M.W. Iqbal, et al.
Enzyme and Microbial Technology 131 (2019) 109427
overexpressed in E. coli BL21. The cells harboring Thto-DaIase were
cultivated in Luria–Brentani (LB) medium with ampicillin at a final
2.5. Effects of pH and temperature on the activity of recombinant Thto-
DaIase
−
1
concentration of 100 μg mL
and incubated at 37 °C until the optical
−1
density (OD600) reached 0.6-0.8. IPTG (1 mM L ) was added for in-
duction, and after 6–10 h, D-AIase expressing cells were grown at 28 °C
with shaking at 200 rpm.
The enzyme activity was determined by using a variety of buffer
solutions with various pH ranges (5–10.5), and the optimal pH values
were detected. Three different buffers with different pH were used:
sodium phosphate buffer (50 mM, pH 5.0–7.0), Tris−HCl (50 mM, pH
7.0–9.0) and glycine-NaOH (50 mM, pH 9.0–10.5). The pH stability was
2
.3. Protein purification and molecular mass determination of recombinant
tested by incubating the enzyme in various pH ranges (5.0–10.5) at 4 °C
and 75 °C for 24 and 1 h, respectively. The optimal temperature for the
enzyme activity with glycine-NaOH buffer at pH 9.0 was evaluated at
35–95 °C. The thermostability of Thto-DaIase was studied by incubating
the enzyme in 50 mM glycine-NaOH buffer at pH 9.0 and various
temperatures 45, 55, 65, 75, 85 and 95 °C within specific intervals of
times. The effects of thermostability on Thto-DaIase were determined
by log k (k: rate constant of thermal denaturation) and 1/T with an
Arrhenius plot using the k = 0.693/t1/2 equation.
Thto-DaIase
The purification steps of D-arabinose isomerase from T. toyohensis
Thto-DaIase) were executed at 4 °C. The recombinant protein-expres-
sing cells were collected from the broth culture by centrifugation at
0,000 × g for 15 min at 4 °C, and then these cells were resuspended in
(
1
lysis buffer (50 mM Tris−HCl buffer, 100 mM NaCl) at pH 7.0. After
mixing, the cell disruption was performed at 4 °C by using an ultra-
sonication Vibra-Cell 72,405 Sonicator (Bio-Block Scientific, Illkirch,
France). The denatured and unlysed cells were discarded by cen-
trifugation (10,000 × g, 20 min, 4 °C), and the supernatant was used as
a crude extract of Thto-DaIase. The crude enzyme was applied to a resin
column (chelating sepharose fast flow 1 cm × 10 cm) charged with
2.6. Effects of metal ions on recombinant Thto-DaIase activity
As previously described, reactions were achieved in 50 mM glycine
buffer (pH 9.0) comprising substrates (D-arabinose/L-fucose) and re-
combinant enzyme (Thto-DaIase) for 10 min at 75 °C. To inspect the
influence of metal ions on the activity of Thto-DaIase, the recombinant
enzyme was tested after treatment with EDTA and individual metal
2
+
Ni
NaCl, pH 7.0),
Novagen). The bound nondenatured protein was detached using a
followed by binding buffer (50 mM Tris−HCl buffer, 500 mM
a
process called nickel-affinity chromatography
(
2
+
2+
2+
2+
2+
2+
2+
2+
washing buffer (50 mM Tris−HCl buffer, 50 mM imidazole, 500 mM
NaCl, pH 7.0). The enzyme was eluted with elution buffer (50 mM
Tris−HCl buffer, 500 mM imidazole, 500 mM NaCl, pH 7.0), and the
solution containing purified enzyme was dialyzed overnight at 4 °C
against dialysis buffers A (50 mM Tris−HCl buffer, 10 mM EDTA, pH
ions, including Ca , Ni , Mn , Mg , Ba , Co , Fe , Zn and
Cu
2+
at concentrations of 1 mM. The measured activities in the pre-
sence of metals were compared with the enzyme activity without the
2
+
addition of a metal ion, which was used as a control. The effect of Mn
concentration for both D-arabinose and L-fucose was determined by
7
.0) and B (50 mM Tris buffer, pH 7.0).
The instinctive molecular mass for Thto-DaIase was determined by
incubating Thto-DaIase with different concentrations (1–5 mM) of
2+
Mn
at pH 9.0 at 75 °C for 10 min.
analyzing the purified enzyme with high-performance liquid chroma-
tography (HPLC, TSK G3000SW) column (Tosoh. Co., Ltd, Tokyo,
Japan). By using differential refractometer (Optilab T-rEX, CA, USA)
with a light-scattering detector (Dawn Heleos II, CA, USA) and a UV
detector (Waters 2489, USA), the purified enzyme was eluted in 50 mM
Tris−HCl buffer (pH 7.0) containing 300 mM NaCl with a flow rate of
2.7. Homology modeling and amino acid sequence comparisons of
recombinant Thto-DaIase
The homology structural and substrate docking of recombinant
termined experimentally by using a template of A. pallidus (PDB ID:
Discovery-studio were used to visualize and access the 3-dimensional
structure.
−1
1
mL min . The subunit molecular mass of purified Thto-DaIase was
calculated by SDS-PAGE (5% stacking gels and 12% resolving gels)
under denaturing conditions using the prestained ladder as a reference
protein (MBI Ferments, Glen Burnie, MD, USA). For the staining of
protein bands, Coomassie Brilliant Blue 250 was used, and the de-
staining solution was used for visualization.
2
.4. Determination of protein concentration and enzyme assay
reported bacteria. A phylogenetic relationship of recombinant Thto-
DaIase and previously identified bacteria with known accession num-
bers was created with MEGA-X using the neighbor-joining method. The
scale of the tree indicates the branch length of the same unit, which is
used to determine evolutionary distances based on phylogenetic trees
formed by the Poisson correction method, and the branch length (op-
timum sum) of the tree was 0.85878910. The phylogenetic tree analysis
involved 6 amino acid sequences [1–3].
The protein concentrations of Thto-DaIase were analysed by the
Bradford method using bovine serum albumin (BSA) as a standard
protein [19]. The activity of Thto-DaIase was determined by calculating
the amount of D-ribulose from D-arabinose and L-fuculose from L-fucose.
Experiments were performed on a high-performance liquid chromato-
graphy (HPLC) system (1200 Series; Agilent Technologies, Santa Clara,
CA, USA) linked with an infrared (IR) detector (Shodex, RI-101), and
the column was a SUGAR-PAK (6.5 × 300 mm) at 85 °C; the mobile
−1
phase was double distilled water at a 0.4 mL min
flow rate. D-Ara-
binose and L-fucose (substrates) were used to examine the activity of
Thto-DaIase, and the substrates were transformed into D-ribulose and L-
fuculose, respectively. The reaction was continued for 10 min at 75 °C
in 50 mM glycine buffer, pH 9.0, with D-arabinose and L-fucose and
purified Thto-DaIase. “The one unit of enzyme activity (U) was defined
as the amount of enzyme required to increase 1 μ mol of D-ribulose/L-
The number of amino acids, isoelectric point, molecular weight,
aliphatic index, instability index, estimated half-life and grand average
w
2.8. Specific activities, substrate specificities and kinetic parameters of
recombinant Thto-DaIase
−
1
fuculose min
at 75 °C and pH 9.0″.
Different substrates were used to analyze the specific activities and
3