Bioinformatics and description of mutations
The three-dimensional structure of proteins was modeled using MODELLER software and the best model for each protein was selected for further studies based on structural factors [21, 15-17]. The structure of the enzymes was examined with the help of Swiss PDB Viewer 4.1.0 software [20]. The results obtained from the Protparam server regarding stability showed that the mutant H403R/L309V was more stable than the wild enzyme and the results of examining the models made for the wild and mutant enzymes in the PIC server showed no disulfide bond in this enzyme. With these interpretations, the role of salt bridge and hydrophobic interactions is more tangible in the stability of this enzyme. This enzyme works mainly as a dimer and can become an octamer [13].
H403R mutation was aimed at increasing protein stability [22]. The second mutation, L309V, which is located near the active site, was applied to the first mutant for the purpose of decreasing the hydrophobicity around the active site by replacing leucine with valine. The results also showed that by reducing a methyl group, the flexibility around the active site is likely to change. T280Q mutation is located at the (α/β)8-barrel for the purpose of the examine the role of N, C, and the central domain of the enzyme in the direction of becoming dimer and dodecameric form. By applying this mutation, it was observed the forming of a van der Waals bond between aspartate 287 at a distance of 5 Angstroms and a hydrogen bond with Tyrosine 286 (Fig. 1).
Examining functional and thermodynamic properties of wild enzyme and mutants
PH effect
Examining the effect of pH changes in the range from 5 to 11 showed that by applying mutations on proteins, no changes happened in the optimal pH of the enzyme, but the rate of activity changed in the same pH. As can be seen from Fig. 2, more than 70% of enzymatic activity occurs in the range pH = 7-8, but enzymatic activity decreased sharply in pHs above 9. At pH 7, the activity of the mutant enzyme H403R/L309V was about 25% higher than that of the wild and mutant enzyme T280Q. The mutant T280Q showed no significant change either.
Temperature effect
The optimal activity of the natural recombinant cyclomaltodextrinase is at 65℃. The results showed that enzymes are active in the temperature range from 20 to 80℃ and in the temperature range from 45 to 70℃ showed a high percentage of activity. Thus, the H403R/L309V mutant showed about 29% increase compared to the wild enzyme activity (Fig. 3). The difference between the activity of the H403R/L309V relative to the T280Q and the wild enzyme at 65℃ was about 23 and 40%, respectively. This means that at the optimal temperature of the natural enzyme, which has 100% activity, the H403R/L309V showed about 134% activity and the T280Q showed about 112% of enzymatic activity.
The increase in the activity of the double mutant at the optimal temperature has a sharp slope. To explain this, one can state that by applying the first mutation of H403R, which was reported to increase activity, the second mutation was applied for increasing flexibility and ease of access to the substrate around the active site, which is the result of a re-increase in enzymatic activity.
Irreversible thermal inactivation
The results of the irreversible thermal inactivation of enzymes at 60℃ show that the H403R/L309V mutant retains about 40% of its activity after 160 min, whereas the wild enzyme and T280Q mutant have retained 20% of their activity (Fig. 4). Thus, it seems that H403R/L309V variant has kept the activity and stability during this time. Also, the activity of all three types of enzymes at 65 and 70℃ was very low after 90 and 20 min respectively.
Effects of metal ions and some organic compounds on enzyme activity
As Table 1 shows, the addition of metal ions K+, Zn2+, and Na+ led to an increase in enzymatic activity. The T280Q mutation in the presence of two concentrations of calcium showed a 5-7-unit increase in the activity, which may have affected the calcium-binding site in the N domain. One study on the effect of calcium on amylase activity showed an increase in temperature stability without increasing activity [23]. In another study, four areas were observed for calcium binding on the enzyme. These regions contain residues necessary for the catalytic activity of the enzyme [24]. The H403R/L309V mutant had a 14-unit increase in enzymatic activity in the presence of a 10 mM calcium concentration. The ion-binding site was affected by the mutation and structural changes as well.
Table 1. The effect of various metal ions/chemicals on the activity of wild and mutant cyclomaltodextrinases
|
(%)
|
activity
|
Relative
|
|
T280Q
|
H403R/L309V
|
WT
|
Concentration(mM)
|
Metal ions/Chemical
|
100.0
|
100.0
|
100.0
|
-
|
Control
|
39.4
|
31.8
|
34.1
|
5
|
Ca2+
|
34.6
|
41.4
|
27.3
|
10
|
|
148.5
|
144.2
|
149.1
|
5
|
K1+
|
135.2
|
135.7
|
136.5
|
10
|
|
0.0
|
0.0
|
0.0
|
5
|
Fe2+
|
3.9
|
6.9
|
2
|
10
|
|
117.5
|
119.8
|
116.9
|
5
|
Zn2+
|
144.3
|
143.4
|
140.5
|
10
|
|
13.7
|
10.9
|
9.7
|
5
|
Co2+
|
20.2
|
20.2
|
15.4
|
10
|
|
0.0
|
0.0
|
0.0
|
5
|
Cu2+
|
0.0
|
0.0
|
0.0
|
10
|
|
131.9
|
131.7
|
134.5
|
5
|
Na1+
|
123.1
|
129.5
|
122.7
|
10
|
|
67.9
|
62.1
|
68.9
|
5
|
Mg2+
|
20.9
|
52.4
|
17.9
|
10
|
|
132.7
|
131
|
134.8
|
5
|
EDTA
|
134.4
|
121.7
|
138.1
|
10
|
|
125.5
|
121.2
|
128.2
|
5
|
PMSF
|
55.8
|
44.7
|
50.5
|
10
|
|
187.4
|
184.5
|
194.9
|
5
|
DTT
|
188.3
|
182.2
|
190.9
|
10
|
|
124.7
|
122.5
|
124
|
5
|
-mercaptoethanolβ
|
150.4
|
146.9
|
148.9
|
10
|
|
Enzymatic reaction products
TLC results showed that the flux ratio (Rf) of glucose and maltose levels was 0.33 and 0.42, respectively, which could indicate that all mutants and the wild-type enzyme produced mostly maltose and a little glucose (Figure 5).
Dimer and dodecamer forms
According to recent studies, most of the enzymes reported with high activity are dimer or mostly tetramer/dodecamer [25, 26, and 10]. Park (2001) showed that the enzyme that breaks down cyclodextrin is more active in the dimeric form rather than the monomeric form. The stability and activity of the enzymes after 72 h of incubation at different pHs (6, 7, and 8) at 4℃ showed that all three enzymes had the highest activity at pH=7 with an increase in H403R/L309V activity relative to the wild enzyme. These results are consistent with those of reversible thermal inactivation. In the case of the T280Q mutant, there is a slight increase relative to the wild enzyme, which may be due to a slight change in the enzyme structure (the hydrogen bonding with tyrosine 286 and van der Waals with bonding aspartate 287) according to the bioinformatics data (Fig. 6).
According to the pH profile, enzyme activity was increased in the H403R/L309V mutant at the optimal pH. Although incubation enzymes in the optimal pH led to a decrease in enzyme activity, it seems that applying this mutation maintains the dodecamer form of the enzyme and creates a kind of stabilizing mechanism against structural changes.
Enzyme stabilizing with nanoparticles
In the present study, SEM images were used to observe the morphology of nanoparticles. Moreover, TEM images were applied to determine the size of synthetic nanoparticles and to ensure the formation of nanoparticle structure (Fig. 7). The results of SDS-PAGE electrophoresis show that Fe3O4 NPs and NiO NPs can be used to purify the enzyme. Also, stabilizing the enzyme on iron oxide nanoparticles was better than stabilizing on nickel oxide such that the concentration of the stabilized enzyme under the same conditions was 1.5 mg/ml for iron oxide and 0.3 mg/ml for nickel oxide (Fig. 8).
Enzyme activity decreased in the presence of magnetic Fe3O4 NPs and NiO NPs. This reduction in enzyme activity continued for several days by incubation (Fig. 9). Overall, it can be concluded that nanoparticles interfere with the process of enzyme supramolecular aggregation, where stabilization interferes with the process of the enzyme being dimer and dodecamer and reduces the enzymatic activity.
Kinetic and thermodynamic parameters
Kcat/Km value was obtained for mutants and wild enzymes at different concentrations of three alpha, beta, and gamma-cyclodextrin substrates (Table 2). According to a previous study, Kcat/Km value for H403R single mutation increased in the presence of gamma-cyclodextrin substrate [22]. The change in the pattern of intermolecular interaction near the active site after the second mutation increases Kcat/Km for the H403R/L309V mutant in the presence of gamma-cyclodextrin substrate relative to the wild enzyme. In the presence of beta-cyclodextrin substrate, Kcat/Km of the H403R/L309V mutant increased, suggesting a decrease in the H403R single mutant [22]. Besides, in the presence of alpha-cyclodextrin substrate, Kcat/Km of the H403R/L309V mutant decreased, which also showed a decrease for the H403R single mutant. All these results show that by applying a second mutation by converting a leucine to a valine for reducing the hydrophobicity of the active site, the tendency to beta substrate increased compared to the H403R single mutant. The value of Kcat/Km for the T280Q mutant decreased in the presence of all three substrates that related to the result of becoming a dodecameric form of the enzyme. Given the lack of change in TΔS and ΔH values in all mutants (Table 3), based on the results of the enzyme-substrate reaction study, it can be stated that the enzyme mechanism did not change due to mutations in wild enzymes.
Table 2. Synthetic parameters of cyclomaltodextrinase of wild type and mutants with three substrates alpha, beta, and gamma-cyclomaltodextrine. (Km is in the unit of mg mL−1, kcat and kcat/Km are in the units of s−1 and [mg mL−1 S]−1, respectively. The observed values of kinetic parameters are the mean of 3–4 replicates)
Substrate
α-CD
|
Enzyme
|
Km
|
kcat
|
kcat/Km
|
WT
|
3.9±0.3
|
580±0.9
|
145±11
|
H403R/L309V
|
5.2±3
|
530±0.5
|
101±2
|
T280Q
|
4.75±1
|
620±0.3
|
130±4
|
β-CD
|
WT
|
3.45±0.01
|
382±0.5
|
111±16
|
H403R/L309V
|
3.69±0.7
|
436±0.7
|
118±3
|
T280Q
|
4.2±0.5
|
412±0.1
|
98±3
|
γ-CD
|
WT
|
2.98±3
|
308±0.8
|
103±14
|
H403R/L309V
|
3.2±0.5
|
437±0.3
|
136±3
|
T280Q
|
5.4±1
|
453±0.5
|
83±7
|
Table 3. Thermodynamic parameters related to the activation reaction of the enzymatic reaction related to the wild protein and each of the mutants at 60℃
|
Ea
(kJ/mol)
|
ΔG#
(kJ/mol)
|
ΔH#
(kJ/mol)
|
TΔS#
((kJ/mol
|
WT
|
49.53±0.8
|
50.53±0.09
|
46.76±0.8
|
-3.77±0.8
|
T280Q
|
49.58±0.4
|
50.49±0.07
|
46.81±0.4
|
-3.68±0.4
|
H403R/ L309V
|
47.95±0.8
|
50.11±0.04
|
45.18±0.8
|
-4.93±0.8
|
Structural studies
By applying the H403R/L309V mutant, the number of photons emitted increased relative to the wild protein which may show the distancing of some of the Tryptophan involved in the suppression of internal protein suppressor distances themselves. About T280Q mutant the reduced fluorescence emissions can be attributed to the closeness of some Tryptophan to internal suppressants and exacerbation of the suppression phenomenon [27] (Fig. 10). Given the presence of 17 Tryptophan residues in the structure of this enzyme, one could take the probability of involvement of some of these chromophores in the suppression. Generally, according to the results of intrinsic fluorescence, one can state that by applying these mutations likely to happen an intramolecular structural rearrangement that would alter the position of some tryptophan without altering the polarity of their surroundings.
The ANS fluorescence results showed that the mutants in the presence of ANS increased the fluorescence intensity (Fig. 11), indicating an increase in the number of protein binding sites for ANS in the mutants. The change in λmax toward the blue wavelength in H403R/L309V mutant shows an increase in binding strength and a better connection environment for ANS, which was similar to the results of H403R mutants [22]. However, in the case of the T280Q mutant, although the number of binding sites increased, the binding power did not change significantly.