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. 2023 Mar 9;8(11):10040–10050. doi: 10.1021/acsomega.2c07380

Binding of Anionic Polyacrylamide with Amidase and Laccase under 298, 303, and 308 K: Docking and Molecular Dynamics Simulation Studies Combined with Experiments

Fanglue Wang †,*, Liwen Zhang ‡, Dongchen Zhang §, Xuefeng Wu ∥, Shengsong Deng ∥
PMCID: PMC10034835  PMID: 36969392

Abstract

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Amidase and laccase play a key role in the degradation process of anionic polyacrylamide (HPAM). However, the largest challenge of HPAM enzymatic degradation is whether the enzyme can bind with a substrate for a period of time. Here, the most suitable complexes, namely, Rh Amidase-HPAM-2 and Bacillus subtilis (B. subtilis) laccase-HPAM-3, were obtained by docking, and they were carried out for molecular dynamics simulation (MDS) under 298, 303, and 308 K. MDS result analysis showed that Rh Amidase-HPAM-2 was the most stable at 298 K mainly due to a salt bridge and a hydrogen bond, and B. subtilis laccase-HPAM-3 was the most stable at 298 K mainly due to two electrostatic and hydrogen bonds. The LYS96 in Rh Amidase-HPAM-2 and LYS135 in B. subtilis laccase-HPAM-3 had been the most important in their binding process. The binding of Rh Amidase-HPAM-2 and B. subtilis laccase-HPAM-3 was optimal at 303 and 298 K, respectively. HPAM was degraded by mixed bacteria, and the optimal conditions were determined to be 308 K, initial pH = 7, and an inoculated dosage of 2 mL. Under these conditions, the degradation ratio reached 39.24%. The effect of parameters on the HPAM degradation ratio followed a decreasing order of temperature > initial pH > inoculated dosage. The HPAM codegradation mechanism was supposed by mixed bacteria according to test data. The mixed bacteria secreted both amidase and laccase, and they interacted jointly with HPAM. These results lay a theoretical foundation to design and modify the enzyme through mutation experiments in the future.

1. Introduction

Anionic polyacrylamide (HPAM) is a linear polymer flocculant that has been used in clarifying slime water.1−4 The carboxyl side chain of HPAM is negatively charged in an alkaline environment, and the suspension is clarified by an adsorption bridge.5 A large amount of HPAM will accumulate in the cycle water of coal dressing, which will reduce the adsorption capacity of coal slime on the flotation reagent.6 In addition, the emission of coal preparation wastewater containing HPAM can cause wide-scale water pollution.7 Hence, it is of great significance to transform HPAM into nontoxic substances.

There are many methods for degrading HPAM, including physical degradation,8 chemical degradation,9 photocatalytic degradation,10 and biodegradation.11 With more and more attention paid to environmental protection and sustainable development, many researchers begin to be apt to biodegradation due to the environmental friendliness, mild condition, and no secondary pollution.12 Nowadays, dominant strains were obtained through screening from the environment polluted by HPAM for a long time. Wen et al.13 found two strains Bacillus cereus and Bacillus flexu from activated sludge and oil soil. They concluded that the bacteria used the amide side chain of HPAM as their nitrogen source. Bao et al.14 isolated and obtained two strains Bacillus cereus and Bacillus sp. from polymer flooding-produced water. They found that the amide group of HPAM was hydrolyzed into carboxyl by amidase, and there was a peak at 2.85 min corresponding to polyacrylate (PAA) in HPLC after degradation. Additionally, there was no peak corresponding to acrylamide, indicating that the toxic acrylamide was not produced. PAA is a kind of intermediate metabolite of HPAM and provided a carbon source.15 The larger PAA fragment was more difficult to be degraded than the smaller ones. According to the difference of starch-cadmium iodide and total organic carbon, the amide side chain was degraded more readily than PAA.16 The key enzymes detected in the HPAM fermentation broth play an important role in biodegradation.17 Zhang et al.12 found that dehydrogenase and urease participated in HPAM degradation. Dehydrogenase promoted electron transfer and oxidation, while urease accelerated the transformation of the amide groups. Song et al.18 found that the laccase activity was not affected by the concentration of HPAM. They pointed out that the amide group of HPAM was easy to be hydrolyzed by amidase, and PAA, the carbon skeleton of HPAM, was formed. Then, PAA was oxidized by oxygenase. However, the key problem for HPAM degradation is whether the enzyme can combine steadily over time with a substrate. Furthermore, the enzyme isolated from the environment has poor stability, which limits its extensive application.19

The formation of the enzyme with the substrate complex is crucial in determining the reaction rate. According to the theory of enzymatic reaction dynamics, the maximum affinity between the enzyme and the substrate indicates that it is the easiest to achieve the maximum reaction rate at a very low substrate concentration. Moreover, the larger the affinity of the enzyme–substrate complex is, the smaller the Km value is. Km is a characteristic constant of an enzyme. Hence, the substrate with the maximum affinity is the optimal substrate for this enzyme. In our previous study, it has been proven that the laccase-HPAM-3 and amidase-HPAM-2 acquired the highest affinity,20 which means that they were the most suitable enzyme–substrate complexes. The protein conformation changes during the simulated time can be studied using molecular dynamics simulation (MDS). By calculating the root-mean-square deviation (RMSD) for backbone atoms and the root-mean-square fluctuation (RMSF) for enzyme residues, the highest flexible regions in protein can be identified. In this study, the binding of the most suitable enzyme–substrate complexes at 298, 303, and 308 K was explored using MDS. However, the combination of the enzyme with HPAM at a time has been reported rarely at the molecular level. This study aims to reveal insights into the mechanistic basis for the degradation process of HPAM.

2. Materials and Methods

2.1. Materials

The structural models of HPAM were generated by the Build Polymer tool of Materials Studio 2017 software, and both terminals were sealed with methyl groups.21 Energy minimization was given by the CHARMm force field, as shown in Figure S1. The 3D crystal structures of amidase22 (PDB code 3A1I) and laccase23 (PDB code 1GSK) were determined by experiments, and their binding sites can be found easily. Both the amidase from Rhodococcus sp. N-771 (Rh Amidase)24 and laccase from Bacillus subtilis (B. subtilis laccase)25 are downloaded from the PDB web (http://www.pdb.org/pdb/home/home.do). Moreover, these two enzymes had the best resolution. Table S1 lists information about selected enzymes. Before docking, the enzymes were prepared using the Prepare Protein tool in Discovery Studio (DS) 2020 to repair the missing loop regions. Meanwhile, waters and all binding ligands were deleted, and hydrogen was added.26

2.2. Docking

Docking can search for the optimal binding conformation of the enzyme with the substrate.27 It was performed using the semiflexible CDOCKER program in DS 2020,28 and the accuracy of this procedure had been previously validated.20 The optimal flexible conformations in active sites were obtained based on high-temperature dynamics, and the first 10 optimal conformations were retained. The docking pose with the highest -CDOCKER_Energy score was used for further analysis. The active site in Rh Amidase was created with coordinates x: −20.835, y: 6.34554, and z: −5.67827 and a radius of 5 Å, while the active site of B. subtilis laccase was produced using coordinates x: 97.3796, y: 61.3005, and z: −8.42748 and a radius of 5 Å. Figure S2 shows the active sites of two prepared enzymes. Dock Ligands (CDOCKER) settings are given in Figure S3.

2.3. MDS

MDS can explore the effect of temperature on the enzyme–substrate over time29,30 and find the residues with the largest fluctuation.31,32 The most suitable complexes were Rh Amidase-HPAM-2 and B. subtilis laccase-HPAM-3, and they were carried out using MDS at 298, 303, and 308 K using the GROMACS software package, version 2021.533 implementing the Amber03 force field.34 In order to make the simulation process closer to reality, we solvated the initial structure in a cubic box of 10 × 10 × 10 nm3 with a single point charge (SPC) water model,35 adding sodium ions and chlorine ions to neutralize the system. Then, energy minimization was performed by steepest-decent, and a 1 ns NPT MDS at a constant pressure of 1 atm was carried out to equilibrate the system. Finally, a 50 ns NPT MDS was conducted, and its dynamic trajectory was collected for analysis. RMSD and RMSF values were calculated using GROMACS tools gmx rmsd and gmx rmsf. Meanwhile, the interaction energy and the contribution of residues to their interaction energy were calculated by the Calculate Interaction Energy tool in DS 2020. The total energy was also calculated by the Analyze Trajectory tool in DS 2020. The initial or final conformations of MDS production were visualized and compared using Visual Molecular Dynamics (VMD) software. During the MDS, the cutoff value for van der Waals was set at 1.4 nm, and long-range electrostatic interactions were calculated using a particle mesh Ewald method.36 The time step was set at 2.0 fs, and other specific parameters were default values. The temperature and the pressure were maintained by a Berendsen thermostat37 and a Berendsen barostat,38 respectively. All the above simulations were conducted under the Max-Flow platform.

2.4. Strains and Media

A Rhodobacter sphaeroides (R. sphaeroides) (ATCC17023) was derived from the China General Microbiological Culture Collection Center, and B. subtilis (GIM1.256) was purchased from the Guangdong Microbial Culture Collection Center. HPAM was obtained from the Water Treatment Material Plant (Gongyi, China), and its molecular weight was about 1.2 × 107. The basal medium was composed of (L–1) glucose, 10 g; peptone, 10 g; yeast extract, 5 g; beef extract, 5 g; NaCl, 5 g; pH = 7. The solid basal medium was basal medium supplemented with 2% agar. The degradation medium was composed of (L–1) K2HPO4·3H2O, 1.6 g; KH2PO4·3H2O, 0.4 g; CaC12, 0.01 g; MgSO4·7H2O, 0.06 g; NaCl, 0.5 g; trace element solution, 5 mL; HPAM, 500 mg; pH = 7. The trace element solution was composed of (L–1) CuSO4·5H2O, 0.8 g; ZnSO4·7H2O, 1.2 g; MnC12·4H2O, 1 g; Fe2(SO4)3, 0.03 g. These two media were sterilized in an autoclave at 120 °C for 30 min before use.

2.5. HPAM Degradation Experiments

The bacterial solution (2 mL) was inoculated into a 250 mL conical flask containing 100 mL of basal medium and enriched at 303 K for 5 days. Then, the enriched 0.5 mL of bacterial solution was inoculated into a 250 mL conical flask containing 100 mL of basal medium again and cultivated at 303 K for 3 days. After that, 2 mL of bacterial solution was inoculated in the degradation medium with a certain HPAM concentration gradient from 100 to 1200 mg/L for domestication. Each stage was incubated at 303 K with an interval of 300 mg/L for 3 days on an incubator shaker. The strain could be induced to secrete the higher active enzyme by domestication.

The mixed bacteria were obtained by combining domesticated R. sphaeroides with B. subtilis in an equal volume. The mixed strain (2 mL) was transferred to 100 mL of the degradation medium and cultured for 7 days under different conditions with no inoculated bacteria as a control. The enzymes play a key role in the degradation process of HPAM. Temperature and pH are the main factors affecting the enzyme properties, while the inoculated dosage can directly affect the enzyme dosage. Hence, an orthogonal test39 of three factors at three different levels was performed to optimize the operation conditions at the given initial pH (6, 7, and 8), temperature (298, 303, and 308 K), and inoculated dosage (1, 2, and 3 mL). The HPAM concentration was measured by the starch-cadmium iodide method at 585 nm,40,41 and its degradation ratio could be calculated to evaluate the enzymatic degradation performance. Moreover, the degradation ratio of mixed bacteria was compared with the single strain, R. sphaeroides and B. subtilis, under optimal conditions.

3. Results and Discussion

3.1. Contribution of Residues to Interaction Energy and The Total Interaction Energy

As can be seen from Figure 1, the residues ILE450, LEU447, TRP328, and ALA195 took part in the hydrophobic actions with HPAM-2 at 298 K. The residues ILE450, ALA332, TRP328, ILE227, and ALA195 produced hydrophobic contacts with HPAM-2 at 303 K. HPAM-2 interacted with residues ILE450, MET449, TRP328, ILE198, and ALA195 by hydrophobic actions at 308 K. These residues contributed to their interaction energies little or even negatively. At the same time, it is also found that the residue LYS96 always contributed the most to the interaction energy at different temperatures. The contribution order of LYS96 to the interaction energy was 303 K > 298 K > 308 K, consistent with the order of total interaction energy, suggesting that the residue LYS96 was the most important residue in the Rh Amidase binding with HPAM-2. From Figure 1d, the total interaction energy was the lowest at 303 K, indicating that the binding of Rh Amidase with HPAM-2 was the best at 303 K.

Figure 1.

Figure 1

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(a–d) Contribution of residues to the interaction energy of Rh Amidase-HPAM-2 and the total interaction energy.

The three residues, namely, LYS135, ARG136, and ASP113, took part in B. subtilis laccase binding with HPAM-3 at 298 K, but only LYS135 was involved in their binding at 303 and 308 K. Thus, it is meaningful to explore the contribution of residues to the interaction energy between B. subtilis laccase and HPAM-3 at 298 K. As shown in Figure S4a, the residue LYS135 always contributed the most to the interaction energy, which suggested that LYS135 is the most important residue in the B. subtilis laccase binding with HPAM-3. From Figure S4b, the total interaction energy was the lowest at 298 K, indicating that the binding of B. subtilis laccase with HPAM-3 was the optimal at 298 K.

3.2. Conformation Transformation

As shown in Figure 2a, the total energies of Rh Amidase-HPAM-2 tended to a constant, indicating that this structure reached equilibrium after MDS optimization. In addition, the total energy of Rh Amidase-HPAM-2 was the largest at 308 K, which further indicated that the stability of this structure is the worst at 308 K. Its total energy had not much difference between 298 and 303 K, and a higher temperature at a certain range was beneficial to the enzymatic reaction. Hence, we selected 303 K as the optimal reaction temperature of Rh Amidase degrading HPAM-2. RMSD is often employed to measure structural stability of the protein backbone atoms. The higher the RMSD value, the worse the stability of the enzyme protein. In order to observe the binding of Rh Amidase to HPAM-2, the variation of RMSD over simulated time was calculated, and the initial conformation was used as a reference, as shown in Figure 2b. The enzyme skeleton vibration was the largest when Rh Amidase was bound to HPAM-2 at 308 K, indicating that their binding was the worst at 308 K. The skeleton vibration of the enzyme between 298 and 303 K was almost the same, and RMSD reached balance earlier at 303 than 298 K when the Rh Amidase was bound with HPAM-2. These further suggested that the binding of Rh Amidase-HPAM-2 was the best at 303 K.

Figure 2.

Figure 2

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Total energy (a) and RMSD of backbone atoms (b) in Rh Amidase-HPAM-2 under 298, 303, and 308 K.

From Figure 3a,b, the conformation of the Rh Amidase-HPAM-2 complex did not change significantly at 298 and 303 K, while the orientation of HPAM-2 changed slightly, indicating that this complex is stable at 298 and 303 K. From Figure 3c, at 308 K, it is worth noting that the enzyme part marked by a dotted line had a larger skeleton fluctuation. This can explain well why the structure of the enzyme acquired the maximum RMSD value when binding to the substrate at 308 K.

Figure 3.

Figure 3

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Conformation transformation of Rh Amidase-HPAM-2 at (a) 298, (b) 303, and (c) 308 K. The stick configuration was HPAM-2, and the secondary structure of Rh Amidase was exhibited in a new cartoon pattern. The solvents were not shown for simplicity. Note: the initial and final frames were only analyzed for the 50 ns dynamics trajectory.

As shown in Figure 4a, the total energies of B. subtilis laccase-HPAM-3 tended to a constant, indicating that this structure reached equilibrium. Additionally, the total energy of B. subtilis laccase-HPAM-3 was the most negative at 298 K, indicating that this structure is the most stable at 298 K. In order to observe the binding of B. subtilis laccase-HPAM-3, the variation of RMSD over simulated time was calculated, and the initial conformation was used as a reference, as shown in Figure 4b. From Figure 4b, the enzyme skeleton vibration was the smallest when B. subtilis laccase was bound to HPAM-3 at 298 K, which suggested that their binding was the optimal at 298 K. As can be seen from Figure S5a,b, the conformation of the B. subtilis laccase-HPAM-3 complex did not change significantly at 298 and 303 K, while the orientation of HPAM-3 changed slightly, indicating that this complex is stable at 298 and 303 K. From Figure S5c, at 308 K, we can find that the HPAM-3 had deviated from the initial docking position of the enzyme, which speculated that it may be due to the larger vibration of the enzyme skeleton, indicating that B. subtilis laccase has the worst binding at 308 K.

Figure 4.

Figure 4

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Total energy (a) and RMSD of backbone atoms (b) in B. subtilis laccase-HPAM-3 under 298, 303, and 308 K.

The higher RMSF reflects the less stability of corresponding individual residues in the enzyme.42−44 These residues may affect the stability of the enzyme binding to the substrate. From what is shown in Figure 5a, the enzyme residue RMSF in Rh Amidase-HPAM-2 had the most fluctuation at 308 K followed by 298 K, while the enzyme residue RMSF in Rh Amidase-HPAM-2 had the smallest fluctuation at 303 K, which further indicated that Rh Amidase binding to HPAM-2 is optimal at 303 K. In addition, at 303 K, Rh Amidase-HPAM-2 enjoyed the highest RMSF only at 0–50 residues of the enzyme, but this value had the smallest variation compared with 298 and 308 K. These indicated that these residues play a dominant role in the stability of Rh Amidase binding to HPAM-2 at 303 K. Locations of 0–50 residues in Rh Amidase are (shown in Figure S6) kept away from the active site of the enzyme, which supposed that these residues only may affect the enzyme binding stability. The calculated enzyme residue RMSF values for B. subtilis laccase-HPAM-3 are shown in Figure 5b. From Figure 5b, we can find that the RMSF values of all residues in B. subtilis laccase gradually increased with the temperature. Moreover, the RMSF values of all residues for the B. subtilis laccase-HPAM-3 at 298 K were the lowest, further indicating that a more stable binding of B. subtilis laccase with HPAM-3 was obtained at 298 K.

Figure 5.

Figure 5

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RMSF of enzyme residues in Rh Amidase-HPAM-2 (a) and B. subtilis laccase-HPAM-3 (b) at 298, 303, and 308 K.

3.3. Effect of Temperatures on the Binding of the Enzyme to the Substrate

Figure 6a shows that at 298 K, HPAM-2 interacted by forming a hydrogen bond with residues LYS96, SER171, PHE146, GLN192, GLY193, GLY194, and ALA195 in the Rh Amidase. Simultaneously, HPAM-2 formed a water hydrogen bond with a water molecule, so a total of eight hydrogen bonds were formed in Rh Amidase-HPAM-2. Moreover, the four hydrophobic actions of residues TRP328, LEU447, ILE450, and ALA195 with HPAM-2 also could stabilize Rh Amidase-HPAM-2. Lastly, HPAM-2 formed a salt bridge with the LYS96 residue. Figure 6b shows that at 303 K, HPAM-2 interacted by forming hydrogen bonds with residues LYS96, SER171, GLN192, and GLY193 in the Rh Amidase, and the HPAM-2 formed two water hydrogen bonds with two water molecules, so a total of six hydrogen bonds were formed in Rh Amidase-HPAM-2. Furthermore, the seven hydrophobic actions of residues ALA195, ILE227, ALA332, ILE450, and TRP328 with HPAM-2 also could stabilize Rh Amidase-HPAM-2. In the end, HPAM-2 formed an electrostatic interaction with the LYS96 residue. Figure 6c shows that at 308 K, HPAM-2 interacted by forming hydrogen bonds with residues GLN192, GLY193, GLY194, ALA195, and SER171 in the Rh Amidase, and the HPAM-2 formed a water hydrogen bond with a water molecule, so a total of seven hydrogen bonds were formed in Rh Amidase-HPAM-2. Additionally, the six hydrophobic actions of residues TRP328, MET449, ILE198, ALA195, and ILE450 with HPAM-2 also contributed to stabilizing Rh Amidase-HPAM-2. Finally, HPAM-2 formed an electrostatic interaction with the LYS96 residue.

Figure 6.

Figure 6

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Effect of (a) 298, (b) 303, and (c) 308 K on the binding of Rh Amidase with HPAM-2 (green: hydrogen bond; light blue: water hydrogen bond; pink: hydrophobic; orange: electrostatic or salt bridge). Note: the final frame of a 50 ns NPT MD production was analyzed their interaction.

The above results are analyzed and summarized in Table 1. From Table 1, we can find that at 298 K, HPAM-2 formed the most number of hydrogen bonds (7) with surrounding residues. Meanwhile, it produced a hydrogen bond and a salt bridge with the LYS96 residue, so Rh Amidase-HPAM-2 was the most stable at 298 K due to a strong hydrogen bond and a salt bridge.

Table 1. Interaction Details of Rh Amidase-HPAM-2 at 298, 303, and 308 K.

temperature interaction residue type number
298 K LYS96, SER171, PHE146, GLN192, GLY193, GLY194, ALA195 hydrogen bond 7
H2O water hydrogen bond 1
TRP328, LEU447, ILE450, ALA195 hydrophobic 4
LYS96 salt bridge 1
303 K LYS96, SER171, GLN192, GLY193 hydrogen bond 4
H2O water hydrogen bond 2
ALA195, ILE227, ALA332, ILE450, TRP328(3) hydrophobic 7
LYS96 electrostatic 1
308 K GLN192, GLY193, GLY194, ALA195, SER171(2) hydrogen bond 6
H2O water hydrogen bond 1
TRP328, MET449, ILE198, ALA195, ILE450(2) hydrophobic 6
LYS96 electrostatic 1
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As shown in Figure 7a, we can find that at 298 K, HPAM-3 formed six H bonds with residues LYS135, ARG136, and ASP113 in the B. subtilis laccase, and it had seven water H bonds with seven water molecules. Moreover, HPAM-3 formed two electrostatic actions with residues LYS135 and ARG136. As shown in Figure 7b, it can be found that at 303 K, the residue LYS135 underwent a H bond and an electrostatic interaction with HPAM-3, and HPAM-3 had 12 water H bonds with 10 water molecules. From Figure 7c, we can find that at 308 K, the HPAM-3 underwent an electrostatic interaction with the residue LYS135 and formed 13 water H bonds with 11 water molecules.

Figure 7.

Figure 7

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Effect of (a) 298, (b) 303, and (c) 308 K on binding of B. subtilis laccase with HPAM-3 (green: hydrogen bond; light blue: water hydrogen bond; orange: electrostatic). Note: the final frame of a 50 ns NPT MD production was analyzed by their interaction.

The above analyzed results are listed in Table 2. From Table 2, we can find that at 298 K, HPAM-3 formed the most number of hydrogen bonds (6) and electrostatics (2) with surrounding residues, so B. subtilis laccase-HPAM-3 was the most stable at 298 K due to strong hydrogen bonds and electrostatic interactions.

Table 2. Interaction Details of B. subtilis Laccase-HPAM-3 at 298, 303, and 308 K.

temperature interaction residue type number
298 K LYS135, ARG136, ASP113 hydrogen bond 6
H2O water hydrogen bond 7
LYS135, ARG136 electrostatic 2
303 K LYS135 hydrogen bond 1
H2O water hydrogen bond 12
LYS135 electrostatic 1
308 K LYS135 electrostatic 1
H2O water hydrogen bond 13
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3.4. Cultivation of Two Bacteria

Both B. subtilis and R. sphaeroides culture results are shown in Figure 8.Figure 9a is an optical microscope image of R. sphaeroides using Gram staining, showing that it is a Gram-negative bacterial strain. Figure 9b is an optical microscope image of B. subtilis using Gram staining, showing that it is a Gram-positive bacterial strain.

Figure 8.

Figure 8

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R. sphaeroides plate colony morphology (a); R. sphaeroides liquid colony morphology (b); B. subtilis plate colony morphology (c); B. subtilis liquid colony morphology (d).

Figure 9.

Figure 9

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R. sphaeroides staining image under light microscopy (a); B. subtilis staining image under light microscopy (b).

3.5. HPAM Degradation Tests

The used level of the main factors and the L9 (33) matrix were employed to assign experiments, as shown in Tables 3 and 4.

Table 3. Factors and Levels of Orthogonal Experiments.

factor A (T/K) B (initial pH) C (inoculated dosage/mL)
level 1 298 6 1
level 2 303 7 2
level 3 308 8 3
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Table 4. The Results of Orthogonal Experiments by Mixed Bacteria.

factor A (T/K) B (initial pH) C (inoculated dosage/mL) HPAM degradation ratio/%
1 1 (298 K) 1 (6) 1 (1 mL) 31.20
2 1 2 (7) 2 (2 mL) 33.69
3 1 3 (8) 3 (3 mL) 32.25
4 2 (303 K) 1 2 33.23
5 2 2 3 34.77
6 2 3 1 33.86
7 3 (308 K) 1 3 34.26
8 3 2 1 35.57
9 3 3 2 34.55
K1 32.38 32.90 33.54  
K2 33.95 34.68 33.82  
K3 34.79 33.55 33.76  
R 2.41 1.78 0.28  
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The HPAM degradation ratio was calculated (Table 4). From Table 4, the optimal factor combination A3B2C2 was obtained according to the range analysis. Consequently, A3B2C2 was selected as the optimal condition to treat 500 mg/L HPAM wastewater. The corresponding specific optimal conditions were 308 K, initial pH = 7, and an inoculated dosage of 2 mL. These conditions resulted in the HPAM degradation ratio of 39.24%. The effect of operation parameters on the HPAM degradation ratio followed a declining order: temperature > initial pH > inoculated dosage, and temperature was the most important effect factor. A high temperature not only can denature and inactivate the enzymes but also increase energy consumption. Thus, 308 K was chosen as the optimal without considering higher temperatures. Subsequent degradation comparison of mixed bacteria with a single strain was carried out at these optimal conditions. From Figure 10a, the enzymatic reaction rate of R. sphaeroides was a little higher than that of B. subtilis. This indicated that the activity of amidase secreted by R. sphaeroides was better that of B. subtilis laccase, and the amide group of HPAM was easier to be degraded by amidase than its carbon chain, which is consistent with experimental results in the literature.16 As shown in Figure 10b, under the optimal conditions obtained above, the degradation rate of mixed bacteria was higher than that of a single bacterial strain. This is because mixed bacteria can secrete both amidase and laccase. Laccase was more likely to oxidize the carbon chain of HPAM to provide a carbon source when the amide side chain of HPAM was degraded by amidase.

Figure 10.

Figure 10

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Comparison of degradation between single strain (a) and mixed bacteria (b). Reaction conditions: T = 308 K, initial pH = 7, inoculated dosage = 2 mL, and time = 7days.

In addition, we can find that HPAM was degraded slowly, and its degradation rate was only 2.58% under natural conditions. This indicated that HPAM can accumulate in sewage for a long time and is difficult to be degraded under natural light irradiation.

3.6. Supposed Codegradation Mechanism by Mixed Bacteria

Previous studies have shown that Rh Amidase was inclined to degrading the short chain of HPAM, and B. subtilis laccase accommodated a certain length of HPAM. MD simulation explored the effect of temperature on the stability of enzyme binding to the substrate, and an approximate temperature range was obtained for further experiments. The optimal stability of complexes and enzyme binding to the substrate was obtained by theoretical calculations, while the optimal degradation performance was obtained at 308 K. This may be because a higher temperature, such as 308 K, can enhance the disturbance of complex and enzyme binding to the substrate, promote mass and heat transfer, and thus obtain the optimal degradation performance.

The degradation effect of R. sphaeroides was better than that of B. subtilis, suggesting that oxidative fracture of the HPAM carbon chain was more difficult than the hydrolysis of its amide group. The degradation effect of mixed bacteria was better than that of a single strain, indicating that the mixed bacteria can simultaneously secrete amidase and laccase; they can jointly degrade HPAM. Figure 11 shows the codegradation mechanism by mixed bacteria, following these two parts: (1) The amide side chain of HPAM was easily hydrolyzed by the amidase secreted by R. sphaeroides into NH4+ as a nitrogen source. (2) Simultaneously, the carbon chain of HPAM was oxidized and broken by laccase secreted by B. subtilis as a carbon source.

Figure 11.

Figure 11

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Supposed HPAM codegradation mechanism by mixed bacteria.

4. Conclusions

The most suitable Rh Amidase-HPAM-2 and B. subtilis laccase-HPAM-3 were obtained by docking to further use MDS at 298, 303, and 308 K to evaluate the binding of the enzyme with the substrate and the stability of complexes. The results of MDS analysis show that both Rh Amidase-HPAM-2 and B. subtilis laccase-HPAM-3 were the most stable at 298 K. The LYS96 in Rh Amidase-HPAM-2 and LYS135 in B. subtilis laccase-HPAM-3 were the key in their binding process at different temperatures. The optimal binding of Rh Amidase-HPAM-2 and B. subtilis laccase-HPAM-3 was at 303 and 298 K, respectively. The optimum conditions of mixed bacteria degrading HPAM were 308 K, initial pH = 7, and an inoculated dosage of 2 mL, and the degradation ratio reached 39.24%. The effect of parameters followed a declining order of temperature > initial pH > inoculated dosage. The HPAM codegradation mechanism was supposed by mixed bacteria based on experimental results. The mixed bacteria concurrently secreted both amidase and laccase, and they acted together on HPAM. These data are not only highly valuable and suggest sites for future experimental mutagenesis studies but also a valuable contribution to the scientific literature and further the field understanding of laccase and amidase.

Acknowledgments

This work was funded by the National Natural Science Foundation of China under grant no. 51274012, the National Natural Science Foundation of China under grant no. 31601465, and the Stable Talent Fund of Huainan Normal University (BSKYQDJ).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c07380.

  • Relevant calculation details; interaction energy that can evaluate how well the enzyme binds to the substrate; conformational change that can intuitively show the stability of the enzyme binding to the substrate; position of the residues with the greatest fluctuation shown in Rh Amidase (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao2c07380_si_001.pdf (538.5KB, pdf)

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