Computational Assessment of CNT–NH₂ and Enzyme-Functionalized Nanomaterials for Polycyclic Aromatic Hydrocarbon Remediation

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Adeleke, Muhammed H. Garuba, Ali A. Aremu, Abubakar M. Ogacheko, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8248323/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 16 You are reading this latest preprint version Abstract Polycyclic aromatic hydrocarbons (PAHs) persist in the environment due to their chemical stability, toxicity, and resistance to conventional remediation processes. This study investigates the electronic reactivity and adsorption potential of selected PAHs such as anthracene, benzo[a]pyrene, fluorene, naphthalene, and pyrene using density functional theory (DFT) and molecular docking approaches. Frontier molecular orbital calculations revealed that PAHs with smaller HOMO–LUMO gaps exhibited greater reactivity and more negative Gibbs free energy values, indicating enhanced thermodynamic favorability for adsorption. Benzo[a]pyrene showed the highest reactivity (HOMO–LUMO gap = 7.26 eV) and the most negative Gibbs free energy (–31.80 kcal/mol), suggesting its strong electron-accepting tendency. Binding energy analysis of PAH adsorption onto amine-functionalized carbon nanotubes (CNT–NH₂) further confirmed benzo[a]pyrene as the most strongly adsorbed molecule (E bind = − 26.75 kcal/mol). Molecular docking of PAHs with a CNT–enzyme ( Bacillus spp . laccase, PDB: 9BD5) complex demonstrated high docking scores and extensive hydrophobic and π–π stacking interactions, indicating a synergistic remediation mechanism driven by nanoparticle adsorption and enzymatic affinity. The combined DFT and Molecular docking results demonstrate that functionalized CNTs coupled with bacterial enzymes offer a highly effective platform for PAH remediation through dual adsorption and catalytic pathways. PAHs DFT binding energy (CNTs) molecular docking Bacillus spp. enzyme environmental remediation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, anthracene, and benzo[a]pyrene are persistent organic pollutants commonly released from petroleum spills, combustion processes, and industrial waste [ 1 ]. Their strong hydrophobicity, bioaccumulation tendency, and mutagenic or carcinogenic effects make them a major environmental concern, particularly in aquatic systems [ 2 , 3 ]. Conventional remediation techniques including chemical oxidation, activated-carbon adsorption, and biodegradation often show limited selectivity and low efficiency under real-world conditions [ 4 , 5 ]. Carbon nanomaterials offer promising alternatives due to their large surface area and tunable surface chemistry. In particular, carbon nanotubes (CNTs) exhibit strong π–π interactions with aromatic pollutants, enabling effective adsorption [ 6 ]. Surface functionalization, especially with amine (− NH₂) groups, improves CNT dispersibility and enhances affinity for aromatic hydrocarbons [ 7 ]. Thus, amine-functionalized CNTs (CNT–NH₂) are increasingly explored as selective adsorbents for PAH remediation in contaminated water. Enzymatic degradation provides another eco-friendly remediation pathway. Laccases (EC 1.10.3.2) from Bacillus licheniformis are thermostable, alkaline-tolerant, and capable of oxidizing a wide range of aromatic pollutants, making them suitable for environmental detoxification [ 8 ]. When immobilized on CNTs, laccase–nanotube hybrids can combine catalytic oxidation with enhanced pollutant adsorption, creating a synergistic nanobiocatalytic remediation system [ 9 ]. Computational tools play a central role in understanding the adsorption and catalytic interactions between PAHs, CNTs, and enzymes [ 10 ]. Density Functional Theory (DFT) provides insights into electronic structure, charge transfer, and adsorption energetics [ 11 ], while molecular docking reveals binding orientation and interaction strength between PAHs and enzyme-functionalized nanomaterials [ 12 ]. DFT methods incorporating dispersion corrections are particularly effective for modeling π–π and van der Waals interactions in PAH–nanomaterial systems [ 13 , 14 ].Therefore, this study based on integrating DFT calculations, molecular docking, and adsorption analysis using CNT–NH₂ and Bacillus licheniformis laccase provides a comprehensive framework for predicting and optimizing PAH remediation efficiency in oil-spill-contaminated environments.. 2. Methodology 2.1 Preparation of ligand structures Five polycyclic aromatic hydrocarbon (PAH) ligands pyrene (PYR), naphthalene (NAP), anthracene (ANT), fluorene (FLU), and benzo[a]pyrene (BEZ) were obtained from the PubChem Chemical Database in .sdf format. The carbon nanotube (CNT) structure and all PAH ligands were sketched and modeled in both two- and three-dimensional (2D and 3D) forms using ChemDraw Professional 16.0®, as shown in Fig. 1 . The generated 3D geometries were subsequently imported into PyRx Virtual Screening Tool® [ 15 ], where they were converted and optimized using Open Babel [ 16 ]. The (3D) three-dimensional crystal structure of laccase from Bacillus licheniformis (PDB ID: 9BD5) was downloaded from the Protein Data Bank (PDB) via the NCBI PubChem interface ( http://pubchem.ncbi.nlm.nih.gov/ ) as presented in Fig. 1 . Prior to docking, the protein structure was prepared by deleting heteroatoms and removing all crystallographic water molecules. The raw files were converted into .pdb format using Discovery Studio Visualizer 2.5.5 [ 17 ]. The PAH ligands were then subjected to energy minimization in UCSF Chimera 1.5.3 using a genetic algorithm with 2000 steps and an optimized grid spacing of 0.5 units. 2.2 Nanoparticle Docking Analysis All in silico docking procedures were carried out using the PyRx Virtual Screening Tool® [ 15 ]. The laccase enzyme was initially docked with the PAH ligands pyrene (PYR), anthracene (ANT), fluorene (FLU), benzo[a]pyrene (BEZ), and naphthalene (NAP) as well as with the carbon-nanotube nanoparticle (CNT-NP). The resulting enzyme–nanotube complex (ENZ–CNT) generated from this first docking stage was exported in .pdb format and subsequently used as the receptor for the second round of docking, in which the five PAH ligands were docked against the ENZ–CNT hybrid, as presented in Fig. 2 . Both receptor and ligand structures were prepared following standard protocols for protein and small-molecule preparation. Docking simulations were executed using PyRx, while visualization and post-docking analysis were performed using BIOVIA Discovery Studio [ 17 ]. Binding affinities for all ligand–receptor pairs were automatically computed in PyRx, and the resulting interactions hydrogen bonding, hydrophobic contacts, π–π stacking, and other noncovalent forces were examined using Discovery Studio Visualizer. Multiple conformations were generated for each PAH ligands during the docking workflow, and the lowest-energy poses were further refined to ensure optimal fitting within the enzyme’s active site. The docking score corresponding to the most stable pose was recorded for each ligand. Final analyses included detailed visualization of receptor–ligand complexes, identification of key active-site residues involved in binding, and generation of high-resolution interaction as presented in the result and discussion section. 2.3 Density Functional Theory (DFT) Density Functional Theory (DFT) calculations [ 18 ] were carried out on Orca software [ 19 ] and the results were visualized using Avogadro software [ 20 ]. Two dimensional (2D) and three dimensional (3D) structures of the studied PAHs and amine-fictionalized carbon-nanotube (CNT-NH 2 ) unit were drawn using ChemSketch [ 21 ] shown in Figure xx, and then the model structures are optimized using DFT theory calculations at B3LYP/LanL2DZ level for the determination of electronic structure properties (HOMO – LUMO) of PAH ligands pyrene (PYR), anthracene (ANT), fluorene (FLU), benzo[a]pyrene (BEZ), and naphthalene (NAP) as well as with the carbon-nanotube nanoparticle (CNT-NP). The binding energies \(\:{E}_{bind}\) of the PAHs and carbon-nanotube nanoparticle (CNT-NP)-complex were calculated from equations 1 [ 22 ] Binding energy was calculated as: $$\:{E}_{bind}={E}_{complex}-\left({E}_{CNT-NH2}+{E}_{PAH}\right)\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:1$$ Where \(\:{E}_{bind}\) is the binding energies, \(\:{E}_{PAH}\) is the binding energy of PAHs, \(\:{E}_{CNT-NH2}\) is the energy of carbon nanotube (CNT-NH 2 ) and \(\:{E}_{complex}\) is the binding energy of the carbon nanotube (CNT-NH 2 complex) doped with PAHs 3. Results and Discussion 3.1 Molecular Docking Analysis Molecular docking was performed to evaluate the binding behavior of the selected PAH ligands within the active site of laccase from Bacillus licheniformis (PDB: 9BD5). Five PAHs—benzo[a]pyrene (BEZ), pyrene (PYR), fluorene (FLU), anthracene (ANT), and naphthalene (NAP)—were retrieved from PubChem and screened against the target enzyme. Docking scores and interaction patterns for all ligands are summarized in Table 5. Ligands exhibiting the most favorable binding affinities [ 23 ], were considered the strongest interactors with the enzyme. To further understand the mechanistic basis of these interactions, a carbon nanotube–ligand docking strategy was employed. This two-stage docking approach allowed assessment of (i) the binding affinity of PAHs toward carbon nanotubes (CNTs), and (ii) the change in enzyme functionality when CNTs were functionalized and subsequently exposed to PAH pollutants. By comparing docking scores of CNT–ligand and CNT–enzyme–ligand complexes, the study highlights potential disruption or enhancement of catalytic properties following PAH exposure. The CNT nanoparticle alone showed measurable affinity toward the PAH ligands with binding energies of − 5.8 (BEZ), − 5.3 (PYR), − 4.5 (FLU), − 4.4 (ANT), and − 3.8 kcal/mol (NAP) as presented in Table 1 . These values indicate a moderate level of adsorption, suggesting that CNTs can interact with PAHs but with varying degrees of preference depending on molecular size and aromaticity. Ligands with energies equal to or more negative than CNT–PAH complexes were considered as stronger candidates for binding to the enzyme–CNT system. The CNT nanoparticle exhibited an even stronger interaction with laccase, yielding a binding energy of − 7.7 kcal/mol, which is considerably more favorable than the PAH–CNT interactions. This indicates that CNT functionalization does not hinder enzyme binding but may facilitate a stable enzyme–nanotube hybrid complex. Table 1 Binding Affinity (docking of Score) of PAH–CNT and complex interactions. PAH Ligand Binding Affinity CNT-NH 2 -enzyme complex CNT-NH 2 PYR –6.7 –5.3 NAP –5.5 –3.8 FLU –6.2 –4.5 ANT –6.7 –4.4 BEZ –7.7 –5.8 To assess the effect of PAH pollutants on this hybrid system, the CNT–enzyme complex was docked with the same PAH ligands. The resulting affinities were: (–7.7, − 6.7, − 6.7, − 6.2 and − 5.5) kcal/mol for BEZ, PYR, ANT, FLU and NAP respectively, as presented in Table 1 .. Among the ligands, benzo[a]pyrene (BEZ) demonstrated the strongest binding to the CNT–enzyme complex, followed by PYR and ANT. These values suggest that high-molecular-weight PAHs have an increased tendency to interact with the catalytic residues of laccase, possibly disrupting normal enzyme function [ 9 ]. Hydrogen bonding, π–π stacking, hydrophobic interactions, and key active-site contacts were observed in the docked complexes, confirming stable ligand accommodation within the enzyme pocket. This observation aligns with the report by Pantsar and Poso [ 23 ], which notes that increasingly negative docking scores typically reflect stronger and more stable molecular interactions. The docking simulations indicate that PAHs particularly BEZ and PYR can form strong interactions with both CNTs and the CNT–enzyme complex. This suggests that PAH pollutants may compete with or inhibit CNT-functionalized enzyme systems, which is essential for understanding their environmental behavior and designing effective nanoparticle-based remediation strategies [ 24 ]. The binding interactions of the PAH ligands within the active site of the CNT-functionalized Bacillus laccase complex showed strong adsorption onto the CNT nanoparticle surface, as demonstrated in the 2D and 3D docking conformations (Fig. 3 ). The dominant interactions involved hydrophobic forces, particularly π–alkyl and π–π stacking, supplemented by non-bonded van der Waals interactions, consistent with typical PAH–nanomaterial binding behavior [ 23 – 25 ]. Benzo[a]pyrene (BEZ), which exhibited the highest docking score, formed multiple hydrophobic contacts with residues Phe205, Pro207, Gln209, Pro210, Asp211, Ala225, Phe226, Cys227, and Asp229, indicating a stable aromatic–aromatic and aromatic–aliphatic interaction network as presented in (Fig. 3 ). The pyrene (PYR) ligand displayed similar hydrophobic interactions with Phe205, Pro207, Gln209, Pro210, Asp211, Val223, Ala225, Phe226, and Cys227, further supporting the high affinity of multi-ring PAHs for the CNT–enzyme complex. Anthracene (ANT) exhibited van der Waals and π–anion interactions involving Ile260, Phe261, Glu262, Val291, Ile318, and Lys466, as presented in (Fig. 3 ), while fluorene (FLU) showed a combination of van der Waals, π–anion, π–alkyl, π–π stacking, and π-donor hydrogen bonding interactions with Pro111, Asp112, Tyr116, Ala119, Phe131, Glu133, Gly484, and Arg485 as presented in (Fig. 3 ). Naphthalene (NAP), the smallest PAH studied, revealed primarily van der Waals and π–alkyl interactions with Pro77, Asp78, Glu97, Val98, Thr122, Lys123, Gln126, and Val127 as presented in (Fig. 3 ), consistent with its weaker aromatic surface area and lower binding affinity [ 26 ]. As previously highlighted, polycyclic aromatic hydrocarbons (PAHs) have received significant environmental attention due to their volatility, persistence, and toxicity. These characteristics make their complete removal challenging through conventional chemical, physical, or biological approaches [ 1 , 3 , 24 ]. Among remediation strategies, biodegradation remains particularly promising because of its practicality and efficiency in reducing PAH contamination. One potential mitigation strategy involves preventing or reducing PAH-induced toxicity by inhibiting their binding to nanoparticles or enzymes. This finding suggests a higher preferential affinity between the functionalized enzyme and the nanoparticle surface. Previous research similarly reports strong adsorption capacities of nanoparticles such as carbon nanomaterials, graphene oxide, TiO₂, and ZnO toward PAHs and related carcinogenic compounds [ 22 ]. 3.2 Density Functional Theory (DFT) Studies 3.2.1 Frontier Orbital Distribution and Surface Electron Density Figure 4 , shows the computed HOMO and LUMO orbital distributions of the PAHs. Distinct electron density patterns were observed across molecules: (1) HOMO distributions were largely localized on the π-conjugated PAHs aromatic rings, indicating potential for π–π interactions with carbon nanotube (CNT–NH₂) surfaces. (2) LUMO density was more delocalized across the molecular surfaces, except in pyrene, which exhibited notable asymmetry due to its compact structure. The electron-density characteristics can be explained by the following considerations: (i) PAH molecules are generally more electrophilic than CNT–NH₂ fragments, therefore electron density flows from CNT–NH₂ to the adsorbed PAH and (ii) The localization of HOMO on PAHs and partial delocalization of LUMO promotes charge-transfer stabilization upon adsorption. 3.2.2 The relationship between the HOMO–LUMO energy and Gibbs free energies Density Functional Theory (DFT) calculations were performed to determine the frontier molecular orbital energies (HOMO and LUMO) and Gibbs free energies (ΔG) of the selected polycyclic aromatic hydrocarbons (PAHs): anthracene, benzo[a]pyrene, fluorene, naphthalene, and pyrene. The obtained results are summarized in Table 2 . Table 2 Computed HOMO, LUMO, and Gibbs free energy values of selected PAHs. PAH Compound HOMO (eV) LUMO (eV) HOMO–LUMO Gap (eV) –ΔG (kcal/mol) Anthracene –8.290 –3.656 4.634 –26.54 Benzo[a]pyrene –9.831 –2.576 7.255 –31.80 Fluorene –9.559 –2.404 7.155 –26.23 Naphthalene –9.652 –0.212 9.440 –23.47 Pyrene –8.441 –1.153 7.288 –27.92 A general trend was observed where PAHs with smaller HOMO–LUMO gaps exhibited higher reactivity and more favorable Gibbs free energies (more negative ΔG values). The relationship between the computed HOMO–LUMO energy gaps and Gibbs free energies is shown in Fig. 5 . The data reveal that benzo[a]pyrene possesses the lowest Gibbs free energy (–31.80 kcal/mol), indicating higher thermodynamic stability and a greater tendency to interact with electron-accepting surfaces, such as amine-functionalized carbon nanotubes (CNTs). This compound also has one of the smallest HOMO–LUMO gaps (7.26 eV), suggesting enhanced charge transfer potential and π–π stacking ability with the CNT surface. In contrast, naphthalene showed the largest energy gap (9.44 eV) and the least negative Gibbs free energy (–23.47 kcal/mol), implying greater electronic stability but weaker adsorption affinity toward the nanomaterial. Pyrene and anthracene displayed intermediate reactivity patterns, suggesting balanced stability and adsorption tendencies, making them suitable reference compounds for evaluating PAH–nanomaterial interactions [ 27 ]. The observed inverse relationship between the HOMO–LUMO gap and Gibbs free energy supports the notion that higher chemical reactivity (smaller gap) correlates with stronger adsorption interactions. Such electronic behavior aligns with previously reported DFT studies on PAH adsorption onto nanostructured materials (Kumar et al., 2022; Zhang et al., 2021). These findings highlight that functionalized CNTs can effectively serve as electron-rich adsorption sites for PAHs, driven by π–π stacking, van der Waals forces, and charge-transfer interactions. When coupled with enzymatic agents such as laccase from Bacillus licheniformis , synergistic remediation is expected due to the enzyme’s oxidative catalytic ability and the nanotube’s adsorption efficiency [ 9 ]. 3.2.3 Electronic Properties and Binding Energies of PAHs Density Functional Theory (DFT) calculations were carried out to investigate the binding interactions of selected polycyclic aromatic hydrocarbons (PAHs) anthracene, benzo[a]pyrene, fluorene, naphthalene, and pyrene on amine-functionalized single-walled carbon nanotubes (CNT–NH₂), as presented in (Table 3 ). The negative value of the E bind indicates that the binding energy of the PAHs are spontaneous and may do not require activation before it occurs [ 22 ]. And when the binding occurs the relaxed structure of the free PAH compounds and adsorbed PAHs will not be nearly identical. Table 3 Computed Gibbs free energy, and binding energy of selected PAHs. PAH Compound –ΔG (kcal/mol) Ebind (kcal/mol) Anthracene –26.54 –18.42 Benzo[a]pyrene –31.80 –26.75 Fluorene –26.23 –19.63 Naphthalene –23.47 –14.21 Pyrene –27.92 –21.34 The calculated binding energies suggest that benzo[a]pyrene exhibited the strongest adsorption onto the CNT–NH₂ surface (E bind = − 26.75 kcal/mol), consistent with its highly negative Gibbs free energy (–31.80 kcal/mol) and relatively narrow HOMO–LUMO gap (7.26 eV). These results indicate strong π–π interactions and possible charge transfer between the aromatic rings of benzo[a]pyrene and the CNT-NH 2 π-system. Pyrene and anthracene also showed significant adsorption strength (E bind ≈ − 21 to − 18 kcal/mol), suggesting stable interactions facilitated by multiple aromatic rings that enhance π–π stacking. In contrast, naphthalene displayed the weakest adsorption (E bind = − 14.21 kcal/mol) due to its smaller aromatic surface and larger energy gap (9.44 eV), reducing its interaction with the nanotube. A clear inverse correlation was observed between the HOMO–LUMO gap and both binding energy and Gibbs free energy, as shown in Fig. 6 . Molecules with smaller energy gaps exhibited higher reactivity and stronger adsorption, in agreement with prior computational studies on PAH–nanomaterial systems [ 27 , 28 ]. The strong binding affinity of benzo[a]pyrene and pyrene suggests that amine-functionalized CNTs are promising materials for the adsorption and removal of high-molecular-weight PAHs in oil spill-contaminated environments. Furthermore, coupling these nanomaterials with laccase from Bacillus licheniformis can enhance biocatalytic degradation of adsorbed PAHs. The synergistic combination of adsorption (CNT–NH₂) and enzymatic oxidation (laccase) offers a dual-function remediation mechanism, improving efficiency in aqueous systems contaminated by hydrophobic organic pollutants [ 29 ]. Conclusion This study employed Density Functional Theory (DFT) calculations and molecular docking analyses to assess the electronic properties, thermodynamic behavior, and adsorption tendencies of selected polycyclic aromatic hydrocarbons (PAHs) anthracene, benzo[a]pyrene, fluorene, naphthalene, and pyrene toward amine-functionalized carbon nanotubes (CNT–NH₂) and a Bacillus spp. enzyme–CNT complex. The combined computational results demonstrate that the reactivity and adsorption strength of PAHs are strongly governed by their frontier molecular orbital energies, Gibbs free energies, and binding energy profiles. Benzo[a]pyrene exhibited the smallest HOMO–LUMO energy gap and the most negative Gibbs free energy, resulting in the strongest interaction with the CNT–NH₂ surface and the CNT–enzyme complex. Pyrene and Anthracene showed moderate reactivity and stable adsorption, while Naphthalene demonstrated the weakest interaction due to its larger energy gap and lower thermodynamic drive for adsorption. Molecular docking analyses further revealed that the CNT–enzyme complex binds PAHs more strongly than CNT alone, highlighting a synergistic remediation potential. The current research work provides computational evidence that amine-functionalized CNTs, particularly in combination with Bacillus spp. enzymes, serve as efficient adsorbent–biocatalyst systems for PAH mitigation. The strong adsorption behavior, favorable thermodynamic properties, and effective binding interactions suggest that CNT–enzyme hybrid systems hold significant promise for future environmental remediation applications. Further experimental validation is recommended to confirm the computational predictions and to optimize their deployment for pollutant removal. Declarations Ethics approval and consent to participate. Not applicable. Consent for publication. Not applicable. Competing interests. The authors declare no competing interests. Funding No External Funding Author Contribution Author contributions Author contribution Richard K. Adeleke: Conceptualization, Visualization, Writing-Review & Editing, computational (DFT) analysis. Muhammed H. Garuba: Methodology, Investigation, Writing-original Draft. Ali A. Aremu: Software, Validation, Software. Abubakar M. Ogacheko and Damilola Ogunleye: Conceptualization, Formal analysis, Writing-Review & Editing. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Feng J, Wang X, Wu D. Environmental persistence of polycyclic aromatic hydrocarbons. Chemosphere. 2010;78(3):260–6. https://doi.org/10.1016/j.chemosphere.2010.01.020 . Zeng X, Lin X, Chen Z. Environmental distribution and risk profile of PAHs. 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Adeleke","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACZgY2MMkHoiUqQGzmBuK0sIFoizMgNiMBLQzIWirbQAIEtJi3Mz978HOPtRwbe1vig5vzaqP524FaflRsw6lF5jCbuWHPs3RjNp5jhw1nbjueO+MwYwNjz5nbOLVIMPOwSfAcOJzYJpHeJi257VhuA1ALM2Mbfi2Sfw4crm+Tf97++++cY7nzidEiDbQlgU2C7RiDZENN7gbCWtjMpGUOpBu28aQlS0gcO5C7EajlIF6/8B9+JvnmgLU8P/sxww8SNXW5884fPvjgRwVuLejgMJg8QLR6IKgjRfEoGAWjYBSMEAAA7B9TVt5gKqYAAAAASUVORK5CYII=","orcid":"","institution":"Kwara State University","correspondingAuthor":true,"prefix":"","firstName":"Richard","middleName":"K.","lastName":"Adeleke","suffix":""},{"id":553272635,"identity":"f674cea0-4707-4121-a0b7-8c20af805686","order_by":1,"name":"Muhammed H. Garuba","email":"","orcid":"","institution":"Kwara State University","correspondingAuthor":false,"prefix":"","firstName":"Muhammed","middleName":"H.","lastName":"Garuba","suffix":""},{"id":553272636,"identity":"2bb8c1fc-0e1e-4382-85e2-026daa8c5c76","order_by":2,"name":"Ali A. Aremu","email":"","orcid":"","institution":"Federal University of Technology Owerri","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"A.","lastName":"Aremu","suffix":""},{"id":553272637,"identity":"246cb578-0d3c-4fd4-a157-fede5abf62da","order_by":3,"name":"Abubakar M. Ogacheko","email":"","orcid":"","institution":"Federal University of Technology Owerri","correspondingAuthor":false,"prefix":"","firstName":"Abubakar","middleName":"M.","lastName":"Ogacheko","suffix":""},{"id":553272638,"identity":"151d11f1-2fd9-45fc-a376-54261d2b9f2a","order_by":4,"name":"Damilola Ogunleye","email":"","orcid":"","institution":"Kwara State University","correspondingAuthor":false,"prefix":"","firstName":"Damilola","middleName":"","lastName":"Ogunleye","suffix":""}],"badges":[],"createdAt":"2025-12-01 09:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8248323/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8248323/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97314261,"identity":"f4b39c5e-f71c-4b60-b9c9-5d858f7fa2df","added_by":"auto","created_at":"2025-12-03 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16:23:12","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89034,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8248323/v1/795fd7c61ee8c88fe79cd988.html"},{"id":97314259,"identity":"caaa4a3f-8055-478e-99b2-2042860b5781","added_by":"auto","created_at":"2025-12-03 06:15:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131194,"visible":true,"origin":"","legend":"\u003cp\u003e2D and 3D of Polycyclic Aromatic Hydrocarbon (PAH) ligands\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8248323/v1/2e5bf0966d6f8efd7eeb0e45.png"},{"id":97369840,"identity":"1f8d5c7f-8723-400d-8935-f259b1286c70","added_by":"auto","created_at":"2025-12-03 16:25:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":341124,"visible":true,"origin":"","legend":"\u003cp\u003eThe carbon-nanotube nanoparticle (CNT-NP), \u003cem\u003eBacillus licheniformis\u003c/em\u003e and the enzyme–nanotube complex (ENZ–CNT)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8248323/v1/0d362023dbc28f9c92b4b527.png"},{"id":97314264,"identity":"826d84f9-46de-427c-91ef-77ee7f9b5fe7","added_by":"auto","created_at":"2025-12-03 06:15:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":455201,"visible":true,"origin":"","legend":"\u003cp\u003e2D and 3D docking conformations\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8248323/v1/97b9bbdc52fc1b0be489b935.png"},{"id":97368968,"identity":"25e7ce7f-b170-4359-81ce-7b64bc438d55","added_by":"auto","created_at":"2025-12-03 16:23:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":388914,"visible":true,"origin":"","legend":"\u003cp\u003eFrontier Orbital Distribution of\u003cstrong\u003e \u003c/strong\u003ePolycyclic Aromatic Hydrocarbons (PAHs)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8248323/v1/e36937be0f38722662f06f08.png"},{"id":97314262,"identity":"779b9187-ef5d-4632-9718-421942c3093e","added_by":"auto","created_at":"2025-12-03 06:15:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62918,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between HOMO–LUMO gap and Gibbs free energy of PAHs.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8248323/v1/cf4d9cc7ebd496d182e10378.png"},{"id":97369502,"identity":"a475e0f8-38ad-49ea-96dc-299c450e885a","added_by":"auto","created_at":"2025-12-03 16:25:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":82827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCorrelation between HOMO–LUMO gap, Gibbs free energy, and binding energy of PAHs.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8248323/v1/8110dbebcfd362f877e2e2d0.png"},{"id":97892974,"identity":"df7fd683-0919-4059-b636-4f94ff01f44f","added_by":"auto","created_at":"2025-12-10 15:25:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2150543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8248323/v1/3506263b-22dc-4e0f-a675-b72045e3e07f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Computational Assessment of CNT–NH₂ and Enzyme-Functionalized Nanomaterials for Polycyclic Aromatic Hydrocarbon Remediation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePolycyclic aromatic hydrocarbons (PAHs) such as naphthalene, anthracene, and benzo[a]pyrene are persistent organic pollutants commonly released from petroleum spills, combustion processes, and industrial waste [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Their strong hydrophobicity, bioaccumulation tendency, and mutagenic or carcinogenic effects make them a major environmental concern, particularly in aquatic systems [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Conventional remediation techniques including chemical oxidation, activated-carbon adsorption, and biodegradation often show limited selectivity and low efficiency under real-world conditions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Carbon nanomaterials offer promising alternatives due to their large surface area and tunable surface chemistry. In particular, carbon nanotubes (CNTs) exhibit strong π\u0026ndash;π interactions with aromatic pollutants, enabling effective adsorption [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Surface functionalization, especially with amine (\u0026minus;\u0026thinsp;NH₂) groups, improves CNT dispersibility and enhances affinity for aromatic hydrocarbons [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus, amine-functionalized CNTs (CNT\u0026ndash;NH₂) are increasingly explored as selective adsorbents for PAH remediation in contaminated water.\u003c/p\u003e\u003cp\u003eEnzymatic degradation provides another eco-friendly remediation pathway. Laccases (EC 1.10.3.2) from \u003cem\u003eBacillus licheniformis\u003c/em\u003e are thermostable, alkaline-tolerant, and capable of oxidizing a wide range of aromatic pollutants, making them suitable for environmental detoxification [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. When immobilized on CNTs, laccase\u0026ndash;nanotube hybrids can combine catalytic oxidation with enhanced pollutant adsorption, creating a synergistic nanobiocatalytic remediation system [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eComputational tools play a central role in understanding the adsorption and catalytic interactions between PAHs, CNTs, and enzymes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Density Functional Theory (DFT) provides insights into electronic structure, charge transfer, and adsorption energetics [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], while molecular docking reveals binding orientation and interaction strength between PAHs and enzyme-functionalized nanomaterials [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. DFT methods incorporating dispersion corrections are particularly effective for modeling π\u0026ndash;π and van der Waals interactions in PAH\u0026ndash;nanomaterial systems [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].Therefore, this study based on integrating DFT calculations, molecular docking, and adsorption analysis using CNT\u0026ndash;NH₂ and \u003cem\u003eBacillus licheniformis\u003c/em\u003e laccase provides a comprehensive framework for predicting and optimizing PAH remediation efficiency in oil-spill-contaminated environments..\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Preparation of ligand structures\u003c/h2\u003e\u003cp\u003eFive polycyclic aromatic hydrocarbon (PAH) ligands pyrene (PYR), naphthalene (NAP), anthracene (ANT), fluorene (FLU), and benzo[a]pyrene (BEZ) were obtained from the PubChem Chemical Database in .sdf format. The carbon nanotube (CNT) structure and all PAH ligands were sketched and modeled in both two- and three-dimensional (2D and 3D) forms using ChemDraw Professional 16.0\u0026reg;, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The generated 3D geometries were subsequently imported into PyRx Virtual Screening Tool\u0026reg; [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], where they were converted and optimized using Open Babel [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe (3D) three-dimensional crystal structure of laccase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e (PDB ID: 9BD5) was downloaded from the Protein Data Bank (PDB) via the NCBI PubChem interface (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Prior to docking, the protein structure was prepared by deleting heteroatoms and removing all crystallographic water molecules. The raw files were converted into .pdb format using Discovery Studio Visualizer 2.5.5 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The PAH ligands were then subjected to energy minimization in UCSF Chimera 1.5.3 using a genetic algorithm with 2000 steps and an optimized grid spacing of 0.5 units.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Nanoparticle Docking Analysis\u003c/h2\u003e\u003cp\u003eAll in silico docking procedures were carried out using the PyRx Virtual Screening Tool\u0026reg; [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The laccase enzyme was initially docked with the PAH ligands pyrene (PYR), anthracene (ANT), fluorene (FLU), benzo[a]pyrene (BEZ), and naphthalene (NAP) as well as with the carbon-nanotube nanoparticle (CNT-NP). The resulting enzyme\u0026ndash;nanotube complex (ENZ\u0026ndash;CNT) generated from this first docking stage was exported in .pdb format and subsequently used as the receptor for the second round of docking, in which the five PAH ligands were docked against the ENZ\u0026ndash;CNT hybrid, as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Both receptor and ligand structures were prepared following standard protocols for protein and small-molecule preparation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDocking simulations were executed using PyRx, while visualization and post-docking analysis were performed using BIOVIA Discovery Studio [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Binding affinities for all ligand\u0026ndash;receptor pairs were automatically computed in PyRx, and the resulting interactions hydrogen bonding, hydrophobic contacts, π\u0026ndash;π stacking, and other noncovalent forces were examined using Discovery Studio Visualizer. Multiple conformations were generated for each PAH ligands during the docking workflow, and the lowest-energy poses were further refined to ensure optimal fitting within the enzyme\u0026rsquo;s active site. The docking score corresponding to the most stable pose was recorded for each ligand. Final analyses included detailed visualization of receptor\u0026ndash;ligand complexes, identification of key active-site residues involved in binding, and generation of high-resolution interaction as presented in the result and discussion section.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Density Functional Theory (DFT)\u003c/h2\u003e\u003cp\u003eDensity Functional Theory (DFT) calculations [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] were carried out on Orca software [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and the results were visualized using Avogadro software [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Two dimensional (2D) and three dimensional (3D) structures of the studied PAHs and amine-fictionalized carbon-nanotube (CNT-NH\u003csub\u003e2\u003c/sub\u003e) unit were drawn using ChemSketch [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] shown in Figure xx, and then the model structures are optimized using DFT theory calculations at B3LYP/LanL2DZ level for the determination of electronic structure properties (HOMO \u0026ndash; LUMO) of PAH ligands pyrene (PYR), anthracene (ANT), fluorene (FLU), benzo[a]pyrene (BEZ), and naphthalene (NAP) as well as with the carbon-nanotube nanoparticle (CNT-NP). The binding energies \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{bind}\\)\u003c/span\u003e\u003c/span\u003e of the PAHs and carbon-nanotube nanoparticle (CNT-NP)-complex were calculated from equations 1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eBinding energy was calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{E}_{bind}={E}_{complex}-\\left({E}_{CNT-NH2}+{E}_{PAH}\\right)\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:1$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{bind}\\)\u003c/span\u003e\u003c/span\u003e is the binding energies, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{PAH}\\)\u003c/span\u003e\u003c/span\u003e is the binding energy of PAHs, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{CNT-NH2}\\)\u003c/span\u003e\u003c/span\u003eis the energy of carbon nanotube (CNT-NH\u003csub\u003e2\u003c/sub\u003e) and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{complex}\\)\u003c/span\u003e\u003c/span\u003eis the binding energy of the carbon nanotube (CNT-NH\u003csub\u003e2\u003c/sub\u003e complex) doped with PAHs\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Molecular Docking Analysis\u003c/h2\u003e\u003cp\u003eMolecular docking was performed to evaluate the binding behavior of the selected PAH ligands within the active site of laccase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e (PDB: 9BD5). Five PAHs\u0026mdash;benzo[a]pyrene (BEZ), pyrene (PYR), fluorene (FLU), anthracene (ANT), and naphthalene (NAP)\u0026mdash;were retrieved from PubChem and screened against the target enzyme. Docking scores and interaction patterns for all ligands are summarized in Table\u0026nbsp;5. Ligands exhibiting the most favorable binding affinities [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], were considered the strongest interactors with the enzyme. To further understand the mechanistic basis of these interactions, a carbon nanotube\u0026ndash;ligand docking strategy was employed. This two-stage docking approach allowed assessment of (i) the binding affinity of PAHs toward carbon nanotubes (CNTs), and (ii) the change in enzyme functionality when CNTs were functionalized and subsequently exposed to PAH pollutants. By comparing docking scores of CNT\u0026ndash;ligand and CNT\u0026ndash;enzyme\u0026ndash;ligand complexes, the study highlights potential disruption or enhancement of catalytic properties following PAH exposure. The CNT nanoparticle alone showed measurable affinity toward the PAH ligands with binding energies of \u0026minus;\u0026thinsp;5.8 (BEZ), \u0026minus;\u0026thinsp;5.3 (PYR), \u0026minus;\u0026thinsp;4.5 (FLU), \u0026minus;\u0026thinsp;4.4 (ANT), and \u0026minus;\u0026thinsp;3.8 kcal/mol (NAP) as presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These values indicate a moderate level of adsorption, suggesting that CNTs can interact with PAHs but with varying degrees of preference depending on molecular size and aromaticity. Ligands with energies equal to or more negative than CNT\u0026ndash;PAH complexes were considered as stronger candidates for binding to the enzyme\u0026ndash;CNT system.\u003c/p\u003e\u003cp\u003eThe CNT nanoparticle exhibited an even stronger interaction with laccase, yielding a binding energy of \u0026minus;\u0026thinsp;7.7 kcal/mol, which is considerably more favorable than the PAH\u0026ndash;CNT interactions. This indicates that CNT functionalization does not hinder enzyme binding but may facilitate a stable enzyme\u0026ndash;nanotube hybrid complex.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBinding Affinity (docking of Score) of PAH\u0026ndash;CNT and complex interactions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePAH Ligand\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eBinding Affinity\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCNT-NH\u003csub\u003e2\u003c/sub\u003e-enzyme complex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCNT-NH\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePYR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;6.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;5.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNAP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;5.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;3.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFLU\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;4.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eANT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;6.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;4.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBEZ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;7.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;5.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo assess the effect of PAH pollutants on this hybrid system, the CNT\u0026ndash;enzyme complex was docked with the same PAH ligands. The resulting affinities were: (\u0026ndash;7.7, \u0026minus;\u0026thinsp;6.7, \u0026minus;\u0026thinsp;6.7, \u0026minus;\u0026thinsp;6.2 and \u0026minus;\u0026thinsp;5.5) kcal/mol for BEZ, PYR, ANT, FLU and NAP respectively, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.. Among the ligands, benzo[a]pyrene (BEZ) demonstrated the strongest binding to the CNT\u0026ndash;enzyme complex, followed by PYR and ANT. These values suggest that high-molecular-weight PAHs have an increased tendency to interact with the catalytic residues of laccase, possibly disrupting normal enzyme function [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHydrogen bonding, π\u0026ndash;π stacking, hydrophobic interactions, and key active-site contacts were observed in the docked complexes, confirming stable ligand accommodation within the enzyme pocket. This observation aligns with the report by Pantsar and Poso [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which notes that increasingly negative docking scores typically reflect stronger and more stable molecular interactions. The docking simulations indicate that PAHs particularly BEZ and PYR can form strong interactions with both CNTs and the CNT\u0026ndash;enzyme complex. This suggests that PAH pollutants may compete with or inhibit CNT-functionalized enzyme systems, which is essential for understanding their environmental behavior and designing effective nanoparticle-based remediation strategies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe binding interactions of the PAH ligands within the active site of the CNT-functionalized \u003cem\u003eBacillus laccase\u003c/em\u003e complex showed strong adsorption onto the CNT nanoparticle surface, as demonstrated in the 2D and 3D docking conformations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The dominant interactions involved hydrophobic forces, particularly π\u0026ndash;alkyl and π\u0026ndash;π stacking, supplemented by non-bonded van der Waals interactions, consistent with typical PAH\u0026ndash;nanomaterial binding behavior [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBenzo[a]pyrene (BEZ), which exhibited the highest docking score, formed multiple hydrophobic contacts with residues Phe205, Pro207, Gln209, Pro210, Asp211, Ala225, Phe226, Cys227, and Asp229, indicating a stable aromatic\u0026ndash;aromatic and aromatic\u0026ndash;aliphatic interaction network as presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The pyrene (PYR) ligand displayed similar hydrophobic interactions with Phe205, Pro207, Gln209, Pro210, Asp211, Val223, Ala225, Phe226, and Cys227, further supporting the high affinity of multi-ring PAHs for the CNT\u0026ndash;enzyme complex.\u003c/p\u003e\u003cp\u003eAnthracene (ANT) exhibited van der Waals and π\u0026ndash;anion interactions involving Ile260, Phe261, Glu262, Val291, Ile318, and Lys466, as presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), while fluorene (FLU) showed a combination of van der Waals, π\u0026ndash;anion, π\u0026ndash;alkyl, π\u0026ndash;π stacking, and π-donor hydrogen bonding interactions with Pro111, Asp112, Tyr116, Ala119, Phe131, Glu133, Gly484, and Arg485 as presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Naphthalene (NAP), the smallest PAH studied, revealed primarily van der Waals and π\u0026ndash;alkyl interactions with Pro77, Asp78, Glu97, Val98, Thr122, Lys123, Gln126, and Val127 as presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), consistent with its weaker aromatic surface area and lower binding affinity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs previously highlighted, polycyclic aromatic hydrocarbons (PAHs) have received significant environmental attention due to their volatility, persistence, and toxicity. These characteristics make their complete removal challenging through conventional chemical, physical, or biological approaches [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Among remediation strategies, biodegradation remains particularly promising because of its practicality and efficiency in reducing PAH contamination. One potential mitigation strategy involves preventing or reducing PAH-induced toxicity by inhibiting their binding to nanoparticles or enzymes. This finding suggests a higher preferential affinity between the functionalized enzyme and the nanoparticle surface. Previous research similarly reports strong adsorption capacities of nanoparticles such as carbon nanomaterials, graphene oxide, TiO₂, and ZnO toward PAHs and related carcinogenic compounds [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Density Functional Theory (DFT) Studies\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Frontier Orbital Distribution and Surface Electron Density\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, shows the computed HOMO and LUMO orbital distributions of the PAHs. Distinct electron density patterns were observed across molecules: (1) HOMO distributions were largely localized on the π-conjugated PAHs aromatic rings, indicating potential for π\u0026ndash;π interactions with carbon nanotube (CNT\u0026ndash;NH₂) surfaces. (2) LUMO density was more delocalized across the molecular surfaces, except in pyrene, which exhibited notable asymmetry due to its compact structure. The electron-density characteristics can be explained by the following considerations: (i) PAH molecules are generally more electrophilic than CNT\u0026ndash;NH₂ fragments, therefore electron density flows from CNT\u0026ndash;NH₂ to the adsorbed PAH and (ii) The localization of HOMO on PAHs and partial delocalization of LUMO promotes charge-transfer stabilization upon adsorption.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 The relationship between the HOMO\u0026ndash;LUMO energy and Gibbs free energies\u003c/h2\u003e\u003cp\u003eDensity Functional Theory (DFT) calculations were performed to determine the frontier molecular orbital energies (HOMO and LUMO) and Gibbs free energies (ΔG) of the selected polycyclic aromatic hydrocarbons (PAHs): anthracene, benzo[a]pyrene, fluorene, naphthalene, and pyrene. The obtained results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComputed HOMO, LUMO, and Gibbs free energy values of selected PAHs.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePAH Compound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHOMO (eV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLUMO (eV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHOMO\u0026ndash;LUMO Gap (eV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;ΔG (kcal/mol)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnthracene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;8.290\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;3.656\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.634\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;26.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBenzo[a]pyrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;9.831\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;2.576\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.255\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;31.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFluorene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;9.559\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;2.404\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;26.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaphthalene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;9.652\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;0.212\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.440\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;23.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePyrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;8.441\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;1.153\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.288\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026ndash;27.92\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA general trend was observed where PAHs with smaller HOMO\u0026ndash;LUMO gaps exhibited higher reactivity and more favorable Gibbs free energies (more negative ΔG values). The relationship between the computed HOMO\u0026ndash;LUMO energy gaps and Gibbs free energies is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe data reveal that benzo[a]pyrene possesses the lowest Gibbs free energy (\u0026ndash;31.80 kcal/mol), indicating higher thermodynamic stability and a greater tendency to interact with electron-accepting surfaces, such as amine-functionalized carbon nanotubes (CNTs). This compound also has one of the smallest HOMO\u0026ndash;LUMO gaps (7.26 eV), suggesting enhanced charge transfer potential and π\u0026ndash;π stacking ability with the CNT surface. In contrast, naphthalene showed the largest energy gap (9.44 eV) and the least negative Gibbs free energy (\u0026ndash;23.47 kcal/mol), implying greater electronic stability but weaker adsorption affinity toward the nanomaterial. Pyrene and anthracene displayed intermediate reactivity patterns, suggesting balanced stability and adsorption tendencies, making them suitable reference compounds for evaluating PAH\u0026ndash;nanomaterial interactions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The observed inverse relationship between the HOMO\u0026ndash;LUMO gap and Gibbs free energy supports the notion that higher chemical reactivity (smaller gap) correlates with stronger adsorption interactions. Such electronic behavior aligns with previously reported DFT studies on PAH adsorption onto nanostructured materials (Kumar et al., 2022; Zhang et al., 2021).\u003c/p\u003e\u003cp\u003eThese findings highlight that functionalized CNTs can effectively serve as electron-rich adsorption sites for PAHs, driven by π\u0026ndash;π stacking, van der Waals forces, and charge-transfer interactions. When coupled with enzymatic agents such as laccase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e, synergistic remediation is expected due to the enzyme\u0026rsquo;s oxidative catalytic ability and the nanotube\u0026rsquo;s adsorption efficiency [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 Electronic Properties and Binding Energies of PAHs\u003c/h2\u003e\u003cp\u003eDensity Functional Theory (DFT) calculations were carried out to investigate the binding interactions of selected polycyclic aromatic hydrocarbons (PAHs) anthracene, benzo[a]pyrene, fluorene, naphthalene, and pyrene on amine-functionalized single-walled carbon nanotubes (CNT\u0026ndash;NH₂), as presented in (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The negative value of the E\u003csub\u003ebind\u003c/sub\u003e indicates that the binding energy of the PAHs are spontaneous and may do not require activation before it occurs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. And when the binding occurs the relaxed structure of the free PAH compounds and adsorbed PAHs will not be nearly identical.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComputed Gibbs free energy, and binding energy of selected PAHs.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePAH Compound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;ΔG (kcal/mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEbind (kcal/mol)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnthracene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;26.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;18.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBenzo[a]pyrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;31.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;26.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFluorene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;26.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;19.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaphthalene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;23.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;14.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePyrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;27.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;21.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe calculated binding energies suggest that benzo[a]pyrene exhibited the strongest adsorption onto the CNT\u0026ndash;NH₂ surface (E\u003csub\u003ebind\u003c/sub\u003e = \u0026minus;\u0026thinsp;26.75 kcal/mol), consistent with its highly negative Gibbs free energy (\u0026ndash;31.80 kcal/mol) and relatively narrow HOMO\u0026ndash;LUMO gap (7.26 eV). These results indicate strong π\u0026ndash;π interactions and possible charge transfer between the aromatic rings of benzo[a]pyrene and the CNT-NH\u003csub\u003e2\u003c/sub\u003e π-system. Pyrene and anthracene also showed significant adsorption strength (E\u003csub\u003ebind\u003c/sub\u003e \u0026asymp; \u0026minus;\u0026thinsp;21 to \u0026minus;\u0026thinsp;18 kcal/mol), suggesting stable interactions facilitated by multiple aromatic rings that enhance π\u0026ndash;π stacking. In contrast, naphthalene displayed the weakest adsorption (E\u003csub\u003ebind\u003c/sub\u003e = \u0026minus;\u0026thinsp;14.21 kcal/mol) due to its smaller aromatic surface and larger energy gap (9.44 eV), reducing its interaction with the nanotube. A clear inverse correlation was observed between the HOMO\u0026ndash;LUMO gap and both binding energy and Gibbs free energy, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Molecules with smaller energy gaps exhibited higher reactivity and stronger adsorption, in agreement with prior computational studies on PAH\u0026ndash;nanomaterial systems [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe strong binding affinity of benzo[a]pyrene and pyrene suggests that amine-functionalized CNTs are promising materials for the adsorption and removal of high-molecular-weight PAHs in oil spill-contaminated environments. Furthermore, coupling these nanomaterials with laccase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e can enhance biocatalytic degradation of adsorbed PAHs. The synergistic combination of adsorption (CNT\u0026ndash;NH₂) and enzymatic oxidation (laccase) offers a dual-function remediation mechanism, improving efficiency in aqueous systems contaminated by hydrophobic organic pollutants [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study employed Density Functional Theory (DFT) calculations and molecular docking analyses to assess the electronic properties, thermodynamic behavior, and adsorption tendencies of selected polycyclic aromatic hydrocarbons (PAHs) anthracene, benzo[a]pyrene, fluorene, naphthalene, and pyrene toward amine-functionalized carbon nanotubes (CNT\u0026ndash;NH₂) and a \u003cem\u003eBacillus spp.\u003c/em\u003e enzyme\u0026ndash;CNT complex. The combined computational results demonstrate that the reactivity and adsorption strength of PAHs are strongly governed by their frontier molecular orbital energies, Gibbs free energies, and binding energy profiles. Benzo[a]pyrene exhibited the smallest HOMO\u0026ndash;LUMO energy gap and the most negative Gibbs free energy, resulting in the strongest interaction with the CNT\u0026ndash;NH₂ surface and the CNT\u0026ndash;enzyme complex. Pyrene and Anthracene showed moderate reactivity and stable adsorption, while Naphthalene demonstrated the weakest interaction due to its larger energy gap and lower thermodynamic drive for adsorption. Molecular docking analyses further revealed that the CNT\u0026ndash;enzyme complex binds PAHs more strongly than CNT alone, highlighting a synergistic remediation potential. The current research work provides computational evidence that amine-functionalized CNTs, particularly in combination with \u003cem\u003eBacillus spp.\u003c/em\u003e enzymes, serve as efficient adsorbent\u0026ndash;biocatalyst systems for PAH mitigation. The strong adsorption behavior, favorable thermodynamic properties, and effective binding interactions suggest that CNT\u0026ndash;enzyme hybrid systems hold significant promise for future environmental remediation applications. Further experimental validation is recommended to confirm the computational predictions and to optimize their deployment for pollutant removal.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate.\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNo External Funding\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAuthor contributions Author contribution Richard K. Adeleke: Conceptualization, Visualization, Writing-Review \u0026amp; Editing, computational (DFT) analysis. Muhammed H. Garuba: Methodology, Investigation, Writing-original Draft. Ali A. Aremu: Software, Validation, Software. Abubakar M. Ogacheko and Damilola Ogunleye: Conceptualization, Formal analysis, Writing-Review \u0026amp; Editing.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFeng J, Wang X, Wu D. Environmental persistence of polycyclic aromatic hydrocarbons. Chemosphere. 2010;78(3):260\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2010.01.020\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2010.01.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng X, Lin X, Chen Z. 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Air Soil Water Res. 2023;16:1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/11786221231170099\u003c/span\u003e\u003cspan address=\"10.1177/11786221231170099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PAHs, DFT, binding energy, (CNTs), molecular docking, Bacillus spp. enzyme, environmental remediation","lastPublishedDoi":"10.21203/rs.3.rs-8248323/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8248323/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolycyclic aromatic hydrocarbons (PAHs) persist in the environment due to their chemical stability, toxicity, and resistance to conventional remediation processes. This study investigates the electronic reactivity and adsorption potential of selected PAHs such as anthracene, benzo[a]pyrene, fluorene, naphthalene, and pyrene using density functional theory (DFT) and molecular docking approaches. Frontier molecular orbital calculations revealed that PAHs with smaller HOMO\u0026ndash;LUMO gaps exhibited greater reactivity and more negative Gibbs free energy values, indicating enhanced thermodynamic favorability for adsorption. Benzo[a]pyrene showed the highest reactivity (HOMO\u0026ndash;LUMO gap\u0026thinsp;=\u0026thinsp;7.26 eV) and the most negative Gibbs free energy (\u0026ndash;31.80 kcal/mol), suggesting its strong electron-accepting tendency. Binding energy analysis of PAH adsorption onto amine-functionalized carbon nanotubes (CNT\u0026ndash;NH₂) further confirmed benzo[a]pyrene as the most strongly adsorbed molecule (E\u003csub\u003ebind\u003c/sub\u003e = \u0026minus;\u0026thinsp;26.75 kcal/mol). Molecular docking of PAHs with a CNT\u0026ndash;enzyme (\u003cem\u003eBacillus spp\u003c/em\u003e. laccase, PDB: 9BD5) complex demonstrated high docking scores and extensive hydrophobic and π\u0026ndash;π stacking interactions, indicating a synergistic remediation mechanism driven by nanoparticle adsorption and enzymatic affinity. The combined DFT and Molecular docking results demonstrate that functionalized CNTs coupled with bacterial enzymes offer a highly effective platform for PAH remediation through dual adsorption and catalytic pathways.\u003c/p\u003e","manuscriptTitle":"Computational Assessment of CNT–NH₂ and Enzyme-Functionalized Nanomaterials for Polycyclic Aromatic Hydrocarbon Remediation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 06:15:29","doi":"10.21203/rs.3.rs-8248323/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-16T03:05:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-16T03:02:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T09:32:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-26T21:51:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-25T15:32:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207512599334348941680000375448923045282","date":"2025-12-22T07:37:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219156390653155429503373068008175461158","date":"2025-12-20T18:31:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T05:02:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92294247470308056047543560894017194039","date":"2025-12-17T10:46:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173696424663607820441652189543954394845","date":"2025-12-17T07:16:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256476937671547453879209847540699069467","date":"2025-12-17T06:56:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-17T04:58:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-05T08:18:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-05T07:49:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-05T07:49:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2025-12-01T08:54:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e3543f3f-468b-4cc7-bd96-b1e2c34b5d2d","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T16:10:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-03 06:15:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8248323","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8248323","identity":"rs-8248323","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}