Q. Du et al. / Journal of Molecular Structure 1219 (2020) 128600
3
flask and was cultured at 37 ꢀC and 200 rpm in a rotary shaker.
When the optical density of the culture at 600 nm reached 0.5, the
expression of DmpA was induced by adding IPTG to a final con-
centration of 0.5 mM, and the culture was incubated at 28 ꢀC and
200 rpm for 8 h. Cells were harvested via 5 min centrifugation at
10,000ꢁg and 4 ꢀC, and washed with 0.2 M phosphate buffer (pH
7.5). Then the obtained cell pellet was maintained at 4 ꢀC for further
studies.
dodecyl sulfate(SDS), Tween-80 and Triton X-100; metal ions
including Mg2þ, Mn2þ, Zn2þ, Co2þ, Ca2þ and Zn2þ
.
3. Results and discussion
3.1. Docking simulation of the DmpA between L-glutamine and D-
glutamine
D-glutamine and L-glutamine were docked into the enzyme,
respectively. The docking results were shown in Fig. 1. The active
center we selected from over 30 supposed receptor cavities for
docking was consistent with the previous report [21], and we knew
that Ser250 at the active site was the most important catalytic
residue. The results of molecular docking showed that the
CDOCKER Interaction Energy (interaction energy between the
ligand and receptor) of the enzyme in combination with L gluta-
mine (ꢂ27.9136 kcal/mol) was lower than that in combination with
D glutamine (ꢂ25.4735 kcal/mol), indicating that L-glutamine was
the preferred binding configuration of the enzyme. Moreover, seen
in Fig. 1B, C, L-glutamine might form two possible hydrogen bonds
with Ser250 (O/HeN: 2.66 Å, 119.566ꢀ; O/HeN: 1.87 Å, 141.012ꢀ),
whereas in Fig. 1E, F, D-glutamine only form one hydrogen bond
with Ser250 (O/HeN: 2.75 Å, 117.525ꢀ) [22]. The analysis of the
above molecular docking results inferred that, it possibly gave
priority to the hydrolysis of L-glutamine theoretically when DmpA
catalyzed the hydrolysis of the substrate DL-glutamine.
2.4. DmpA activity assay
The whole-cell strains expressing DmpA were kept in store in
the freezing condition. Before the activity assay, the whole-cell
strains were weighted to control the steadiness, and then initially
recovered with a 20-min incubation. This pre-incubation has been
convinced to recover the high DmpA activity. Afterwards, the
recovered whole-cell strains were used directly as an enzyme
source to produce L-glutamic acid from DL-glutamine. Activities of
DmpA was measured by detecting the formation of L-glutamic acid.
The standard assay solution (final volume, 2 mL) containing
200 mM DL-glutamine and the whole-cell (0.02 g) were further
incubated for 20 min at 30 ꢀC, pH 8. One unit (U) of enzyme activity
was defined as the amount of whole-cell strain catalyzing the
conversion of substrate to the product at a rate of 1 mmol/min [18].
2.5. Analytical methods
The chemical structures of components synthesized in Scheme 1
were determined by Bruker DRX-600 MHz spectrometer. D-gluta-
mine and L-glutamine were separated and detected by HPLC after
the derivatization with the chiral derivative reagent (1-Fluoro-2, 4-
dinitrophenyl)-5-L-alaninamide (FDAA) according to the literature
[19]. The elution conditions were as follows: solvent A, 20 mM
ammonium acetate in aqueous solution; solvent B, acetonitrile;
flow rate, 0.6 mL/min; 0e8 min 95% A and 5% B; 8e30 min 87% A
3.2. Effect of temperature and pH on DmpA activity
The preferences of DmpA under different temperature and pH
conditions exhibited consistency with that of the previous report
[23]. The reaction was carried out in a 2 mL mixture containing
200 mM DL-glutamine and 0.02 g recovered whole-cell strain. The
optimal temperature of the enzyme was evaluated using the
Na2CO3/NaHCO3 (pH 8). The temperature stability of DmpA was
determined after pre-incubation of the recovered whole-cell strain
at a broad temperature range of 20e45 ꢀC. The enzymic activity was
measured after 2 h. The highest relative activity and residual ac-
tivity of 100% denoted 66.7% and 55.9% conversion of L-glutamine.
The data were presented as mean standard deviation (SD) from
three independent experiments. Subsequently, the effect of tem-
perature on DmpA activity was investigated within a range from 20
to 45 ꢀC. Along with the increase of temperature, the activity of
DmpA also enhanced. The highest activity of DmpA was observed at
30 ꢀC and then its activity decreased significantly with a further
increase of temperature (Fig. 2A). The thermostability of DmpA was
examined by determination of its residual activity after incubation
at 20, 30 and 40 ꢀC, respectively. Accordingly, 30 ꢀC was chosen as a
favorable temperature for glutamine synthesis (Fig. 2B).
In terms of pH, the reaction was carried out the same as Fig. 2.
The optimal pH of the enzyme was evaluated using the following
buffers (50 mM): sodium phosphate (pH 6e7), Na2CO3/NaHCO3 (pH
8e10). The pH stability of DmpA was determined after pre-
incubation of the recovered whole-cell strain at a broad pH range
of 6e10. The enzyme activity was measured after 2 h. The highest
relative activity and residual activity of 100% denoted 18.8% and
50.1% L-glutamine, respectively. The mixture was kept in a shaking
plastic tube at 30 ꢀC. The data were presented as mean standard
deviation (SD) from three independent experiments. The
maximum activity of DmpA for the hydrolysis of L-glutamine was
observed at pH 8 in 50 mM Na2CO3/NaHCO3 buffer, while it
decreased significantly with pH lower than 7 or higher than 9, as
shown in Fig. 3A. Therefore the optimum pH of this reaction was
considered as pH 8. Similarly, the pH stability of the enzyme was
also the best at pH 8 (Fig. 3B).
and 13% B. Ultimate XB-C18 (4.6 ꢁ 100 mm, 5
mm) was used for
compound separation. The compounds were monitored at
UV ¼ 340 nm.
2.6. Molecular docking analysis
In order to hint the mechanism of DmpA preferentially hydro-
lysis L-glutamine than D-glutamine, we performed molecular
docking analysis thus the possible patterns could be visualized
before a further exploration. The three-dimensional structures of
D-glutamine and L-glutamine were constructed using Chem. 3D
ultra 19.0 software, then they were energetically minimized by
using Minimize Small Moleculars function under CharMm force-
field by Discovery Stutio (version 3.5). The crystal structure of
DmpA (PDB code: 1B65) complex was downloaded from the RCSB
Protein Data Bank. All bound waters and ligands were eliminated
from the protein and the polar hydrogen was added to the proteins
after the Prepare Protein procedure in the same software [20].
Molecular docking of compounds L-glutamine and D-glutamine
into the three-dimensional X-ray structure of DmpA was carried
out using the CDOCKER protocol Discovery Stutio (version 3.5).
2.7. Enzymatic resolution of DL-glutamine by DmpA
Enzymatic resolution reaction was in 10 mL tube filled with
2 mL of reaction mixtures containing Na2CO3/NaHCO3 buffer
(50 mM), the whole-cell catalyzing system, and 200 mM DL-
glutamine. Reaction condition optimization was performed under
the following parameter variation: temperature variation from 20
to 45 ꢀC; pH variation from 6 to 11; surfactants including Sodium