Enhanced Plant-microbe Remediation of PCBs in Soil using Enzyme Modification Technique Combined with Molecular Docking and Molecular Dynamics Site-Directed Mutagenesis;

: The study on the enhanced mechanisms of the enzymes involved in plant absorption, plant degradation, and microbial mineralization in the remediation of soils contaminated with polychlorinated biphenyls (PCBs) is of great significance for the application of plant-microbe combined remediation technique in PCB-contaminated soils. The present study first used a combination of molecular docking and molecular dynamics methods to calculate the effects of the plant absorption enzyme, plant degradation enzyme, and microbial mineralization enzyme on the PCBs in the soil environment. A multifunctional plant degradation enzyme was constructed with three functional roles of absorption, degradation, and mineralization using amino acid sequence recombination and site-directed mutagenesis to modify the template of plant degradation enzyme. Finally, using the Taguchi experimental design-assisted molecular dynamics simulation method, the suitable external environmental conditions of plant-microbe combined remediation of the PCB-contaminated soil were determined. In total, six multifunctional plant degradation enzymes were designed, which exhibited a significantly improved efficiency of PCB degradation. In comparison to the complex of plant absorption enzyme, plant degradation enzyme, and microorganism mineralization enzyme (6QIM-3GZX-1B85), the six multifunctional plant degradation enzymes exhibited significantly higher efficiency (2.10–2.38 times) in degrading the PCBs, with a maximum of 2.69 times under suitable external environmental conditions. The present study combined molecular docking and molecular dynamics methods to explore the remediation capacity of the plant absorption enzyme, plant degradation enzyme, and microbial mineralization enzyme for PCB-contaminated soil. The results revealed that the binding energy of the combined action of the seven PCBs as a whole on the plant degradation enzyme (3GZX) was the highest, which indicated that the plant degradation enzyme (3GZX) is more suitable for application in the remediation of PCB-contaminated soils. Therefore, the plant degradation enzyme was selected as the template for enzyme modification using amino acid recombination and site-directed mutagenesis. we have achieved the design of six multifunctional plant degradation enzymes which can degrade the PCBs significantly effective. Molecular dynamics assisted by the Taguchi experimental design method was used for determining the suitable external environmental conditions (such as pH, temperature, organic matter content (nitrogen and phosphorus dosing ratio), oxygen promoter concentration, and catalyst concentration) that would promote the plant-microbe combined remediation of the PCB-contaminated soil. The multifunctional plant degradation enzymes constructed in the present study and the suitable external environmental conditions determined for the different remediation methods can meet the design requirements for enhanced efficiency of the enzymes in degrading PCBs. an improved


Introduction
Polychlorinated biphenyls (PCBs) have 209 congeners based on the different positions and number of chlorine atom substitutions. Previously, PCBs were used widely as dielectrics and lubricants in capacitors and transformers and also as additives in paints or plastics due to their highly stable physical and chemical properties, good electrical insulation, high heat resistance, and low flammability [1]. However, PCBs exhibit environmental persistence, high biological toxicity, high bioconcentration, and long-range migration, because of which they are now listed among the 12 environmental persistent organic pollutants (POPs) to be controlled under the Stockholm Convention [2][3]. The period between the 1920s and 1970s is considered the golden age of PCB production and application. Since the 1980s, countries worldwide have gradually banned the production and application of commercial mixtures containing PCBs. Nonetheless, large quantities of environmentally persistent commercial mixtures of PCBs continue to exist in the electrical systems, land, and landfills [4]. Besides the global pollution caused by PCBs, the incineration of chlorinated compounds and the use of chlorinated chemicals continues to introduce PCBs into the soil environment. Humans would be exposed to soil environments containing these contaminants for decades and even centuries to come [5].
The estimated cumulative global production of commercial PCBs is 1.3×10 6 tons, out of which, approximately 440-92,000 tons of PCBs are being released into the environmental media.
Since PCBs have low volatility and low water solubility, the soil environment becomes their largest sink, accounting for approximately 93.1% of the PCBs existing in the environment [6].
Numerous studies have demonstrated that PCBs in contaminated soils have reached levels that could severely affect human health [7][8]. The concentrations of PCBs are observed to be in the range of 0.0626-97 ng·g -1 , with an average of 5.41 ng·g -1 in the 191 topsoil sites investigated worldwide; PCBs have even been detected in the soil samples from the Antarctic regions [9][10].
The evaluation of 7 typical PCBs in the surface soil samples collected from highly industrialized areas around the Yellow Sea and the Bohai Sea revealed residues of PCBs in a concentration of up to 385.67 ng·g -1 dw, with a mean value of 19.89 ng·g -1 dw [11]. The  of 49.75 ng·g -1 and 246.86 ng·g -1 , respectively [12]. The data for the urban soils of the different regions of China collected between 2004 and 2018, which included the PCBs detection data, reveals that the average concentration of PCBs in these soil samples was 4984 mg/kg and that 64% of the study areas had the total carcinogenic risk values for PCBs above the individual lifetime acceptable risk level of 10 -4 mg/kg. Meanwhile, the non-carcinogenic risk values exceeded the target risk level (10 -1 mg/kg) in 53% of the areas, and via ingestion, inhalation, and dermal exposure routes, the lifetime carcinogenic and non-carcinogenic risks of PCBs in the urban soils of China have exceeded the safe levels in most cases [8]. Therefore, it is clear that the soil is contaminated with a variety of PCBs and the residual concentration of the PCBs in the contaminated soils is considerably high, which is harmful to human health as well as for the ecological environment in the long-term perspective. Therefore, identifying an efficient and secondary contamination-free method for the remediation of PCB-contaminated soils is of great significance.
The traditional remediation approach for soil pollution include physical, chemical, and biological methods. It has been reported that PCBs can be adsorbed by adding activated carbon to the soil, and the fixation and treatment of PCBs in polluted soil can be completed [13]. However, physical methods cannot degrade PCBs completely, so they need to be combined with other treatment methods. This method has the disadvantage of easy to produce secondary pollutants. It has been shown that the combined use of H 2 O 2 and KMnO 4 as chemical oxidants had a certain effect on the remediation of PCBs contaminated soil [14]. The chemicals used may cause damage to the health of the soil, and the cost is relatively high. It has been reported that the concentration of PCBs decreased by 53.1% after a combination treatment with ZY1, a strain isolated from the rhizobia of Astragalus rhizogenic, which exerted a synergistic effect and promoted the extraction and degradation of PCBs [15]. Compared with the physical method and chemical method, biological method is widely used in soil remediation of organic pollutants because of its simple operation, low cost, and no secondary pollution. Plant-microbe remediation technology is a highly efficient biological treatment method [16].
In order to improve the efficiency of bioremediation, genetically modified plants and microorganisms are applied in the bioremediation of PCBs contaminated soil. For instance, the isolation of cytochrome P450 monooxygenase genes and their functional expression in Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210104/908807/bcj-2021-0104.pdf by guest on 27 April 2021 recombinant enzyme enabled efficient oxidation of cyclohexane to cyclohexanol [17]. Biphenyl dioxygenase was modified to enhance its ability to degrade contaminants in the plant along with enabling its secretion from the root system into the soil environment to promote contaminant degradation in the soil as well [18]. The main processes involved in the plant-microbe combined remediation of organic soil contamination are plant absorption, plant degradation, and microbial mineralization [19]. The water channel enzyme in the plant roots mediates the transport of PCBs within the plant and consequently influences the ability of the plant to absorb PCBs [20]. Plants secrete the enzyme biphenyl dioxygenase, which is associated with the degradation of PCBs, and the ability of this biphenyl dioxygenase to degrade PCBs influences the effectiveness of plant-based remediation of PCB-contaminated soil [18]. The mineralization of the PCBs by plant rhizosphere is one of the approaches of plant-microbe combined remediation of PCB-contaminated soil. The microorganisms in the plant rhizosphere include both fungi and bacteria. A typical species of the white-rot fungus, viz., Phanerochaete chrysosporium, was investigated, and it was observed that its secreted lignin peroxidase that mainly mineralized the organic pollutants [21]. Since the plant absorption enzyme, plant degradation enzyme, and microbial mineralization enzyme are involved directly in soil remediation, if we modify an enzyme with three functions of absorption, degradation and mineralization. When multifunctional enzyme is applied to the plant-microbe combined remediation technology, the remediation ability of the technology will be greatly improved.
Enzyme modification allows the identification of the key amino acid residues in the plant absorption enzyme, plant degradation enzyme, and microbial mineralization enzyme that could be functionally relevant to the action of these enzymes on PCBs. Further, precisely designing the mutation sites in these amino acids helps in altering these specific residues in the target enzyme to improve their functional properties. For instance, biphenyl dioxygenase was modified to enhance its ability to degrade contaminants in the plant along with enabling its secretion from the root system into the soil environment to promote contaminant degradation in the soil as well [18]. Zhang et al. (2010) studied the effects of amino acid residues at the Cel6A active site of Thermobifida fusca cellulase, and mutants modified with the amino acid residues were found to exhibit improved hydrolytic activity towards carboxymethyl cellulose [22]. To promote the biodegradation of aromatic hydrocarbons in contaminated soils, the key acid residues of Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210104/908807/bcj-2021-0104.pdf by guest on 27 April 2021 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20210104 naphthalene dioxygenase (NDO) was modified [23]. However, there are few reports on the application of enzyme modification to reconstitute the key amino acid fragments of plant absorption enzyme, plant degradation enzyme, and microbial mineralization enzyme with the aim of obtaining a multifunctional enzyme with the combined ability of absorption, degradation, and mineralization to facilitate the remediation of PCB-contaminated soils. It is reported that the physicochemical properties of the soil are a major factor affecting the plants-microorganisms combined remediation of soil contaminants [24]. In this context, the present study identified the internal modification scheme of these three enzymes and their external environmental conditions to enhance their ability to realize plant-microbe remediation.
The present paper describes the study of the ability of plants to absorb, degrade, and mineralize the PCBs in the soil environment by using a combination of molecular docking and molecular dynamics methods. Furthermore, the enzyme identified to have the strongest efficiency against PCBs was selected as the template for generating the modified enzyme with a combination of the three functions roles of absorption, degradation, and mineralization. The multifunctional enzymes for enhanced PCB-contaminated soil remediation were constructed under consistent external environmental stimulation conditions. The suitable external environmental stimulation conditions for enhanced plant-microbial combined remediation of PCB-contaminated soil were determined by evaluating different external environmental stimulation conditions of plant absorption enzyme, plant degradation enzyme, and microbial mineralization enzyme. In addition, the preferential and applicability analysis of the modified enzymes for the degradation of different PCBs was conducted to provide a basis for the enhanced plant-microbe combined remediation of PCB-contaminated soils. The findings would provide a research perspective for enhancing enzyme repair ability to enhance the plant-microbe combined remediation of PCB-contaminated soils indirectly.

Characterization of CBs binding to plant-microbe enzyme using molecular docking
The present study investigated the seven PCB contaminants (PCB-28, PCB-52, PCB-101, PCB-118, PCB-138, PCB-153, PCB-180) present commonly in soil, among which PCB-118 is a member of the class of dioxin-like polychlorinated biphenyls [25]. Three representative functional enzymes (water channel enzyme (6QIM), biphenyl dioxygenase (3GZX) and lignin peroxidase can perform rational design in functional enzymes [26]. Therefore, the corresponding positions of the third functional enzyme were replaced by the key amino acid residues of the other two functional enzymes. The purpose is to keep the function of the third enzyme unchanged and to combine the functions of the other two enzymes. The insertion of key amino acid residues corresponding to the active regions of two enzymes into the third enzyme exerts a significant effect on PCBs, and therefore, this approach was used for designing and constructing a multifunctional enzyme with a combination of the three functional roles of plant absorption, plant degradation, and microbial mineralization.
According to the similar phase solubility principle, PCBs are hydrophobic molecules, which bind stably to the hydrophobic region formed by hydrophobic amino acids residues [27][28].
Therefore, an attempt was made to replace hydrophilic molecules with hydrophobic amino acids residues (the hydrophobicity of the amino acid residues in descending order: Val > Ile > Leu > Cys) by the site-directed mutagenesis method [29]. The key amino acid residues in the active region of the multifunctional enzyme were mutated to produce the multifunctional enzyme. The structural reliability of the designed multifunctional enzyme was evaluated using the Ramachandran conformational map in the online evaluation server PROCHECK (https://saves.mbi.ucla.edu/). When the percentage of the amino acid residues in the core area + allowable area + maximum allowable areas was greater than 95%, it was considered that the model meets the quality requirements [30][31].
Furthermore, CHARMm force field was applied to the designed multifunctional enzyme using the "Apply Forcefield" module in the Discovery Studio 2020 software. In addition, the affinity of the multifunctional enzyme toward the mutated amino acids and the thermal stability of the enzyme structure were evaluated using the "Calculate Mutation Energy (Binding)" and "Calculate Mutation Energy (Stability)" modules, respectively, in the same software. The thermal stability mutation energy of an enzyme is an important parameter determining its, that is, the enzyme's ability to maintain its functional activity under higher temperature conditions. The heat resistance of an enzyme is a necessity for its application in complex real-world external environments [32]. The principle for calculating the affinity of the multifunctional enzyme toward the mutated amino acids and the thermal stability of the multifunctional enzyme structure is similar, as both are determined by the mutation energy of the enzyme structure. The only difference is that the mutation energy representing affinity is the difference in the binding free energy of the structure prior to and after mutation, while the mutation energy representing thermal stability is calculated as the difference in the folding free energy of the structure prior to and after mutation. The mutation energy (ΔG mut ) representing the thermal stability of an enzyme is calculated using the following equation [33][34][35]:

Characterization of the key enzymes influencing the plant-microbe combined remediation using enzyme-enzyme docking
In order to separately determine the conditions that would promote the simultaneous absorption, degradation, and mineralization of the PCBs in the plant-microbes combined remediation of soil in different external environment conditions, enzyme-enzyme docking was performed in the "ZDOCK" module in the Discovery Studio 2020 software. One of the three enzymes was randomly selected as the target enzyme, and its structure was loaded into the Discovery Studio 2020 software as the receptor, while the other two enzymes were designated as ligands 1 and 2 using the "ZDOCK" module. The "RMSD Cutoff" was set to a cluster radius of 6.0 Å, the "Interface Cutoff" was set to 9.0 Å, and the "ZRank" was set to false.

Determination of the external environment conditions to promote the plant-microbe combined remediation using Taguchi experimental design
In this analysis, the following five different external environmental conditions influencing the degradation of PCBs by plants and microorganisms in the soil environment were used as factors for generating the Taguchi experimental design table: pH, temperature (°C), organic matter content (nitrogen and phosphorus dosing ratio), oxygen promoter concentration (mg/L), and catalyst concentration (mg/L). The two levels of external environmental conditions were selected as described below.
As the pH of soil varies widely, different pH levels influence the efficiency of PCB biodegradation. The biodegradation of PCBs is better at the pH range of 6-8, and the degradation decreases when the pH drops below 5 or is above 9 [36]. Therefore, the physically lower (pH = 5) and higher (pH = 9) pH values were selected as the two levels of the external environmental factor pH.
Temperature is one factor that directly influences the biodegradation of aromatic hydrocarbon pollutants. The optimum temperature range for the biodegradation of PCBs is 22-40 °C, although certain cold-tolerant bacteria are capable of degrading the PCBs pollutants even at low temperatures in the range of 3-20 °C [37]. Therefore, the physically lower (15 °C) and higher (35 °C) values of temperature were selected as the two levels of the external environmental factor temperature.
Since organic matter content has a huge impact on the rate of biodegradation of pollutants, this can only be improved if the nutrient requirements of the microbial metabolism are fulfilled.
The best degradation of pollutants by bacteria is achieved at the appropriate nitrogen to phosphorus ratio of 10:1 [38]. Therefore, the lower (1:1) and higher (10:1) ratio values were selected as the two levels of the external environmental factor of organic matter content.
Sufficient levels of oxygen are required for the oxidation process with which the PCBs are degraded. Therefore, Oxygen promoter concentration is an important external environmental factor. H 2 O 2 concentration of 50 mg/L promotes the degradation of PCBs, while the concentrations above 300 mg/L result in slower degradation [39]. Therefore, the lower (50 mg/L) and higher (350 mg/L) physical values of H 2 O 2 concentrations were selected as the two levels of the factor of oxygen promoter concentration. A catalyst enhances the activity of the degradation enzymes. The concentration of resveratrol, a catalyst, at 150 mg/L promotes the degradation of pollutants by the degradation enzymes, while at 200 mg/L, the degradation slows down [19]. Therefore, in the present study, the lower (150 mg/L) and higher (200 mg/L) values of resveratrol concentration were selected as the two levels of the factor catalyst.

PCB-contaminated soil
The Taguchi experimental design method is a particular-orthogonal method of experimentation [40]. This method could analyze many variables using a relatively small number  (Table 2).
In summary, the binding energy of the combined action of the seven PCBs as a whole on the plant degradation enzyme (3GZX) was the largest. It was also 2.57 times and 1.21 times higher than the binding energy of the combined action of the seven PCBs as a whole on the plant absorption enzyme (6QIM) and the microbial mineralization enzyme (1B85), respectively. This, indicated that the plant degradation enzyme (3GZX) is more suitable for application in the remediation of PCB-contaminated soils.  The Ramachandran conformational map obtained using the online server of PDBsum (http://www.ebi.ac.uk) was used for evaluating the structural quality of the enzymes (Figure 3). It was revealed that all the amino acid residues in the six multifunctional plant degradation enzymes were located in the "allowed" region. The sum of the percentage amounts of the bases located in the core, allowed, and generously allowed regions within the six multifunctional plant degradation enzymes was 99.8%, which met the quality rationality requirement of greater than 95%.

Modification and evaluation of the multifunctional plant degradation enzymes for
Evaluation of the affinity and the thermal stability of the six multifunctional plant degradation enzyme structures (Table 4). When the mutation energy was between -0.5 kcal/mol and +0.5 kcal/mol, the mutation had no effect on the affinity between the multifunctional plant degradation enzyme and the mutant amino acid or on the thermal stability of the multifunctional enzyme. When the mutation energy was less than -0.5 kcal/mol, the mutation enhanced the affinity between the multifunctional plant degradation enzyme and the mutant amino acid, and also the thermal stability of the multifunctional enzyme. As presented in Table 4, the affinity mutation energy and the thermal stability mutation energy of the six multifunctional plant degradation enzymes were less than -0.5 kcal/mol, indicating that the corresponding mutant amino acids had enhanced the affinity for the multifunctional plant degradation enzymes and improved the structural stability of the six multifunctional enzymes [41].  (Table 5).
It was observed that the suitable conditions for improving the binding effect of the 7 PCBs were different (Table 6) Table 8).
Analysis of the suitable external environmental conditions for the binding effect of the seven PCBs as a whole docked with the 6QIM-3GZX-1B85 enzyme receptor. As presented in Table 9,  (Table 10).
In summary, the binding effect of the seven PCB molecules and the six multifunctional plant degradation enzymes under the same external environmental conditions exhibited significant improvement. It indicated that the multifunctional plant degradation enzymes designed in the present study, possessing a combination of the three functional roles, could be used for the plant-microbe combined remediation of PCB-contaminated soils instead of other enzymes.

Selective analysis of the degradation of PCBs by the multifunctional plant degradation enzymes
In order to study the preference of different PCBs degraded by multifunctional enzymes, four 5.37%-9.50%, respectively) was not significant. In summary, it indicated that there was no significant difference in the degradation selectivity for the other PCBs.

Applicability analysis of the multifunctional plant degradation enzymes in degrading PCBs
To study the applicability of multifunctional enzyme degradation of different PCBs, using the  In this paper, we studied a method to strengthen the repair ability of enzymes, and the multifunctional plant degradation enzyme has a combination of the three functions roles of absorption, degradation, and mineralization. It has not been studied before to solve the three functions with one enzyme. The presented method is design concept of transgenic plants and microbial improvement. The application of this method has practical significance for developing an improved plant-microbe remediation technique in the soil environment.