T. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 225–232
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2.7. Substrate specificity
only 11–17% sequence identity with the characterized thermophilic
carboxylesterases (data not shown). BLAST-P analysis revealed the
highest similarity to other hypothetical proteins and a putative
esterase. The deduced amino acid sequence of ST2026 showed
58, 58, 59, 47, 43, and 42% sequence identity with the putative
esterases from Sulfolobus acidocaldarius DSM 639 (Saci 0120), Sul-
folobus solfataricus (SSO2979), Acidianus hospitalis W1 (Ahos 0898),
Metallosphaera cuprina Ar-4 (Mcup 0788), Thermaerobacter mar-
ianensis DSM 12885 (Tmar 0396), and Thermobaculum terrenum
ATCC BAA-798 (Tter 0425). In addition, the multiple sequence
alignment revealed that EstSt7 contains a typical catalytic triad
composed of Ser131-Asp263-His299 and the conserved motif Gly-
X-Ser-Ser-Gly-Gly-Trp-Gly containing the active site residue. Based
on the results of the secondary structure prediction and conserved
domain search, EstSt7 consists of a large domain with a classical
␣/ hydrolase core including seven ␣-helices (␣1–␣4 and ␣8–␣10)
and seven-stranded -sheets (1–7), and a smaller domain com-
prising three ␣-helices (␣5–␣7) (Fig. 1). The hypothetical model of
EstSt7 was constructed using the 3D-structural threading program
Phyre. The Esta protein from Streptococcus pneumonia (PDB: 2uz0)
was used to build the model of EstSt7 (Supplementary Fig. 1). The
overall hypothetical structure covered the ␣/ hydrolase core of
EstSt7 (amino acid residues 1–162 and 220–332), but the smaller
domain has not been modeled.
Substrate specificity was determined by hydrolysis of differ-
ent pNP-esters including pNP-acetate (C2), pNP-C4, pNP-octanoate
(C8), pNP-decanoate (C10), pNP-laurate (C12), pNP-myristate
(C14), and pNP-palmitate (C16). The Km and Vmax values were deter-
mined by measuring the initial velocity of hydrolysis at different
substrate concentrations (0.001–1.0 mM) in three independent tri-
als. The corresponding Km and Vmax values were computed using
Hanes–Wolff plots and the Michaelis–Menten equation.
2.8. Effect of temperature and pH on enzyme activity and stability
The effects of temperature and pH on the esterase activity
were examined using pNP-C4 as the substrate. The optimum pH
was studied at 80 ◦C in the pH range of 5.0–11.0. The following
buffers (50 mM) were used: sodium acetate (pH 5.0–6.0), sodium
phosphate (pH 6.0–7.5), Tris–HCl (pH 7.5–9.5), and N-cyclohexyl-
3-aminopropanesulfonic acid (CAPS, pH 9.5–11.0). The pHs of the
different buffers were adjusted at 80 ◦C. The pH stability was mea-
sured after incubation of the purified enzyme (23 g/ml) in the
reaction mixture at 80 ◦C for 60 min, and then the residual enzyme
activity was measured by the standard method. The optimum tem-
perature was determined at temperatures ranging from 30 ◦C to
100 ◦C in 50 mM of Tris–HCl buffer (pH 9.0). The buffer solutions
were adjusted to pH 9.0 at each assayed temperature. In addi-
tion, the thermostability of the purified esterase (23 g/ml) was
examined in 50 mM Tris–HCl buffer (pH 9.0) at three different tem-
peratures (70 ◦C, 80 ◦C, and 90 ◦C). Each sample (50 l) was assayed
after incubation for 60, 120, 180 or 240 min. The residual activities
were assayed under the standard conditions.
To clarify the possible evolutionary position of EstSt7 and its
fication of bacterial esterases and lipases proposed by Arpigny and
Jaeger [3]. Phylogenetic tree analysis showed that EstSt7 and its
homologs, Saci 0120 from S. acidocaldarius DSM 639 and SSO2979
from S. solfataricus are not grouped in any of the eight families and
instead formed a distinct group (Fig. 2). This divergence from the
current families can be verified by aligning the conserved motif
(Supplementary Fig. 2). A highly conserved motif GXSSGGYG (X
indicating any amino acid), which is different from the consensus
of the current esterase and lipase families, was identified in EstSt7
and its homologs. Taken together, these data suggest that EstSt7
and its homologs should form a new bacterial esterase and lipase
family that diverges from the eight families proposed by Arpigny
and Jaeger. The enzymes belonging to this new family have not
from other thermophilic bacteria have been grouped into new car-
boxylesterase families including PhaZ7 from Paucimonas lemoignei,
EstD from Thermotoga maritima, Est30 from Geobacillus stearother-
mophilus, and EstGtA2 from Geobacillus thermodenitrificans, rather
than one of the eight families [31–34].
2.9. Effect of metal ions, organic solvents, inhibitors and
detergents on enzyme activity
The effects of metal ions on the esterase activity were investi-
gated by adding 5 mM of various metal salts (CaCl2, CoCl2, CuCl2,
FeSO4, NaCl, KCl, NiSO4, MgCl2, MnCl2, and ZnCl2) directly to
the assay solution containing the purified enzyme (23 g/ml) and
50 mM Tris–HCl buffer (pH 9.0) for 60 min at room temperature. The
stability of the esterase against organic solvents was determined
using methanol, ethanol, acetone, propanol, chloroform, DMF, n-
hexane, heptane, formaldehyde and toluene at final concentrations
of 20% or 50% (v/v) in 50 mM Tris–HCl buffer (pH 9.0). The mixture
containing the purified enzyme (23 g/ml) and each organic sol-
vent of interest was incubated at 30 ◦C with constant shaking at
160 rpm for 12 h. Potential inhibitors (EDTA, PMSF, DEPC, and DTT)
at a final concentration of 5 mM were incubated with the enzyme
(23 g/ml) in 50 mM Tris–HCl buffer (pH 9.0), and then the solu-
tions were assayed for enzyme activity. The effect of detergents
on enzyme activity was determined by incubating the enzyme
(23 g/ml) in 50 mM Tris–HCl buffer (pH 9.0) with 1% or 5% (w/v)
of Tween 20, Tween 80, Triton X-100 and SDS at room temperature
for 60 min. In all of these assays, the residual enzyme activity was
measured under the standard assay conditions and was initiated
by addition of the substrate pNP-C4.
3.3. Expression and purification of EstSt7
The recombinant plasmid pET15b/EstSt7 was constructed to
determine the biochemical properties of EstSt7. EstSt7 was
expressed in E. coli BL21-CodenPlus (DE3) and purified to homo-
geneity after heat treatment, Ni-NTA affinity and Superdex-200
gel filtration chromatography. The purified recombinant protein
(Fig. 3). The specific activity of the purified EstSt7 reached 354 U/mg
using pNP-C4 as a substrate in Tris–HCl buffer at pH 9.0 and 80 ◦C.
The enzyme was finally purified 11.7-fold with a yield of 43.2%
(Table 1). The SDS-PAGE analysis of the EstSt7 is shown in Fig. 3.
3. Results and discussion
3.1. Amino acid sequence comparison
An ORF (ST2026) of 969 bp encoding a hypothetical protein
(named EstSt7) of 322 amino acids was identified from the genomic
DNA of S. tokodaii strain 7. The nucleotide sequence reported herein
was submitted to the GenBank database with the accession number
NP 378015. Sequence alignment demonstrated that EstSt7 shares