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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
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