 4 ◦C. These cells were stored at −20 ◦C and
used as the genomic DNA donor for cloning of the esterase gene.
E. coli DH5␣ cells were used as host cells for the cloning of the S.
solfataricus esterase gene, whereas E. coli BL21(DE3) harboring the
recombinant plasmid was used for gene expression. The vectors
utilized were pUC118 for cloning and pET-11d (Novagen) for
expression. These E. coli DH5␣ and E. coli BL21(DE3) cells were
routinely grown at 37 ◦C on Luria–Bertani (LB) medium with
ampicillin (100 ␮g/ml). Yeast extract from Difco Laboratories and
Q-Sepharose Fast Flow from Amersham Biosciences were used.
p-Nitrophenyl (PNP) caprylate was purchased from Fluka, and
paraoxon (diethyl-p-nitrophenyl phosphate) was from Merck. All
restriction enzymes, isopropyl-l-d-thiogalactoside (IPTG), and

J.-K. Nam et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
97
the S. solfataricus esterase gene in the insert DNA was analyzed
and verified by comparison with sequence data retrieved from the
Entrez server at NCBI (http://www.ncbi.nlm.nih.gov/Entrez/).
prepared and prewarmed PNP-caprate solution (5 mM), as the sub-
strate, to 0.1 ml of the purified esterase enzyme solution (15 ␮g/ml)
and 0.8 ml of prewarmed 100 mM Tris–HCl buffer (pH 8.0) contain-
ing sodium taurocholate (2 mg/ml) and gum arabic (1 mg/ml), as
emulsifiers, in cells held in the cell holder at 60 ◦C or at a desired
temperature. The initial velocity of the reaction was measured by
monitoring changes in absorbance at 405 nm using a spectropho-
tometer (Shimatzu UV2401PC) fitted with a temperature controlled
cell holder. One unit of esterase was defined as the amount of the
enzyme releasing 1 ␮mol of p-nitrophenol per min. The extinction
coefficients of p-nitrophenol were determined prior to the mea-
surements under each condition. In every measurement, the effect
of nonenzymatic hydrolysis of substrates was taken into consider-
ation and subtracted from the value measured when the enzyme
was added. Measurements were carried out at least three times.
Standard deviations never exceeded 10% of the mean values.
For the determination of substrate specificity, other kinds of p-
nitrophenyl (PNP) esters such as PNP-butyrate (C4, 0.02–5 mM),
PNP-caproate (C6, 0.01–1 mM), PNP-caprylate (C8, 0.01–1 mM),
PNP-caprate (C10, 0.01–1 mM), PNP-laurate (C12, 0.02–2 mM), and
PNP-palmitate (C16, 0.02–5 mM) were also used as substrates.
Activities for all substrates were determined as described above.
2.4. Overexpression and purification of the S. solfataricus esterase
For the overexpression of S. solfataricus esterase, two primers
were designed based on the complete coding sequence of the
esterase gene identified previously, and in order to facilitate
the insertion of the gene into the NcoI site and BamHI site of
the expression vector, pET-11d. The constructed primers were
5ꢀ-CGCCCATGGAAATGAACAGGAGAGACGCT-3ꢀ (forward) and 5ꢀ-
CGCGGATCCTTATCCTAGGAAGGACGTCA-3ꢀ (reverse), where the
underlined letters indicate the bases inserted to create the cloning
sites of NcoI and BamHI, respectively. The S. solfataricus esterase
gene containing NcoI and BamHI sites at both ends was ampli-
fied in a 30-cycle PCR (at 92 ◦C for 1 min, 55 ◦C for 1 min, and 72 ◦C
for 1 min) using as the template the insert DNA, containing the S.
solfataricus esterase gene, from the recombinant plasmid isolated
from transformed E. coli DH5␣ cells in the first gene cloning step
as described previously. The PCR products were separated by elec-
trophoresis in a 0.7% (wt/vol) agarose gel, and the DNA fragments
were purified using the gel purification kit. The PCR product was
digested with NcoI and BamHI, and ligated into the expression vec-
tor, pET-11d, digested with the same enzymes. Following this, the
constructed recombinant DNA plasmid (pSsoEst) was transformed
into E. coli BL21(DE3) cells for overexpression.
2.6. Activity staining
Two kinds of activity staining methods were employed pref-
erentially for tracing esterase activity during cloning, subcloning,
and purification procedures. One was carried out on tributyrin-
emulsified agar plates containing 1.5% (wt/vol) agar powder, 0.01%
(wt/vol) rhodamine B, and 1% (vol/vol) tributyrin in buffer A, as
described previously [15,33]. The presence of active esterase in the
sample was evidenced by the formation of a clear zone on the turbid
tributyrin-emulsified agar plate at 75 ◦C. Triolein (C18:1)-emulsified
agar plates were also prepared for the substrate specificity test.
Another activity staining method [15,24] was employed after
SDS-PAGE, because esterases from hyperthermophilic archaea are
remarkably stable at high temperature and high concentrations of
general denaturants such as alcohols and SDS. A renaturation pro-
cedure was carried out to recover enzyme activity in a gel. The gel
was washed twice in 2-propanol–50 mM Tris–HCl (pH 7.0) (1:4,
vol/vol) for 15 min, rinsed three times in 50 mM Tris–HCl (pH 7.0)
buffer after each 15-min wash, and finally rinsed with water. The
gel was stained in the dark at 37 ◦C by immersing it in a solution
consisting of 100 ml of 50 mM sodium phosphate buffer (pH 7.5)
and 50 mg of Fast Blue BB with 1 ml of acetone solution containing
10 mg ␣-naphthyl acetate (␣-NA), until a dark-gray band appeared
on the gel. The stained gel was rinsed with water and fixed in 3%
(vol/vol) acetic acid.
The transformant was cultivated at 37 ◦C in LB medium with
ampicillin (100 ␮g/ml). When the optical density of the cultured
cells reached 1.0 at 600 nm, 1 mM IPTG was added to induce gene
expression. After induction for 6 h, the OD600 reached approxi-
mately 3.0. The cells were precipitated by centrifugation at 3000 × g
for 40 min at 4 ◦C. The precipitated cells were rinsed with 10 mM
Tris/HCl buffer (pH 8.0) (buffer A) and precipitated again by cen-
trifugation at the same conditions. Cells suspended in the same
buffer A to make a 10% (wt/vol) homogenate were sonicated with
maximum power on ice three times every 30 s, with 30-s inter-
vals, heated at 70 ◦C for 30 min, and then centrifuged at 100,000 × g
for 1 h at 4 ◦C to remove the denatured E. coli proteins. The exis-
tence of the expressed S. solfataricus esterase in the supernatant
was examined using activity staining methods. To purify this
expressed S. solfataricus esterase, the supernatant was adjusted
to 2 M by powdered NaCl and loaded onto a butyl Sepharose col-
umn (3.0 cm × 15 cm), equilibrated with buffer A containing 2 M
NaCl. After washing with buffer A and subsequent elution with a
series of step gradients of 150 ml of buffer A containing 30%, 40%,
50%, 60%, or 70% (vol/vol) ethylene glycol, the fractions containing
esterase activity were pooled. The pooled solution was dialyzed
twice to remove ethylene glycol against 5 L of buffer A for 18 h.
The dialyzed protein solution was applied to a Q-Sepharose col-
umn (3.0 cm × 15 cm) equilibrated with buffer A. After washing
with buffer A and subsequent elution with 200 ml of a linear gradi-
ent of 0–0.3 M NaCl in buffer A, the purified S. solfataricus esterase
was collected from the fractions containing esterase activity. The
presence of active esterase in each collected fraction during enzyme
purification using columns was also primarily judged using activity
staining methods.
2.7. Determination of molecular weight of the purified S.
solfataricus esterase
The molecular weight of the purified esterase was deter-
mined on 12.5% (wt/vol) SDS-polyacrylamide gel with and without
mercaptoethanol in the sample buffer using low-molecular-
weight standards [34]. Gels were stained with silver [35]. The
molecular weight of the native protein was also determined
by high-pressure liquid chromatography (HPLC) [GPC column
(Protein-PakTM 300SW, 7.8 by 300 mm, Waters)] using molecular
weight standards (Sigma MW-GF-200 kit).
The protein concentration was determined by the Bradford
method [32] using bovine serum albumin as a standard.
2.5. Enzyme assay
2.8. Catalytic properties of the S. solfataricus esterase
The standard esterase assay was carried out with an artificial
chromogenic substrate, p-nitrophenyl (PNP) caprate (C10), with a
modified version of the Stöcklein method using emulsifiers [15,18].
The enzyme reaction was started by the addition of 0.1 ml of freshly
The optimum pH and temperature for purified esterase activity
were determined by the standard esterase assay using PNP-caprate.
The effect of pH on esterase activity was examined at 60 ◦C in the pH

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J.-K. Nam et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
Table 1
range 4.0–11.0. The following buffers (100 mM) were used: sodium
citrate (pH 4.0–6.0), sodium phosphate (pH 6.0–8.0), Tris/HCl (pH
8.0–9.0), and sodium bicarbonate (pH 9.0–11.0). The desired pH of
each buffer was adjusted at 60 ◦C. The effect of temperature (rang-
ing from 20 ◦C to 95 ◦C) on esterase activity was investigated at the
optimum pH (pH 8.0).
The thermal stability of the purified esterase was examined
using the standard esterase assay after incubating the enzyme for
the designated time periods (0–120 h) at two different tempera-
tures (50 ◦C and 80 ◦C).
Complementary mutagenic primers for the generation of mutant esterases.
Primera
Sequenceb
Ser144Ala-f
Ser144Ala-r
Ser144Cys-f
Ser144Cys-r
Asp266Asn-f
Asp266Asn-r
His295Asn-f
His295Asn-r
5ꢀ-AATGTTCTAGGCTGGGCAATGGGTGGTTTTG-3ꢀ
5ꢀ-CAAAACCACCCATTGCCCAGCCTAGAACATT-3ꢀ
5ꢀ-AATGTTCTAGGCTGGTGCATGGGTGGTTTTG-3ꢀ
5ꢀ-CAAAACCACCCATGCACCAGCCTAGAACATT-3ꢀ
5ꢀ-ATCGGAGGTGACAGTAATCTTTTACTGCCTC-3ꢀ
5ꢀ-GAGGCAGTAAAAGATTACTGTCACCTGCGAT-3ꢀ
5ꢀ-GCCCTGATGCGGGTAATGGACTGATATAC-3ꢀ
5ꢀ-GTATATCAGTCCATTACCCGCATCAGGGC-3ꢀ
The stability of the esterase against several compounds [water-
miscible organic solvents (methanol, ethanol, and 2-propanol),
detergent (SDS), and urea] was examined by measuring the residual
activity using the standard esterase assay immediately after each
compound was mixed with the enzyme and incubated for 30 min
at 30 ◦C. Blank samples were prepared with the buffer solution
instead of the enzyme, and incubated in the same way as described
above. Control experiments were performed in the absence of the
compound. Each measurement was carried out with two different
concentrations of the compounds: 50% and 90% (vol/vol) organic
solvents, 1% and 5% (wt/vol) SDS, and 4 M and 8 M urea.
The inhibitory effect of chemical modifiers that are specific to
particular amino acids (such as pyridoxal 5ꢀ-phosphate [PLP] to Lys,
phenylglyoxal [PGO] to Arg, HgCl2 and p-chloromercuribenzoate
[PCMB] to Cys, diethyl pyrocarbonate [DPC] to His, and diisopropy-
lfluorophosphate [DFP] and phenylmethylsulfonyl fluoride [PMSF]
to Ser) on the purified esterase was examined. Enzyme activity
was measured using the standard esterase assay after incubating
the enzyme with 0.01 mM or 5 mM of each inhibitor for 30 min
at 30 ◦C. In addition, 0.5 mM and 5 mM paraoxon or eserine, which
are known as indicators for the classification of esterases, were also
examined under the same conditions as described above. To inves-
tigate the effect of divalent cations on esterase activity, the enzyme
was incubated separately with 5 mM of each divalent cation (CaCl2,
CuSO4, FeSO4, MnCl2, MgCl2, and ZnSO4) for 30 min at 30 ◦C. In
order to clarify whether divalent cations are required for the reac-
tion, the enzyme was incubated with 10 mM EDTA for 60 min at
75 ◦C. The residual activities were measured using the standard
esterase assay after incubation.
a
f and r, forward and reverse primers, respectively.
Boldface indicates the mutated nucleotide residues.
b
S. solfataricus esterase genes from these mutant plasmids were
sequenced to confirm the presence of the correct mutations.
2.10. Expression and purification of mutant esterases
The mutant plasmids pSsoEstSer144Ala, pSsoEstSer144Cys,
pSsoEstAsp266Asn, and pSsoEstHis295Asn were transformed into
E. coli BL21(DE3) competent cells for expression and cultivated
in 1 L of LB broth supplemented with ampicillin (100 mg/L) at
37 ◦C. Transformed E. coli BL21(DE3) cells containing the expression
vector plasmid, pET-11d, and the recombinant plasmid, pSsoEst,
were used as controls. When the OD600 of the culture reached
0.5, expression was induced by addition of 1 mM isopropyl-␤-d-
thiogalactopyranoside (IPTG). Mutant esterases were subsequently
purified by the same method as mentioned in the purification of S.
solfataricus esterase.
3. Results and discussion
3.1. Cloning, subcloning, and sequence analysis of S. solfataricus
esterase gene
To isolate the S. solfataricus P1 esterase gene, 3–5 kb fragments
of genomic DNA partially digested with Bam HI were inserted into
the cloning vector pUC118. E. coli DH5␣ transformants containing
the S. solfataricus esterase gene in the recombinant plasmid were
screened on LB-ampicillin-X-gal-IPTG plates and on tributyrin-
emulsified agar plates. Among 800 white colonies, two positive
colonies were selected on tributyrin-emulsified agar plates. The
size of the insert DNA containing the S. solfataricus esterase gene
in the plasmid from one of the two positive transformants was
approximately 4.5 kb. A restriction map of the insert DNA of the
plasmid for subcloning was generated using restriction enzymes in
the polylinker region of the cloning vector pUC118, and SacI and
PstI sites were identified in the insert DNA. Various DNA fragments
of the insert DNA produced by these two enzymes were subcloned
into the cloning vector pUC118, and then the recombinant plasmids
were transformed into E. coli DH5␣ cells. A transformant harboring
the recombinant plasmid, which contained a BamHI/SacI fragment
as the insert DNA, was identified as containing the S. solfataricus
esterase gene and found to produce the active S. solfataricus esterase
on tributyrin-emulsified agar plates and on SDS-PAGE gels using
the activity staining method. This subfragment (2846 nucleotides)
was fully sequenced and analyzed for the identification of the S.
solfataricus esterase gene. The identified open reading frame (ORF)
of the S. solfataricus esterase gene (accession number HF586419)
was composed of 942 nucleotides encoding 314 amino acids.
2.9. Construction of mutant S. solfataricus esterase plasmids
To understand the catalytic mechanism of the esterase, the
constructed recombinant DNA plasmid, pSsoEst, containing the S.
solfataricus esterase gene in the expression vector, pET-11d, was
used as the template for the generation of mutations (Ser144Ala,
Ser144Cys, Asp266Asn, and His295Asn) in the expected catalytic
triad, Ser-Asp-His, of the S. solfataricus esterase gene by site-
directed mutagenesis. Mutagenic oligonucleotide primers were
designed, and PCR based on the QuikChange II site-directed muta-
genesis kit (Stratagene) for the construction of S. solfataricus
esterase mutant plasmids (pSsoEstSer144Ala, pSsoEstSer144Cys,
pSsoEstAsp266Asn, and pSsoEstHis295Asn) was carried out using
pSsoEst as the template DNA, and forward and reverse primers
with lengths of 29–31 bases (Table 1) mutated at the desired
sites (Ser144Ala, Ser144Cys, Asp266Asn, and His295Asn) in the
S. solfataricus esterase gene, according to the guidelines and
protocol recommended by the manufacturer. The PCR condi-
tions were as follows: 1 cycle of 30 s at 95 ◦C, followed by 12
cycles of 30 s at 95 ◦C, 1 min at 55 ◦C, and 7 min at 72 ◦C. The
products were digested with 1 ␮l of DpnI (10 U/␮l) at 37 ◦C
for 1 h to digest the template DNA, and then the PCR prod-
ucts were transformed into E. coli DH5␣ ultra-competent cells to
obtain the mutant plasmids pSsoEstSer144Ala, pSsoEstSer144Cys,
pSsoEstAsp266Asn, and pSsoEstHis295Asn. Finally, the inserted
3.2. Amino acid sequence comparison
The identified amino acid sequence of S. solfataricus esterase was
aligned with those of other known archaeal and bacterial esterases

J.-K. Nam et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
99
MNRRDAIKTLGAGAIAGALATRIPLSISQTPPQINYSNLVPKEFGWAPVNNIHIYYEIYG: 60 SsoP1Est
MNRRDAIKILGAGAIAGALATRIPLSISQTPPQINYSNLVPKEFGWAPVNNIHIYYEIYG: 60 SacEst
--------------------------------------MALPPPETCTIGDVTLAYRTYG: 22 MboEst
-------------------MKQIPRSIS---PEIAASQL--------QVMDITMNCRVLG: 30 CphEst
------------------------------------------------------------: 00 SsoP2Est
SGEPLIMIMGYLGNLESWGPIIINGLASQYEVIIFDNRGTGRSGTVGQDPLHDALTYTIP: 120 SsoP1Est
SGEPLIMIMGYLGNLESWGPIIINGLASQYEVIIFDNRGTGRSGTVGQDPLHDALTYTIP: 120 SacEst
TGFPLVLINGLASAMDTWNPPVLDALARHFRVIVFDHRGTGYSGASAE-------SFSIP: 75 MboEst
SGHPLLMIMGYGSTMNLWESTLLEKLAEHFKVIVFDNRGIGGTQT-GTQP------FSIG: 83 CphEst
-----MLIHHLAGSYKSW-KFVIPKLSLDNTVVAYDLRGHGRSSTPNS-------PYNIE: 47 SsoP2Est
LYASDTIGLLNYLGYSNLNVLGWSMGGFVAQQIAIDYPSYVNKLVL---L---CTAPNIY: 174 SsoP1Est
LYASDTIGLLNYLGYSNLNVLGWSMGGFVAQQIAIDYPSYVNKLVL---L---CTAPNIY: 174 SacEst
LFSRDTAGLMEHLGITRAHVLGFSMGTCIAQELELAFPEKVDRLVL---VSGDCGGTEAV: 132 MboEst
QFAEDSAALLQSLDVEHAHVLGWSMGSLIAQELALRHPSLISKLIL---Y---AAHCDAK: 137 CphEst
DHSNDLRRLLVQLGIEKPVLIGHSIGSLIAIDYALKYP--IEKLVLIGAL---YRAPSSE: 102 SsoP2Est
LYPPKVSPQSIITGFTASDPTVTVETIIPYLVPSDWLQAHPDVAKYVLFTLEKYPISYTS: 234 SsoP1Est
LYPPKVSPQSIITGFTASDPTVTVETIIPYLVPSDWLQAHPDVAKYVLFTLEKYPISYTS: 234 SacEst
RTDPDVFARLIDRSGTIDD---VVNRMLPLLFPSFWLATH-DPFRYCPAVEE-----ITS: 183 MboEst
MFPPSPEVLTMLTD-TSGTPEERGMRYISTLFPENWLHGNGQRMKEIFFR-PMGTIPEES: 195 CphEst
VYEKYVRI-AVNFGMRALAEYRRFHKEFTETLASNYQAWNSLLEVYE----ETTPIGYKN: 157 SsoP2Est
VLKQTNALATF----N-SVGQLQNITAPTLVIGGDSDLLLPPQNSQYLAENIPNAQLYIF: 289 SsoP1Est
VLKQTNALATF----N-SVGQLQNITAPTLVIGGDSDLLLPPQNSQYLAENIPNAQLYIF: 289 SacEst
DENAARQLAAFLSWPG-TFSRLENIRSRTLVITGDADAVVPCENSRLLAGKIPGAKLVEF: 242 MboEst
VGRQAAAIDEW----NGTTGKLGEITCPTLLITGSEDKLTVPDNARYMSERIPDAELIII: 251 CphEst
AVE--GLLKAK----D-YSDELMGINAKTLIVYGTYDGLIV—-NLNVFKNNMKNVEIKTI: 208 SsoP2Est
SPDAGHGLIYQYPTQFINLVTSFLG : 314 SsoP1Est
SPDAGHGLIYQYPTQFINLVTSFLG : 314 SacEst
-PGAGHGLMYQCPDRFGETVLDFL- : 265 MboEst
E-NGGHGLMFQYPEIFRDSVIGFLG : 275 CphEst
D-GYGHFLNFENPLLLSEIIKNFL- : 231 SsoP2Est
Fig. 1. Multiple-sequence alignment of S. solfataricus P1 esterase (SsoP1Est, HF586419), S. acidocaldarius DSM639 esterase (SacEst, AF071233), Methanoregula boonei 6A8
esterase (MboEst, YP 001403315), Chlorobium phaeobacteroides DSM266 esterase (CphEst, YP 92040), and S. solfataricus P2 putative esterase (SsoP2Est, NP 341668). The
catalytic triad (Ser144, Asp266, and His295) in S. solfataricus P1 esterase deduced from the alignment is indicated by an arrow. The consensus sequence motif Gly-x-Ser-
Met-Gly-Gly around the active site Ser144 is boxed. The characteristic conserved sequence motifs around the catalytic triad of esterases grouped in family V are indicated
by closed circles.
showing high similarity from the BLAST search (Fig. 1). Among
archaeal esterases, the amino acid sequence of the S. solfataricus P1
esterase was the most similar to that of S. acidocaldarius DSM639
esterase (accession number YP 256792), with 99.7% identity, and
next to that of Methanoregula boonei 6A8 (YP 001403315, 36% iden-
tity). Among bacterial esterases, the amino acid sequence of S.
solfataricus P1 esterase showed the highest identity (36%) with that
of Chlorobium phaeobacteroides DSM266 esterase (YP 92040). On
the contrary, the amino acid sequence of S. solfataricus P1 esterase
showed relatively low similarities to those of several esterases
reported from S. solfataricus P2 belonging to same species and,
among these esterases, the highest identity (25%) was seen with
a putative esterase (NP 341668) composed of 231 amino acids.
However, we could not find any proteins whose amino acid
sequences were completely coincident with that of S. solfataricus
P1 esterase from the BLAST search. Furthermore, the amino acid
sequence of this S. solfataricus P1 esterase is completely different
from that of another S. solfataricus P1 (DSM1616) esterase com-
posed of 305 amino acids, reported as the only carboxylesterase
from this S. solfataricus P1 strain [15], and no significant similarity
between the two was found in a BLAST search.
that the esterase may also be classified into family V, because S.
acidocaldarius esterase is the only archaeal esterase grouped in fam-
ily V among the eight families (I to VIII) according to the initial
classification of bacterial lipolytic enzymes proposed by Arpigny
and Jaeger [3]. Since only two archaeal esterases, Archaeoglobus
fulgidus carboxylesterase (NP 070544) grouped in family IV and S.
acidocaldarius esterase (YP 256792) classified into family V, were
presented by them, a number of archaeal esterases have been iso-
lated and characterized. Regardless, most archaeal esterase families
are not well established; in particular, family V, probably the least-
known family of archaeal esterases, has not been well studied. All
archaeal esterases such as A. fulgidus carboxylesterase (NP 070544),
Pyrobaculum calidifontis carboxylesterase (BAC06606), S. toko-
daii carboxylesterase (BAB65028), S. shibatae carboxylesterase
(BAD82944), S. solfataricus P1 carboxylesterase [15], S. solfatar-
icus MT4 carboxylesterase [24] and S. solfataricus P2 esterase
(NP 343858) reported later were grouped into family IV (HSL fam-
ily) and also relatively well characterized. Moreover, the crystal
structures of A. fulgidus carboxylesterase and S. tokodaii car-
boxylesterase are well known [36,37]. On the contrary, there has
been no additional archaeal esterase grouped in family V. To the
best of our knowledge, S. acidocaldarius esterase (YP 256792) was
the only archaeal easterase classified into family V on the basis
Here, the 99.7% amino acid sequence identity of the S. sol-
fataricus P1 esterase with the S. acidocaldarius esterase suggests

100
J.-K. Nam et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
120
(A)
100
80
60
40
20
0
4
5
6
7
8
9
10
11
pH
120
100
80
60
40
20
0
(B)
Fig. 2. Electrophoretic analysis of recombinant S. solfataricus P1 esterase purified
from E. coli. Samples were loaded on a SDS-PAGE gel in duplicate and after being
run the gel was cut. One half was stained with silver (A) and the other was stained by
the activity staining method using the chromogenic substrate, ␣-naphthyl acetate,
after renaturation (B).
of amino acid sequence comparison. However, S. acidocaldarius
esterase was neither purified nor were its biochemical and catalyt-
ical properties well characterized. Hence, S. solfataricus P1 esterase
(HF586419) in this study is an archaeal esterase classified into
family V that was purified and characterized with respect to its
biochemical and catalytical properties.
20 30 40 50 60 70 80 90 100
Temperature (ºC)
Fig. 3. Effects of pH and temperature on the activity of the purified S. solfataricus P1
esterase. The effects of pH and temperature on the activity of the purified esterase
were determined spectrophotometrically using PNP-caprate as a substrate. (A and
B) The enzyme activity was examined at 60 ◦C and the indicated pH values (A) or
at pH 8.0 and the indicated temperatures (B). The pH was obtained using 100 mM
sodium citrate buffer (ꢀ) for pH 4.0–6.0, 100 mM sodium phosphate buffer (ꢁ) for pH
6.0–8.0, 100 mM Tris–HCl buffer (᭹) for pH 8.0–9.0, and 100 mM sodium bicarbonate
buffer (ꢁ) for pH 9.0–11.0. Activity at optimal pH (A) or at optimal temperature (B)
was taken as 100%. Error bars indicate standard deviations.
In addition, enzymes grouped in family V possess the typical
␣/␤ hydrolase fold, the catalytic triad (Ser, Asp, and His), and the
characteristic conserved sequence motifs around the catalytic triad.
Hence, the above suggestion that the S. solfataricus P1 esterase
belongs to family V esterases is strengthened by the observation
that it had typically conserved sequence motifs, marked with closed
circles around the Ser, Asp, and His as shown in Fig. 1: Met is
conserved just next the Ser in a consensus sequence, Gly-x-Ser-
Met-Gly-Gly; Pro-Thr-Leu-Val and Gly-Asp are conserved before
the Asp; Gly is conserved just before the His, etc. Sequence align-
ment also revealed that S. solfataricus esterase contains a typical
catalytic triad composed of Ser144, Asp266, and His295, and the
consensus sequence (Gly-x-Ser-x-Gly) around the active site serine.
In particular, S. solfataricus P1 esterase contains a typical conserved
sequence (Gly-x-Ser-Met-Gly-Gly) of family V esterases around the
active site Ser144 residue, unlike the previously described typical
consensus sequence (Gly-x-Ser-Ala-Gly-Gly) of archaeal esterases
grouped in family IV (HSL family) [3].
identical to the molecular mass obtained from SDS-PAGE and
deduced from the amino acid sequence (34,260 Da). These results
indicate that the enzyme is monomeric. In addition, SDS-PAGE
followed by activity staining using ␣-NA showed that the purified
enzyme displays esterase activity (Fig. 2B). This result indicates
that heating the sample buffer containing the purified enzyme,
1% mercaptoethanol, and 1% SDS for 1 min in boiling water before
performing the SDS-PAGE does not appear to significantly affect
enzyme activity. This enzyme stability against boiling water and
1% SDS was confirmed by examining the effect of temperature and
various denaturing agents on enzyme activity (see below).
3.3. Overexpression and purification of S. solfataricus esterase
3.4. Catalytic properties of S. solfataricus esterase
For the overexpression of S. solfataricus esterase, the recom-
binant plasmid containing the esterase gene inserted into the
NcoI and BamHI sites of the expression vector, pET-11d, was
transformed into E. coli BL21(DE3), and its expression was induced
by the addition of IPTG. The S. solfataricus esterase was found to be
stably expressed in E. coli BL21(DE3) cells. The expressed esterase
was purified to apparent homogeneity in five steps starting from
the cell extract as shown in Table 2. The enzyme was purified
12-fold with a yield of 80%. The apparent homogeneity of the
purified enzyme was confirmed by SDS-PAGE, with a single band
of approximately 35 kDa (Fig. 2A). The molecular mass of this
enzyme, determined using a HPLC sizing column, was almost
The effect of pH and temperature on the activity of S. solfatari-
cus esterase was determined using the standard enzyme assay. The
enzyme activity was assayed at several different pH values at 60 ◦C,
and at different temperatures at the established optimum pH. S. sol-
fataricus esterase displayed maximal activity at pH 8.0 (Fig. 3A) and
at 80 ◦C (Fig. 3B).
The thermostability of S. solfataricus esterase was evaluated
at 50 ◦C and 80 ◦C with increasing incubation time up to 120 h
(Fig. 4). No significant change in enzyme activity was observed up
to 5 days at 50 ◦C. Even at 80 ◦C, 31% of it was preserved after 5
days, although enzyme activity as a function of time proportion-
ally decreased with increasing temperature. The S. solfataricus P1

J.-K. Nam et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
101
Table 2
Purification of the S. solfataricus P1 esterase.
Procedure
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purification (fold)
Cell extract
38
20
14
5.8
2.5
4354
4155
4016
3810
3480
115
208
287
657
1392
100
95
92
88
80
1
1st heat treatment
2nd heat treatment
Butyl-sepharose
Q-sepharose
1.8
2.5
5.7
12.1
enzyme activity. The enzyme still retained 35% residual activity in
5% SDS. Sixty percent of the enzyme activity was retained at 4 M
urea. Even with 8 M urea, 25% of the enzyme activity was retained.
In addition to thermostability, the S. solfataricus P1 esterase displays
high stability against high concentrations of various compounds
such as organic solvents (methanol, ethanol, and 2-propanol),
detergent (SDS), and urea, which significantly affect protein fold-
ing in the case of mesophilic enzymes. For example, the stability
of S. solfataricus P1 esterase against these alcohols or SDS is much
higher than those of mesophilic enzymes like Penicillium expansum
lipase [18]. In the case of archaeal esterases, the stability of S. solfa-
taricus P1 esterase against these alcohols is similar to those of the
carboxylesterases from the archaeon P. calidifontis [17] and S. sol-
fataricus P1 [15] and the arylesterase from S. solfataricus P1 [25].
However, the S. solfataricus P1 esterase displays much higher sta-
bility against these alcohols than does the carboxylesterase from S.
solfataricus P2, composed of 308 amino acids [16]. The enzyme also
displays high stability against high concentrations of SDS (5%) and
urea (8 M); however, its stability against 5% SDS is higher than that
of S. solfataricus P1 arylesterase [25] and similar to that of S. solfatar-
icus P1 carboxylesterase [15], and its stability against 8 M urea is a
little lower than those of S. solfataricus P1 arylesterase [25] and the
S. solfataricus P1 carboxylesterase [15]. The stability of S. solfataricus
esterase against high temperature and high concentrations of alco-
hols, SDS, and urea suggests that strong hydrophobic interactions
may make up the stable core of the enzyme. The stability in the
presence of various compounds, along with the thermal stability of
this esterase, can make it very attractive for future applications in
industry as well as in biotechnology.
120
100
80
60
40
20
0
0
20
40
60
Time (h)
80
100
120
Fig. 4. Stability of purified S. solfataricus P1 esterase at different temperatures. The
residual activity was determined after incubation of the enzyme for the indicated
times at 50 ◦C (᭹) or 80 ◦C (ꢁ). Activity measurement was carried out using the
standard enzyme assay. Activity at zero time was taken as 100%. Error bars indicate
standard deviations.
esterase exhibits remarkable stability against high temperatures
like all enzymes including esterases from thermophilic archaea
[15–27,38,39]. We can assume that this stability against high tem-
perature can be expected from the natural thermal habitats of
hyperthermophilic archaea and also derived from strong interac-
tions, including hydrophobic interactions, inside the enzyme.
The effect of various chemicals [water-miscible organic solvents
(methanol, ethanol, and 2-propanol), SDS and urea] on S. solfatar-
icus esterase after incubation of the enzyme with each chemical
for 30 min at 30 ◦C is shown in Fig. 5. Although an increase in the
concentration of each compound resulted in decreasing enzyme
activity, in general the enzyme displayed considerable stability in
the presence of these compounds. In the presence of up to 90%
of each organic solvent, the enzyme was not so much inactivated
as stabilized. The addition of 1% SDS did not significantly affect
The substrate specificity of S. solfataricus esterase was investi-
gated using PNP esters with various acyl chain lengths (C4–C16). As
shown in Table 3, the Km value was the lowest with PNP-caprate
(C10) but the kcat value was the highest with PNP-caprate. Hence,
among the PNP esters tested, PNP-caprate (C10) was observed to
have the highest specificity constant (97.4 s−1 ␮M), and the Km
and kcat values for it were 24.0 ␮M and 2337 s−1, respectively.
Concerning the substrate specificity of S. solfataricus P1 esterase,
kinetic studies using PNP esters showed that PNP-caprate (C10
)
was the most suitable substrate for the enzyme, identical with
results obtained using Thermus thermophilus esterase [40]. Among
the PNP esters, the most suitable substrate was PNP-valerate (C5)
or PNP-caproate (C6) for most archaeal carboxylesterases reported
[16,17,19,22–27]. On the contrary, esterases from Picrophilus tor-
ridus [41] and Pyrococcus furiosus [20] were active with PNP-acetate
as the most favored substrate. These data suggest that the S. solfa-
taricus P1 esterase is a medium-length acyl esterase containing a
140
120
100
80
60
40
20
0
Table 3
MeOH
EtOH
2-PrOH
SDS
Urea
Kinetic parameters for the hydrolysis of various PNP esters.
kcat (s−1
)
kcat/Km (s−1 ␮M−1
)
Chemical compounds
p-Nitrophenyl substrate
Km (␮M)
Butyrate (C4)
Caproate (C6)
Caprylate (C8)
221.5 19.1
50.8 3.2
28.8 1.5
24.0 1.1
36.5 2.8
130.1 8.9
861 27
1476 59
1491 76
2337 92
1312 80
1138 68
3.9
29.1
51.8
97.4
35.9
8.7
Fig. 5. Effects of various chemical compounds, alcohols (methanol, ethanol, and 2-
propanol), SDS and urea, on the stability of S. solfataricus P1 esterase at 30 ◦C. MeOH,
EtOH, and 2-PrOH indicate methanol, ethanol, and 2-propanol, respectively. White
bars indicate controls of alcohols, SDS, and urea, respectively. Gray bars indicate
the concentrations of 50% alcohols, 1% SDS, and 4 M urea, respectively. Black bars
indicate the concentrations of 90% alcohols, 5% SDS, and 8 M urea, respectively.
Caprate (C10
Laurate (C12
Palmitate (C16
)
)
)

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J.-K. Nam et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
Table 4
Table 5
Effect of various inhibitors on the activity of S. solfataricus P1 esterase.
Specific activities of native and mutant S. solfataricus P1 esterases.
Concentration (mM)
Relative residual
activity after
Sp act (U/mg)
Relative activity (%)
100
Native
1192.99
8.58
24.38
25.74
58.60
incubation for 30 min
Ser144Ala
Asp266Asn
His295Asn
Ser144Cys
0.72
at 30 ◦C (%)
2.04
2.16
4.91
Control
PLP
PGO
PMSF
DFP
PCMB
HgCl2
DPC
0
5
5
5
0.01
5
5
5
5
0.5
5
0.5
5
100
115
8
5
1
2
4
4
2
5
1
2
4
6
95
28
25
98
100
23
87
28
10
91
82
3.5. Examination of amino acid residues related to the catalytic
activity of S. solfataricus esterase
EDTA
Paraoxon
Table 4 shows that the modification of Ser or His with a specific
chemical modifier resulted in the loss of enzyme activity. Inhibition
of the enzyme by chemical modifications of Ser and His residues
could be expected from the sequence alignment data. As shown
in Fig. 1, the S. solfataricus esterase was presumed to contain the
catalytic triad composed of Ser144, Asp266, and His295. Hence,
these three amino acid residues, Ser144, Asp266, and His295,
were replaced using site-directed mutagenesis to create Ser144Ala,
Asp266Asn, and His295Asn, in order to confirm whether these
three amino acids are related to the catalytic mechanism in the
active site of this enzyme. The mutant proteins behaved identical to
the wild type on SDS-PAGE (data not shown). The results are shown
in Table 5. The specific activities of all three mutant esterases,
Ser144Ala, Asp266Asn, and His295Asn, were significantly lower
than that of the wild type. Moreover, all three mutant esterases
showed little activity. These results strongly suggest that Ser144,
Asp266, and His295 comprise a catalytic triad in the active site of
the S. solfataricus esterase. In particular, the substitution of the ser-
ine residue in the active site of the enzyme with alanine severely
affects enzyme activity. The result indicates that the enzyme is
a serine esterase. In addition, the exchange of Ser to Cys in the
active site of S. solfataricus P1 esterase (Ser144Cys mutant) was
also examined because the Ser or Cys residue can function as a
nucleophile to the substrate in the active sites of ␣/␤-hydrolase fold
enzymes. However, the activity of the Ser144Cys mutant esterase
was also significantly lower, although it was much higher than that
of Ser144Ala. This result was similar to results obtained with the
serine proteases subtilisin [45,46] or trypsin [47], which belong
to the same family of ␣/␤-hydrolase fold enzymes. These results
indicate that these three amino acids, Ser144, Asp266, and His295,
forming the catalytic triad, are crucial for the activity of the S. sol-
fataricus P1 esterase.
Eserine
hydrophobic binding pocket at its active site. Short (C4) and long
chain esters (C12 and C16) were hydrolyzed as well, albeit at a lower
rate. In addition, the S. solfataricus P1 esterase generated a clear
zone on a turbid tributyrin (C4:0)-emulsified agar plate but not
on a triolein (C18:1)-emulsified agar plate (data not shown). These
results indicate that the S. solfataricus P1 esterase shows broad
substrate specificity possessing high esterase activity toward PNP
esters with medium chains and lipase activity toward short acyl
chain triacylglycerols such as tributyrin.
To investigate the amino acids involved in the catalytic mecha-
nism, the effects of various chemical modifiers that are specific to
particular amino acids were measured using the standard enzyme
assay after incubation for 30 min at 30 ◦C. As shown in Table 4,
the purified enzyme was inhibited by 0.01 mM DFP, 5 mM PMSF,
5 mM DPC, and 5 mM paraoxon, respectively. However, PLP, PGO,
PCMB, HgCl2, and eserine had no significant effect on enzyme
activity. These results of enzyme inhibition by serine-specific
inhibitors (DFP and PMSF) and a histidine-specific inhibitor (DPC)
suggested that Ser and His residues are located at or near the
active site and are involved in the catalytic activity of the S.
solfataricus esterase, and also indicated that the esterase might
contain a ‘catalytic triad’ consisting of Ser, His, and Asp, typically
found in active sites of bacterial and archaeal esterases as shown
in Fig. 1. In addition, the S. solfataricus esterase was inhibited
by organophosphates such as paraoxon and DFP, but not inhib-
ited significantly by eserine (Table 4). Esterases can be divided
into three groups on the basis of their substrate specificities and
sensitivities to various inhibitors [42,43], although they exhibit
overlapping substrate specificities, leading to problems in identi-
fication and classification [44]. Arylesterases (A-esterases) are not
inhibited by organophosphates such as paraoxon, and hydrolyze
aromatic esters. Carboxylesterases (B-esterases/serine esterases)
are inhibited by organophosphates and have wide substrate speci-
ficity, hydrolyzing aliphatic and aromatic esters. Acetylesterases
(C-esterases) are inhibited by organophosphates and eserine and
hydrolyze choline esters. From this, the S. solfataricus P1 esterase in
this study can be classified as a B-esterase (carboxylesterase/serine
esterase), similar to the carboxyleasterases from A. fulgidus [19]
and S. solfataricus P1 [15]. However, the S. solfataricus P1 esterase is
completely different from the A. fulgidus and S. solfataricus P1 car-
boxyleasterases, because the former is grouped in family V, but the
latter are grouped in family IV.
4. Conclusion
The present study reports that an esterase gene (accession
number HF586419) was isolated from 3 to 5 kb fragments of S.
solfataricus P1 (DSM1616) genomic DNA partially digested with
BamHI by cloning, subcloning, and expression in E. coli, and that the
thermostable S. solfataricus esterase was stably expressed in E. coli
BL21(DE3) cells, purified to apparent homogeneity, and its bio-
chemical and catalytical properties characterized. The S. solfataricus
P1 esterase was different from other known archaeal esterases in
its amino acid sequence as well as some properties. This esterase
from S. solfataricus P1 is an esterase classified as a member of
B-esterases/serine esterases, which exhibit the typical behavior
of carboxylesterases including partial lipase activity, and is also
grouped into family V esterases, which possess the typical ␣/␤-
hydrolase fold and a catalytic triad composed of Ser144, Asp266,
and His295. The enzyme showed remarkable stability against high
temperatures and various chemicals such as alcohols, SDS, and urea.
Enzyme activity was not affected by incubation with 5 mM of
various divalent cations for 30 min at 30 ◦C (data not shown) or
by incubation with 10 mM EDTA for 30 min at 75 ◦C. These results
suggest that the S. solfataricus esterase does not require any divalent
cation for activity.

J.-K. Nam et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 95–103
103
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