Antimicrobial potential and rhodamine B dye degradation using graphitic carbon nitride and polyvinylpyrrolidone doped bismuth tungstate supported with in silico molecular docking studies

The environmental-friendly hydrothermal method has been carried out to synthesize Bi2WO6 and g-C3N4/PVP doped Bi2WO6 nanorods (NRs) by incorporating different concentrations of graphitic carbon nitride (g-C3N4) as well as a specified quantity of polyvinylpyrrolidone (PVP). Bi2WO6 doped with g-C3N4 provides structural and chemical stability, reduces charge carriers, degrades dyes, and, owing to lower bandgap energy, is effective for antibacterial, catalytic activity, and molecular docking analysis. The purpose of this research is the treatment of polluted water and to investigate the bactericidal behavior of a ternary system. The catalytic degradation was performed to remove the harmful rhodamine B (RhB) dye using NaBH4 in conjunction with prepared NRs. The specimen compound demonstrated antibacterial activity against Escherichia coli (E. coli) at both high and low concentrations. Higher doped specimens of g-C3N4/PVP-doped Bi2WO6 exhibited a significant improvement in efficient bactericidal potential against E. coli (4.55 mm inhibition zone). In silico experiments were carried out on enoyl-[acylcarrier-protein] reductase (FabI) and β-lactamase enzyme for E. coli to assess the potential of Bi2WO6, PVP doped Bi2WO6, and g-C3N4/PVP-doped Bi2WO6 NRs as their inhibitors and to justify their possible mechanism of action.


Catalytic activity
To determine how much RhB dye is degraded, the investigators examined the catalytic activity (CA) of pristine and g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs.The dye is broken down through a series of oxidation and reduction processes during the redox reaction that is part of the catalytic degradation process.Catalysis activity was used to determine the efficacy of synthetic dyes for degradation in the presence of sodium borohydride (NaBH 4 ) and the synthesized nanocatalyst.RhB and NaBH 4 were both freshly prepared before being used in the catalytic reactions to ensure that the data from the experiments were accurate.3 mL of RhB dye was added to a mixture containing 400 µL of NaBH 4 and 400 µL of synthesized NRs as a catalyst.The primary function of a catalyst has been used to reduce the activation energy (Ea) of a reaction, which ultimately increases both the reaction's rate and its degree of stability.The surface activation energy of the reaction has been reduced by using a catalyst, which has increased both the reaction's stability and its effectiveness.A UV-Vis spectrophotometer was used to perform spectrophotometric measurements at regular intervals to obtain absorption variation spectra.A reduction of RhB took place, bringing about a color transformation from pink to colorless.% Degradation was calculated using the formula, % degradation = (C o − C t )/C o × 100, where C o is the initial and C t is the final absorbance at a specific time after the incorporation of materials.

Sample collection and isolation
Direct milking into sterile glassware collected raw milk samples from lactating cows marketed at different markets, including veterinary hospitals and farms in Punjab, Pakistan.The raw milk samples were promptly transported to the laboratory at 4 °C temperature.MacConkey agar was utilized to count the number of coliform bacteria that were present in the raw milk.To isolate the bacteria, all plates were incubated at 37 °C for 48 h.

Identification and characterization of bacterial isolates
The first identification of E. coli was made by Bergey's Manual of Determinative Bacteriology using colonial morphology, Gram stain, and many different biochemical tests 29 .Antibiotic susceptibility.The disk diffusion method was utilized to perform the antibiotic susceptibility test, and Mueller Hinton agar (MHA) was the medium preferred for the microbial test 30 .The experiment was carried out to determine whether or not E. coli was resistant to the following antibiotics (classes): Gentamicin (CN) 10 µg (Aminoglycosides), Ceftriaxone (CRO) 30 µg (Cephalosporins), Amoxycillin (AX) 25 µg (Penicillin), Imipenem (IPM) 10 µg Carbapenem), Tetracycline (TE) 30 µg (Tetracyclines), Azithromycin (AZM) 15 µg (Macrolides), Ciprofloxacin (CIP) 5 µg (Quinolones), Septran (SXT) 25 µg and Augmentin (AMC) 30 µg 31 .The purified Cultures of E. coli had been established and brought to a turbidity of 0.5 MacFarland.They were spread-plated on MHA (Oxoid Limited, Basingstoke, UK), and the antibiotic disks were positioned sufficiently separated from one another on the surface of the inoculated plate to avoid overlying the zones of inhibition.The Clinical and Laboratory Standard Institute 32 evaluated the results after 24 h of incubation on plates at 37 °C.MDR was a designation given to bacteria that tested positive for resistance to at least three drugs 33 .
Antimicrobial activity.The in vitro antibacterial activity of pristine and (2, 4%) g-C 3 N 4 /PVP-doped Bi 2 WO 6 has been assessed via the agar well diffusion method on 10 characteristic isolates of MDR E. coli collected from mastitis milk.These isolates were tested on 10 different mastitis milk samples.Petri dishes have been inoculated with MDR E. coli at a concentration of 1.5 × 10 8 CFU/mL (0.5 McFarland standard) using MacConkey agar.The 6 mm wells have been drilled using a sterile cork borer.The low and high concentrations of synthetically produced pristine and (2, 4%) g-C 3 N 4 /PVP-doped Bi 2 WO 6 were utilized as (0.5 mg/50 µL) and (1.0 mg/50 µL) respectively.Ciprofloxacin at a concentration of (0.005 mg/50 µL) was used as the positive control 34 , while DIW (50 µL) was used as the negative control 35 .
Statistical analysis.One-way analysis of variance (ANOVA) was performed in SPSS 20 36 using the inhibition zone as the unit of measurement for antimicrobial efficacy.Inhibition zone diameters were analyzed, and the results were declared statistically significant at p < 0.05.www.nature.com/scientificreports/

Molecular docking analysis
Molecular docking simulations were performed on enoyl-[acylcarrier-protein] reductase (FabI) and β-lactamase enzyme since both are necessary for bacterial survival 37 .The 3D structural dimensions of chosen enzyme targets were obtained from the protein data source (https:// www.rcsb.org/), with the PDB Codes 4D46 for FabI E.coli 38 and 4KZ6 for β-lactamase E. coli 39 .SYBYL-X 2.0 software was used for the docking analysis as reported in our previous studies 40,41 .The Sybyl-X2.0/SKETCHmodule was used to generate three-dimensional structures of specific compounds.Subsequently, energy reduction was performed using the Tripos force fieldb by employing Gasteiger Hückel atomic charges.The Surflex-Dock module, a component of the SYBYL-X 2.0 molecular modelling software package, was used to conduct flexible molecular docking simulations.These simulations aimed to investigate the binding interactions between nanoparticles and the active site residues of certain proteins.Hydrogens that were absent were incorporated.In accordance with the AMBER 7 FF99 force field, the allocation of atomic types and the application of atomic charges were performed.Ultimately, the Powell technique was used, using a convergence gradient of 0.5 kcal/ (mol・A) over a span of 1000 cycles, in order to mitigate steric conflicts and effectively minimize the energy.In each ligand-receptor complex system, a minimum of 20 highly refined docked postures were definitively preserved.The Hammerhead scoring system was used to evaluate the optimal putative ligand poses.The Surflex dock module utilizes an empirically derived consensus scoring function called cScore to generate and rank potential poses of ligand fragments.This scoring function combines various empirical scoring components, including D-score (dock score), G-score (gold score), Chem-Score, potential mean force (PMF) score, and/or complete score.Additionally, the module incorporates a molecular similarity method known as morphological similarity.

Results and discussion
The Bi 2 WO 6 and g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs have been synthesized using the hydrothermal method.g-C 3 N 4 has been prepared in the laboratory by pyrolysis of urea (CH 4 N 2 O).Then various concentrations of g-C 3 N 4 were incorporated in PVP-doped Bi 2 WO 6 (Fig. 1).
The constructive nature, crystal structures, phase purities, crystallinity, and crystal size of pristine and various (2, 4%) g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs were examined through XRD analysis that has been conducted in the 2θ mode as represented in Fig. 2a.The results were analyzed with X-Pert High score software, which proclaimed that the unit cell of synthesized NRs has an orthorhombic structure with Pna21 and Pca21 and space group numbers 33 and 29.The two phases of bismuth tungstate oxide have been confirmed, which are Bi 2 WO 6 and Bi 2 W 2 O 9 .The pristine sample (Bi 2 WO 6 ) exhibits the diffraction peaks at 28.3°, 34.6°, 36.7°,45.2°,47.0°, 52.4°, and 55.6° correspond to crystal planes (131), ( 022), ( 160), ( 171), ( 260), ( 242) and ( 191) respectively are well matched with the JCPDS card number 01-079-2381.Bi 2 W 2 O 9 exhibits the diffraction peaks at 14.9°, 22.5°, 30.0°,37.9°,49.9°, and 59.5° correspond to crystal planes (004), ( 006), ( 008), ( 0010), ( 224) and ( 229 To understand the chemical structures and functional groups of synthesized Bi 2 WO 6 and (2 and 4%) g-C 3 N 4 / PVP-doped Bi 2 WO 6 NRs.The FT-IR spectroscopy revealed many bands that announced the presence of functional groups in the frequency range of 4000-400 cm −1 , as represented in Fig. 2b.The bending vibrations of O-H are responsible for the stronger band observed, with the peak intensity at approximately 3335 cm −1 .The presence of moisture in the atmosphere is responsible for the appearance of a weak band with a peak intensity of 1658 cm −1 .These characteristic absorption peaks are assigned to the bending vibration of the H-O-H band 42 .The band appeared at 546 cm −1 , associated with the stretching vibration of the W-O and the W-O-W 43 .The band found at 730 cm −1 belongs to the stretching vibration of the Bi-O 43 .The observed band confirms the presence of the synthesized material with the incorporation of g-C 3 N 4; a peak observed at 1240 cm −1 has been attributed to C-N, to confirm the presence of doped g-C 3 N 4 /PVP-doped Bi 2 WO 6 44 .The SAED patterns of both the pristine and the (2 and 4%) g-C 3 N 4 //PVP-Bi 2 WO 6 NRs exhibit a distinguishable bright spot and a bright ring, as shown in Fig. 2c-f.The pristine Bi 2 WO 6 points to the bright spots, laid out in a systematic pattern and taken up of individual crystals.Bright spot combinations generate bright rings of g-C 3 N 4 /PVP-Bi 2 WO 6 NRs, which strongly indicates the polycrystalline nature of the synthesized materials.The rings offered indisputable evidence for pristine and doped NRs, have a highly crystalline composition, and the rings were aligned with the XRD observations.SEM micrographs have been used to identify the surface morphology of the prepared specimen indicated in Fig. 3a-d  using EDS analysis displayed in Fig. 6a-d.The EDS analysis was used to investigate the elemental distribution to establish whether or not there is any additional interfacial contact.The appearance of significant peaks of the elements Bi, O, and W in the nanopowders provide conclusive evidence that Bi 2 WO 6 NRs presented in the sample.The presence of carbon (C) peaks attributed to the PVP was successfully integrated.Some additional peaks appeared that are sodium (Na), copper (Cu), and gold (Au) 45 .The sample had a gold coating sprayed on it to reduce the charging impact of the sample so that the peaks of Au appeared in the spectrum.The peaks of Na have been found in the EDS analysis assigned to the NaOH used to maintain the pH of synthesized NRs.The appearance of the Cu peak can be attributed to the presence of Cu tape in the sample holder.Possible evidence of contamination includes the appearance of new peaks of technetium (Tc).
Mapping analysis has been used to detect the presence of elements in the synthesized samples.The elemental mapping analysis of pristine Bi 2 WO 6 and (2 and 4%) g-C 3 N 4 /PVP have been represented in Fig. 7a-g, which indicates the presence of W, O, and Bi in the pure sample after incorporation of g-C 3 N 4 /PVP indicate the appearance of C and N. The presence of sodium (Na) in the synthesized NRs, plays a role in both the regulation of pH and the formation of precipitates 45 .
Ultra-visible absorption spectroscopy was utilized to examine the optical absorption properties and the band gap in the 240-500 nm wavelength range.The optical properties of synthesized pristine and g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs were indicated in Fig. 8a.The pristine sample exhibits an absorption peak at 305 nm 46 .Upon doping with PVP and g-C 3 N 4, absorption peaks shift gradually toward longer wavelengths indicating a redshift.Absorption spectra were employed for pure and doped samples to compute the band gap energy (Eg).The band gap was calculated via Tauc's plot method using the extracted data from UV-Visible spectra can be represented in Fig. 8b.The energy band gap of pure Bi 2 WO 6 is 4.0 eV.The band gap decreased from 4.0 to 3.5 eV on doping, as evident by the absorption spectra's redshift.
Catalytic activity (CA) of pristine and (2 and 4%) g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs for the degradation of RhB dye in the presence of NaBH 4 at different pH values was measured using UV-vis spectrophotometer.The CA of synthesized catalysts depends on the pH, which is crucial in determining the extent of degraded dyes.Dye pollutants have been typically discharged at different pH levels.0.5 M solution of H 2 SO 4 was dissolved with RhB dye to maintain the pH of solution ∼2.5.The samples yield significant results as 98.9%, 99.9%, 96%, and 97.7% degradation were observed for 10 min.The catalytic degradation decreased with the incorporation of g-C 3 N 4 /PVP in the acidic dye solution because the redox reaction did not occur between the dye and the catalyst.In a basic medium, a 0.5 M solution of NaOH was utilized to achieve the desired pH∼12 in the dye solution.www.nature.com/scientificreports/ The findings demonstrate that the basic medium exhibited the highest rate of dye degradation, with recorded degradation percentages of 90%, 98.5%, 98.8%, and 96.7% for pristine and doped samples, respectively, after a 10-min duration.The catalytic degradation increased by incorporating a doped sample up to the optimum amount because of the redox reaction between the dye and the synthesized materials.In a neutral medium at pH ~ 7 results measured as 93%, 98.7%, 97.4%, and 99.1% for pristine and doped NRs respectively can be represented in Fig. 9a-c.The degradation or reduction of the potentially hazardous dye is triggered when a catalyst is added to the mixture of oxidizing and reducing agents.Surprisingly, an abundance of BH 4 − donor species in the solution increases adsorption, whereas the transfer of electrons from the donor species to the accepter species through an oxidation and reduction reaction is required for the CA.The enhanced rate of reactions and attainment of the maximum degrading efficiency can be attributed to the particle's comparatively large surface area to its overall size 47 .The degradation of RhB was observed for all of the samples, demonstrating an increase in degradation efficiency for both pristine and doped samples due to the availability of active sites to adsorb.However, upon higher concentration doping, a decrease in the efficiency of RhB was observed due to an imbalance between the dye molecules and the nanocatalyst active site availability, which caused a weak adsorption capacity on the surface of the nanocatalyst.In general, the larger the surface area of the catalyst or the smaller its size, the more active sites it can provide to boost its CA.The enhanced efficiency of the catalyst can be attributed to the microporosity, which hinders the diffusion of the reactant into the active sites.Increasing the surface area of the catalyst leads to higher catalytic activity, potentially resulting from the production of a porous structure with a larger surface area 48 .
The catalytic mechanism is depicted in Fig. 10, where the reducing agent and nanocatalyst are key factors in the degradation of RhB dye.The e-transfer from the reductant to the oxidant was involved in the redox reaction.As a result, the dye was broken down via electron absorption.The reaction was prolonged and less effective in the presence of NaBH 4 .To address these issues, nanocatalysts were incorporated into oxidation-reduction reactions.The catalyst acts as an electron relay by transferring electrons from NaBH 4 to RhB, while NaBH 4 acts as a reducing agent for the degradation of RhB.NRs increased the adsorption of BH 4 − ions alongside dye molecules through many active sites, resulting in dye degradation that was faster and more efficient 49 .
The agar well diffusion method was used to investigate the in vitro antimicrobial activity of pristine and (2, 4%) g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs by measuring inhibition zones.NRs possess antimicrobial activity that is effective against various strains of bacteria.Inhibition zones were statistically significant against E. coli found at low and high concentrations for pristine and doped Bi 2 WO 6 , respectively.The measurement of the inhibition zone for E. coli was compared with negative control, which consisted of DIW and had an inhibition zone evaluation of 0 mm, and positive control, which consisted of ciprofloxacin and had an inhibition zone measurement of 8.65 mm.Both the negative control and the positive control were used as comparisons.The pristine specimen produced low inhibition zones throughout the measurements when it was present in low quantities.However (2, 4%) g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs were present in both high and low concentrations, it produced a large number of inhibition zones.In addition, the concentration of doped material is directly related to the zone of inhibition as the dopant concentration increased, which led to an expansion of the zone area.The inhibition zone measured between 1.35 and 2.45 mm in E. coli at low doses and between 3.25 and 4.55 mm in E. coli at high doses, as shown in Table 1.
The photographic evidence of antimicrobial activity for low and high concentration of synthesized materials can be represented in the Fig. 11a,b.The graphical representation of the inhibition zone measurement for  The antimicrobial activity is caused by the interaction of OH groups in the PVP.Because of this increase in permeability and cytoplasmic leakage, the functional groups of components have the potential to form direct bonds with the phospholipids that make up the cell membrane 50 .NRs react with the cell wall and form a helical, disorganized spring on the cell wall and then enter the cell membrane, where they form interlinks with the structure of deoxyribonucleic acid (DNA) molecules 51 .www.nature.com/scientificreports/ The small particles actively produce reactive oxygen species (ROS), which cause the decomposition of the bacterial membrane and cytoplasmic contents, resulting in the bacteria's extinction 52 .The reaction that follows 53,54 can be used to address ROS formation seen in Fig. 12.
The ability to eradicate bacteria is tied to the production of ROS, which would be influenced by a wide range of factors the crystallinity of the specimen, its surface area, and the quantity of oxygen-containing functional groups on its surface.These factors are responsible for the ability to kill bacteria 55 .The membrane of the bacteria is negatively charged, whereas the NRs have a positive charge.The interaction of a strong cationic (Bi 3+ ) charge with a bacterial membrane, which results in increased bactericidal potential with increasing concentration of NRs, promotes lysis and bacterial cell collapse, ultimately resulting in bacterial cell death 56 .
The potential involvement of NMs as bactericidal agents has been widely documented; nevertheless, the reason for their potency remains a mystery that requires investigation.The disruption of many cellular processes by inhibiting enzyme targets has been regarded as a viable technique for discovering novel antibacterial drugs 57,58 .Computational methods, especially molecular docking, are useful for predicting the processes that could underlie certain biological functions.In the instance of FabI E.coli , these synthesized NRs showed strong binding interactions with critical amino acid residues of the active site where the best-docked complex was formed for Bi 2 WO 6 NMs with a binding score of 4.24.As illustrated in Fig. 13a, two H-bond interactions, namely S91 and G93, were found.PVP-doped Bi 2 WO 6 exhibited H-bond interactions with L144, S145, Y156, and K163, with a docking score of 5.26.Furthermore, the binding score found for g-C 3 N 4 /PVP-doped Bi 2 WO 6 inside the active pocket of FabI E.coli was rather high (7.0),indicating the participation of several H-bonds like S19 and I20, as shown in Fig. 13b-d.
. Pristine samples indicate the agglomeration and aggregation of nanorods (NRs), and the NRs exhibit chunk-like morphology.Incorporating PVP in the control sample showed the overlapping of PVP around nanorods because PVP acts as a capping agent and also reduces the size and increases the surface area.The addition of various concentrations of g-C 3 N 4 into the binary system PVP-Bi 2 WO 6 represented the overlapping of 2D material (g-C 3 N 4 ) to nanorods and nanoparticles, and accumulation increased with increasing amounts of g-C 3 N 4 .The topography and morphology of the synthesized materials shown in Fig.4a-dwere determined through TEM analysis.The pristine sample reveals a rod-like morphology characterized by aggregation and agglomeration.Incorporating polymer (PVP), a function of the capping agent, demonstrates that the nanorods (NRs) have been capped.The incorporation of PVP indicates a decrease in agglomeration and an increase in aggregation due to the formation of covalent bonds or metallic bonds that are difficult to break.Incorporating g-C 3 N 4 /PVP increased the agglomeration of NRs, leading to an increase in surface area and a reduction in the sizes of the NRs.The surface became non-uniform as the doping increased gradually, and NRs started to appear.The interlayer d-spacing of pristine and g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs have been measured utilizing the HR-TEM micrograph with the assistance of Gatan digital software seen in Fig.5a-d.The d-spacing for pristine and g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs have been measured as 0.316, 0.310, 0.312, and 0.314 nm, respectively.The results obtained are consistent with the XRD crystallographic plane (131).EDS analysis has been utilized to validate the presence of bismuth, oxygen, and tungsten in the synthesized material.The elemental composition of pristine and g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs has been investigated

Figure 10 .
Figure 10.Catalytic mechanism for degradation of RhB dye.

Figure 11 .
Figure 11.Photographic evidence and graphical representation of inhibition zones (a) low concentration, (b) high concentration, (c) comparative study of inhibition zones and (d) % age efficiency of pristine and g-C 3 N 4 / PVP-doped Bi 2 WO 6 for E. coli.
Bi 2 WO 6 and g-C 3 N 4 /PVP-doped Bi 2 WO 6 nanorods (NRs) have been synthesized using the hydrothermal technique to investigate the catalytic and antibacterial activity as well as molecular docking analysis.Several structural and optical characterization techniques were employed to examine the characteristics of synthesized NRs.The XRD pattern endorsed the orthorhombic crystal structure of the pristine sample with different composition phases appearing as Bi 2 WO 6 and Bi 2 W 2 O 9 and improved crystallinity by incorporating dopants.The presence of functional groups in synthesized NRs and the stretching vibration of the Bi-O and W-O have been identified at 730 and 546 cm -1 , respectively, using FTIR.SAED confirms the polycrystalline nature of synthesized NRs.TEM micrographs revealed the agglomeration of nanorods and particles overlapped by dopants.The interlayer d-spacing has been investigated using HR-TEM analysis and measured as 0.316, 0.310, 0.312, and 0.314 nm for Bi 2 WO 6 and g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs.The highest catalytic degradation of RhB can be measured as 99.9, 98.8, and 99.1% for acidic, basic, and neutral mediums.The inhibition zones for low and high NRs 2.45 and 4.55 mm can be used to investigate the antimicrobial activity against E. coli bacteria.The effectiveness of Bi 2 WO 6 , PVP-doped Bi 2 WO 6 , and g-C 3 N 4 /PVP-doped Bi 2 WO 6 NRs as inhibitors of enoyl-[acylcarrierprotein] reductase (FabI) and the β-lactamase enzyme was evaluated, and their mechanisms of action were clarified by in silico research.