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J. Yao et al. / Journal of Molecular Catalysis B: Enzymatic 95 (2013) 55–61
ligase and DNA polymerase were purchased from Takara (Dalian,
China). E.Z.N.A® Plasmid Mini Kit and E.Z.N.A® Gel Extraction Kit
were purchased from Omega (Norcross, USA). E. coli DH5␣ was used
as the host for gene cloning and E. coli BL21 (DE3) (Novagen, Madi-
son, USA) for protein expression. pUC118 (Takara, Dalian, China)
and pET-28a (+) (Novagen, Madison, USA) were used to construct
the metagenomic libraries and express the target protein, respec-
tively.
dNTP, 0.2 M of each primer, 1 unit of Taq polymerase, and 3 L of
DNA template. The reaction conditions were 4 min of initial denatu-
ration at 95 ◦C followed by 30 cycles, 1 min of denaturation at 94 ◦C,
1 min of annealing at 60 ◦C, 1.5 min of extension at 72 ◦C and a final
extension step of 10 min at 72 ◦C. Amplified DNA was digested by
BamHI/HindIII, ligated into the BamHI–HindIII- linearized pET-28a
(+), and then introduced into E. coli BL21 (DE3) cells. Transformed
cells were grown in a 250-mL flask containing 50 mL LB at 37 ◦C
until the cell concentration reached an OD600 of 0.7, and then they
were induced with 0.5 mM IPTG. After incubation at 25 ◦C for 12 h
with shaking at 220 rpm, cells were harvested by centrifugation
at 6000 × g for 10 min at 4 ◦C. The cells were sonicated and the
supernatant was collected by centrifugation (16,000 × g, 20 min)
at 4 ◦C. The purification of FAE was carried out by using Ni-NTA
His-Bind resin column chromatography according to the manufac-
turer’s instructions.
2.2. Metagenomic library construction and screening of feruloyl
esterase activity
DNA from a cotton field sample was extracted based on a
method described previously with minor modifications [26]. About
4 g of soil sample was saturated with 13.5 mL of DNA extraction
buffer (100 mM Tris–HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0],
100 mM sodium phosphate [pH 8.0], 1.5 M NaCl, 1% CTAB, 2% SDS)
and was completely homogenized after shaking at 220 rpm for
30 min. Then the mixture was incubated at 65 ◦C with mixing every
15–20 min. After 2 h incubation, the supernatants were harvested
by centrifugation at 6000 × g for 10 min, and then were poured into
a new tube containing 15 mL chloroform. After complete mixing,
the aqueous phase was recovered by centrifugation and precipi-
tated with 0.6 volume of isopropanol at room temperature for 1 h.
The pellet of crude nucleic acids was obtained by centrifugation
(16,000 × g, 20 min) at 4 ◦C, washed twice with cold 75% ethanol and
suspended in an appropriate volume of sterile deionized water. To
purify the crude DNA, low-melting-temperature agarose gels were
prepared in 1× TAE buffer. After electrophoresis at 40 V for 10 h,
the gel containing DNA fragments ≥20 kb was cut off and the DNA
was purified using an E.Z.N.A® Gel Extraction Kit. The isolated DNA
was digested with BamHI. DNA fragments of 2.5–10 kb were ligated
into BamHI-digested pUC118 and the ligated products were trans-
formed into E. coli DH5␣. In order to screen for FAE activity, the
transformed cells were plated onto Luria-Bertani (LB) agar plates
containing 100 mM X-caprylate as a substrate. After incubation at
37 ◦C for 24 h, clones with blue color were further tested for the
ability to hydrolyze EFA. The positive clones were reconfirmed and
sub-cloned.
2.5. Protein determination and SDS-page analysis
Protein concentrations were determined according to Brad-
ford’s method [29]. Bovine serum albumin (BSA) was used as
the standard. SDS-PAGE analysis was carried out according to the
method of Laemmli [30]. A 12% separating gel and 5% concentrating
gel was used for this study. Gels were stained with the Coomassie
brilliant blue (G-250) method. The molecular mass of native protein
was determined by gel filtration on a Superose 12HR 5/30 column.
Gamma globulin (160 kDa), bovine serum albumin (67 kDa), oval-
bumin (43 kDa) and carbonic anhydrase (30 kDa) were used as the
reference proteins. Isoelectric point (pI) was estimated by PAGE
with 6.25% ampholine (pH 3.5–10) in a gel rod (0.5 cm × 10 cm)
using a kit for isoelectric focusing calibration (Pharmacia LKB)
according to the recommendations provided by the supplier.
2.6. Enzyme assays
The FAE activity of the enzyme was measured by analyzing
the free p-nitrophenyl released from p-nitrophenyl ferulate. The
assay was carried out in 100 mM phosphate buffer (pH 7.0) con-
taining 1 mM p-nitrophenyl ferulate at 35 ◦C for 20 min, and then
the liberated free p-nitrophenyl was measured at 405 nm. One unit
of FAE activity was defined as the amount of enzyme required
to release 1 mol of p-nitrophenyl in one minute under specific
conditions.
2.3. Sequence and phylogenetic analysis
DNA sequencing was carried out by using an ABI Prism 377
DNA sequencer (Applied Biosystems, Inc.). The deduced amino
acid sequence analysis and open reading frame (ORF) search
were performed with the BLAST program provided by NCBI
Feruloyl esterase sequences for comparative study were retrieved
from protein and nucleotide databases on the NCBI Entrez server
searches were performed with the BLAST 2.0 program [27]. Amino
acid sequence alignments of Tan410 with homologous proteins and
phylogenetic analyses were performed with the Align X program
[28]. DNA restriction analysis was performed with the DNAman5.2
program.
2.7. Effect of pH and temperature on Tan410 activity
Purified enzyme was used to determine the optimum pH and
temperature. In order to determine the optimal pH of Tan410 activ-
ity, the following buffers were used for the different pH ranges:
100 mM sodium acetate buffer (pH 4.0–6.0); 100 mM phosphate
buffer (pH 5.5–8.0); 100 mM Tris–HCl buffer (pH 7.5–9.0); 100 mM
glycine–NaOH buffer (pH 8.5–10.0). The effect of temperature on
FAE activity was carried out at 20, 25, 30, 35, 40, 45, 50, 55, 60,
65 and 70 ◦C. Tan410 activity assays were performed as described
above to determine the optimal pH and temperature. For the ther-
mal stability measurements, the enzyme was incubated in 100 mM
phosphate buffer (pH 7.0) at 30, 40, 50 and 60 ◦C for 15 min, 30 min,
1 h, 2 h, 4 h and 13 h. After incubation, the residual activity was mea-
sured as described above. Each experiment was done twice and
measured in triplicate, and the standard deviation was less than 1%
of the mean.
2.4. Subcloning and expression of the recombinant FAE
The positive FAE gene was amplified from the pUC118-
tan410 plasmid using the primers P1 (5ꢀ-CGCGGATCCATGCCCGC-
AAAAACCCGCCT G-3ꢀ) and P2 (5ꢀ-CCCAAGCTTATTCCCGTTAGTAAA-
GCCGTC-3ꢀ) which contained restriction enzyme sites (underlined)
for BamHI and HindIII. The amplifications were carried out in a total
volume of 50 L. The reaction mixture contained 1× PCR buffer
(20 mM Tris–HCl, pH 8.4, 50 mM KCl), 2 mM MgCl2, 0.2 mM of each
2.8. Effect of different chemicals on Tan410 activity
Various chemicals (ZnSO4, MgSO4, (NH4)2SO4, NiSO4, AlCl3,
CaCl2, CuCl2, MnCl2, FeCl3, EDTA and urea) at final concentrations