N. Mokoena et al. / Biochemical and Biophysical Research Communications 437 (2013) 342–348
343
Production Kit (Epicentre Biotechnologies, USA) [9]. Functional
screening of the recombinant esterase positive clones in Escherichia
coli EPI100-T1R was performed on LB agar plates supplemented
colorimetric method measuring the release of p-nitrophenol from
p-nitrophenyl esters at 410 nm [9], using a Beckman DU850 UV/
visible spectrophotometer with a temperature controller. De-
scribed enzyme activity was measured at 30 °C in 20 mM Tris–
HCl, pH 7.5 with 1 mM p-nitrophenyl butyrate (dissolved in iso-
propanol) as the substrate. The extinction coefficient of p-nitro-
with isopropyl-b-D-thiogalactopyranoside (IPTG, 0.1 mM), chlor-
ꢀ1
amphenicol (12.5 lg ml ), tributyrin 1% (v/v) and Gum Arabic
0
.1% (w/v), followed by incubation at 37 °C. Esterase positive
ꢀ1
ꢀ1
clones were identified by the presence of zone of clearance around
phenol under these conditions was 13,800 M cm .
the colony margins.
2
.5.2. b-Lactamase assay
The b-lactam hydrolytic activity of Est22 was determined spec-
trophotometrically using 0.1 mM Nitrocefin [3-(2, 4 dinitrostyrl)-
6R, 7R-7-(2-thienylacetamido)-ceph-3-em-4-carboxylic acid, E-
isomer)] as a substrate (Oxoid kit manual, 6th edition 1990, Uni-
path Ltd., Basingstoke, UK). The activity of Est22 against non-chro-
mogenic b-lactam included the following substrates: 7-
Aminocephalosporanic acid, Cephalosporin C and Cephalotin. The
purified Est22 and a positive control b-lactamase from Bacillus cer-
eus (Sigma) were incubated with antibiotic substrates at 1 mM in
2.2. DNA manipulation and sequencing
Recombinant DNA techniques were carried out as described by
(
Sambrook and Russell [10]. DNA sequencing using 454-pyroseq-
uncing technology and oligonucleotide synthesis services were
provided by Inqaba Biotech (South Africa). Sequence analysis and
manipulation were performed using CLC Combine Workbench
software (CLCBIO, Denmark) and Bioedit [11] with the aid of
5
0 mM Tris–HCl (pH 8.0) for 1 h at 30 °C. The reaction mixtures
were analysed by HPLC, using a Hewlett Packard 1100 HPLC (Agi-
lent Technologies Incorporated, Lovedale, CO, USA) equipped with
a binary pump autosampler, column oven, UV diode array detector
and ChemStation Chromatography Management software (For de-
tailed HPLC conditions, refer to the Supplementary File).
2
.3. Phylogenetic analysis
The evolutionary relationship between Est22, family VIII ester-
ases and class C b-lactamases was inferred using the Neighbor-
Joining method conducted with MEGA5 [14]. The tree was drawn
to scale, with branch lengths in the same units as those of the evo-
lutionary distances used to infer the phylogenetic tree. The evolu-
tionary distances were computed using the Poisson correction
method [15], and are in the units of the number of amino acid sub-
stitutions per site.
2.7. Biochemical characterisation
To investigate substrate specificity of Est22, enzyme activity
was determined using standard esterase assay in the presence of
mM of the specified p-nitrophenyl esters of various chain
1
lengths: p-nitrophenyl acetate (C2), p-nitrophenyl butyrate (C4),
p-nitrophenyl caprylate (C8) and p-nitrophenyl laurate (C12).
Optimum temperature of the Est22 was determined by measur-
ing the rate of p-nitrophenyl butyrate hydrolysis over a tempera-
ture range (30–70 °C). Where necessary the pH adjustments at
set temperatures were performed to take into account the effect
that changing temperature has on pH.
A thermostability profile of Est22 was generated by incubating
the enzyme at three temperature points 30-, 50- and 70 °C, fol-
lowed by measuring residual activities after every 30 min using
the standard esterase assay. The influence of pH on the p-nitro-
phenyl butyrate hydrolysis was tested from pH 4.0–9.5 at 30 °C.
2
.4. Recombinant protein expression and purification
The est22 gene was amplified with and without signal peptide
using F8F/F8R and F8FF/F8R primer pairs, targeting the full length
and truncated (without leader peptide) version of the gene respec-
tively (Supplementary Data: Table S1). Both primer pairs intro-
duced the NdeI and XhoI sites at the 5 - and -3 end of the gene
respectively. Amplified PCR products were digested with NdeI/XhoI,
followed by ligation into pET28a linearised with the same
enzymes. The recombinant est22 gene (with and without leader
peptide) was placed in-frame with the 6x-His tag sequence at
0
0
0
the 3 -end of the gene. Expression vectors were propagated in E.
coli BL21 (DE3) cells and the recombinant clones were selected
on Kanamycin (50 lg/ml). Protein Expression studies were per-
2.8. Accession number
formed using the EnBase technology (BioSilta, Finland). The soluble
esterase (Est22) protein fraction was loaded onto the immobilised
metal affinity column (IMAC) packed with Protino Ni-TED resin
The esterase (est22) gene nucleotide coding sequence has been
deposited in the GenBank under the Accession number: KF052088.
(
2
Macherey-Nagel, Germany). The bound Est22 was eluted with
50 mM imidazole. Eluted fractions were passed through VIVA-
3
. Results
SPIN 10 kDa cut-off spin columns (Vivascience, U.K.) for imidazole
removal and protein concentration. Protein concentration of the
purified samples was determined by the Bradford [16] method,
using bovine serum albumin (BSA) as a standard, while the purity
of the samples was analysed on denaturing sodium dodecyl sul-
phate–polyacrylamide gel electrophoresis (SDS–PAGE) [17].
Molecular weight of Est22 was determined with Superdex™ 200
3
.1. Library screening and sequencing
A fosmid library from leachate environment (library size, aver-
age insert size) was previously constructed using a copy-controlled
pEpiFOS-5 vector and subsequently screened for esterase positive
colonies resulting in one fosmid (FOS8) with esterolytic activity
(
For detailed screening protocol adopted in this study refer to Sup-
1
0/30 GL using 30% Acetonitrile with 0.1% TFA buffer. The gel filtra-
tion standard (Bio–Rad, USA) contained the mixture of thyroglob-
ulin, -globulin, ovalbumin, myoglobin, and vitamin B12.
plementary File). In order to locate the gene(s) encoding an ester-
ase phenotype within the pFOS8 fosmid, a random shotgun
sequencing of a complete insert DNA was performed.
c
2
2
.5. Functional assays
3.2. Primary structure analysis and phylogenetic classification
.5.1. Esterase Assays
Unless otherwise stated all enzyme assays were performed in
The est22 gene (ORF22 which is located at the -3 strand at
triplicate. Esterase activity assays were performed by a standard
position 31174-29906) is 1269 pb has a GC content of 52%.