Biomolecular strategy for designing antibiotic–silver nanoparticles conjugate via nitrate reductase mediated β-lactamase inhibition with molecular docking insights

Biomolecular strategy for designing antibiotic–silver nanoparticles conjugate via nitrate reductase mediated β-lactamase inhibition with molecular docking insights | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biomolecular strategy for designing antibiotic–silver nanoparticles conjugate via nitrate reductase mediated β-lactamase inhibition with molecular docking insights Gerges Gad Faheem, Bahig A. El Deeb, Mohamed Ismeal, Mahmoud S. Bakhit This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7042236/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract In this study, mechanistic analysis using SDS-PAGE identified a 58 kDa protein as the biomolecule responsible for AgNPs biosynthesis and capping. Colorimetric microplate-based assay confirmed the protein as nitrate reductase, with structural preservation evidenced by a 29.7% activity increase (1.856 to 2.407 U/g) following AgNPs synthesis. Functionalization of AgNPs with ampicillin was indicated by SPR shift from 422.5 to 340.5 nm and disappearance of the FTIR band at 1736 cm⁻¹. Amp–AgNPs conjugate was stable (3 months), spherical, mono-dispersed (PDI: 0.037), average diameter of 27.26 nm, Zeta potential of − 24.9 mV, and showed broad pH (1–9) and thermal (5–55°C) stability. Docking analysis revealed strong binding of ampicillin within the nitrate reductase catalytic pocket through hydrogen bonding, hydrophobic, and electrostatic interactions, confirming the conjugate stability. Amp–AgNPs (50 µg/mL) exhibited potent antibacterial activity against β-lactamase-producing bacteria with inhibition zones of 27.3 mm (Escherichia coli), 25.0 mm (Enterococcus faecalis), and 26.3 mm (Staphylococcus aureus), and MICs of 3.3, 4.7, and 4.3 µg/mL, respectively. SEM analysis revealed severe structural changes, indicating synergistic membrane disruption and antibiotic delivery. Amp–AgNPs showed potent β-lactamase inhibition in the iodometric assay, supporting their potential as alternative therapeutic agents. Future studies should focus on in vivo efficacy and expand this strategy to additional drug delivery applications. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Drug discovery Biological sciences/Microbiology Biosynthesis mechanism Amp–AgNPs characterizations In silico docking Iodometric assay β-lactamase inhibition Drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Nanoparticles have become transformative tools in medicine, biotechnology, and environmental applications due to their unique physicochemical properties at the nanoscale 1 . Their synthesis can be achieved through physical, chemical, or green methods categorized into top-down and bottom-up approaches based on the formation mechanism 2 . Top-down methods involve breaking down bulk materials into nano-scale particles using mechanical or thermal forces, producing uniform nanoparticles but requiring complex equipment, additional energy inputs, and lacking stabilizers to prevent agglomeration 3,4 . The bottom-up approach assembles nanoparticles from molecular precursors through nucleation and growth, typically using chemical or biological synthesis pathways involving metal salts 5 . While chemical synthesis allows for rapid production, it may involve hazardous substances, which can restrict medical applications 6 . In contrast, biological synthesis offers an environmentally friendly alternative, utilizing molecules derived from plants and microorganisms 7,8 . Fungi, particularly endophytic fungi, are efficient biological agents for nanoparticle synthesis due to their ease of cultivation and high secretion of enzymes and proteins that enhance nanoparticle stability 9,10 . Endophytes produce diverse bioactive compounds and extracellular enzymes with industrial and therapeutic relevance 11-13 . Among these, nitrate reductase (NR) is an enzyme implicated in the extracellular synthesis of AgNPs, facilitating the reduction of metal ions 14-16 . Nitrate reductase, a molybdo-flavoprotein, catalyzes the nitrate reduction to nitrite using pyridine nucleotides as electron donors 17 . Nitrate reductase is categorized based on its coenzyme specificity: NADH-specific in higher plants, NADH/NADPH in algae, and NADPH-specific in fungi 18,19 . It is a multidomain enzyme comprising the prosthetic groups molybdopterin, Fe-heme, and FAD (flavin adenine dinucleotide) in a 1:1:1 stoichiometry that mediates an electron transfer from NAD(P)H to nitrate 20 . The biosynthesis of AgNPs is proposed to involve NADH as an electron donor and NADH-dependent nitrate reductase as a catalytic mediator in the reduction of silver ions to metallic silver, a function consistent with the enzyme’s electron transfer capabilities and structural composition 21,22 . Capping agents are molecules that encase and stabilize AgNPs, preventing agglomeration, and ensuring their stability 23 . Proteins function effectively as green capping agents in the synthesis of nanoparticles due to their capacity to bind to metal surfaces through functional groups like amine, carboxyl, and thiol residues 24 . These biomolecules not only stabilize AgNPs by curtailing agglomeration but also play a crucial role in influencing their surface charge (Zeta potential), size, and shape 25 . Protein-capped nanoparticles enhance biocompatibility, offering a biodegradable, non-toxic alternative to synthetic stabilizers, and enable surface modifications for various medical applications 26 . The stabilization of nanoparticles by capping agents is mediated through various mechanisms, including steric hindrance, depletion stabilization, electrostatic interactions, hydration forces, and van der Waals forces 27 . Nitrate reductase primarily facilitates the bio-reduction of silver ions while associated proteins possibly including the enzyme itself may adsorb onto the nanoparticle surface functioning as capping agents that contribute to their stabilization 28,29 . Functionalization of AgNPs involves the modification of the nanoparticle surface with specific ligands, functional groups, or biomolecules to improve physicochemical properties and biological interactions 30 . This strategy improves stability, enables targeted delivery, enhances stability, and facilitates controlled drug release 31 . The synthesis of AgNPs functionalized with antibiotics holds potential medical applications, making the elucidation of the binding mechanisms on their surface crucial for further study 32 . Conjugates of antibiotics and AgNPs may be considered an alternative treatment to resistant bacterial strains because combined formulations of antibiotics-nanoparticles not only reduce the dose of medicine but also minimize the chances of toxicity 33 . Multidrug-resistant (MDR) microorganisms are being targeted through the functionalized nanoparticles with less potent antibiotics to enhance their antimicrobial efficacy 34 . β-lactamase-producing bacteria pose a significant challenge in modern medicine, as they produce enzymes that hydrolyze the β-lactam ring of antibiotics, thus rendering them ineffective 35 . These enzymes are characteristic of MDR strains, which severely reduce the efficacy of β-lactam antibiotics, including penicillin, ampicillin, and cephalosporin 36 . Addressing β-lactam resistance is essential for improving the therapeutic potential of antibiotic-conjugated nanoparticles 37 . In silico molecular docking has emerged as a pivotal tool in fundamental and applied biological research, enabling the rational investigation of molecular interactions 38 . These computational studies facilitate the elucidation of interaction mechanisms between functionalized nanoparticles and biomolecular targets, providing structural and energetic insights that complement experimental observations 39 . Advanced bioinformatics platforms allow for the prediction of binding affinities, identification of active site preferences, and assessment of structural compatibility among AgNPs capping proteins and antibiotics, thereby uncovering potential modes of action and synergistic effects 40 . Computational approaches provide in-depth insights into nanoparticle behavior and interactions within complex biological systems 41 . This study presents an integrated experimental and computational approach to investigate the role of fungal nitrate reductase in the green synthesis, capping, and functionalization of AgNPs produced by Talaromyces funiculosus (SUMCC 22011). The novelty of this research lies in recognizing nitrate reductase not only as a biocatalyst for the biosynthesis of AgNPs but also as a capping agent. Ampicillin (Amp) was conjugated with the biosynthesized AgNPs in a defined stoichiometric ratio, resulting in a stable nano-antibiotic conjugate (Amp–AgNPs). Comprehensive characterization techniques, including ultraviolet–visible spectroscopy (UV–Vis), dynamic light scattering (DLS), Zeta potential analysis, Fourier transform infrared spectroscopy (FTIR), and high-resolution transmission electron microscopy (HR-TEM) confirmed the successful formation and structural integrity of ampicillin–AgNPs conjugate (Amp–AgNPs). The physicochemical stability of Amp–AgNPs was assessed under varying pH and temperature conditions. Molecular docking was utilized to model the active site architecture of nitrate reductase and its interactions with ampicillin, providing mechanistic insights into the formation of Amp–AgNPs conjugate. The antibacterial activity of Amp–AgNPs was assessed, and the associated morphological alterations in bacterial cells were confirmed by scanning electron microscopy (SEM). The β-lactamase inhibition ability of Amp–AgNPs was evaluated using the iodometric assay. This integrative experimental and computational approach provides a sustainable and innovative platform for AgNPs biosynthesis and functionalization for biomedical applications. Results and Discussion Functional verification of nitrate reductase in the biosynthesis and capping of AgNPs Talaromyces funiculosus (SUMCC 22011) is an endophytic fungus isolated from the wild medicinal plant Euphorbia hirta and identified based on morphological characteristics and phylogenetic analysis, as reported in our previous study El deeb et al. 8 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of T. funiculosus filtrates and the corresponding biosynthesized AgNPs revealed distinct protein profiles (Fig. 1). Fungal filtrates showed protein bands at 58, 75, and 100 kDa. A prominent band consistently observed at 58 kDa across all fungal filtrates and AgNPs derived from three independent fungal patches indicates the presence of a protein involved in the synthesis and capping of AgNPs. A colorimetric microplate-based assay for NR activity was performed to confirm the identity of the observed protein band. The results validated the enzymatic activity associated with nitrate reduction in the fungal filtrate and the AgNPs-capped protein. The NR activity of the fungal filtrates was 1.856 ± 0.004 U/g biomass, and the activity increased to 2.407 ± 0.006 U/g for the AgNPs, indicating 29.68 % enhancement following biosynthesis. The detection of enzymatic activity at the same molecular weight position in the SDS-PAGE further supports the presence of functionally active NR in both fractions. The results confirm that NR activity is preserved during AgNPs biosynthesis and remains catalytically active when associated with the nanoparticle surface. Several studies have highlighted the involvement of proteins in AgNPs biosynthesis. For instance, Macrophomina phaseolina exhibited an 85 kDa protein band, identified as a stabilizing agent for AgNPs 42 . Similarly, extracellular proteins with molecular masses of 45 kDa were reported in Aspergillus niger cell filtrates 28 , while Aspergillus flavus secreted proteins of 32 and 35 kDa, in the culture filtrate and bound to AgNPs 43 . Trichoderma asperellum was shown to produce protein bands at 70 and 55 kDa, which were directly linked to nitrate reductase activity and AgNPs biosynthesis 44 . For further confirmation that the 58 kDa band belongs to NR, A colorimetric assay was employed for the fungal filtrates and the AgNPs after drying at room temperature and suspended in ionized distilled water. To the best of our knowledge, this is the first quantitative study to report a significant enhancement in NR activity following the biosynthesis of AgNPs in a fungal system. Nitrate reductase is an enzyme produced by various microorganisms, plays a crucial role in the extracellular biosynthesis of AgNPs by catalyzing the reduction of silver ions to elemental silver via an NADH-dependent electron transfer mechanism 45 . While it typically facilitates the conversion of nitrate to nitrite, the capacity of the enzyme to mediate electron transfer is effectively utilized in AgNPs biosynthesis to reduce silver ions, leading to their nucleation and subsequent formation 46 . This mechanism is conserved across a diverse range of microbial taxa including fungi, yeasts, and bacteria 14,47,48 . Among filamentous fungi, Fusarium oxysporum has been extensively characterized for its NR-mediated nanoparticle synthesis, with reported enzymatic rates up to 220 nmol/h/mL 49 . Similarly, Aspergillus oryzae and Trichoderma reesei have been shown to utilize nitrate reductase effectively in AgNPs biosynthesis 15,50 . Intra-genus variability in NR activity has also been documented among Aspergillus species, with A. fumigatus exhibiting the highest activity, followed by A. clavatus and A. niger , while A. flavus demonstrated comparatively lower enzymatic performance 51 . Other fungal genera have also displayed considerable NR activity. For instance, Penicillium spp. exhibited significant enzymatic activity quantified at 270 nmol/h/mL 52 . Among yeasts, Cryptococcus laurentii and Rhodotorula glutinis showed NR activities of 266.56 nmol/h/mL and 216.85 nmol/h/mL, respectively 48 . In bacterial systems, Bacillus subtilis demonstrated nitrate reductase activity of 152 nmol/h/mL 47 , while crude metabolite extracts from Escherichia coli displayed activity levels equivalent to 2.18 U/mL 53 . The significant increase in nitrate reductase activity in the AgNPs-associated fraction of T. funiculosus highlights a functional integration between the enzyme and the nanoparticle matrix. This interaction enhances catalytic efficiency, suggesting a stable nano–bio configuration with potential applications in developing high-performance bio-catalytic systems for therapeutic and industrial use. Physicochemical and morphological validation of Amp–AgNPs formation UV–visible spectroscopy provided initial confirmation of the successful formation of Amp–AgNPs conjugate. As shown in Fig. 2a, increasing the ampicillin concentration (0.2–4.0 mg/mL) resulted in a gradual shift of the characteristic AgNPs SPR peak at 422.5 nm, along with the appearance of a secondary peak at 340.5 nm. These spectral changes indicate strong interactions between the β-lactam antibiotic and the nanoparticle surface. Similarly, varying the AgNPs concentration from 0.2 to 1.0 mg/mL (Fig. 2b) led to enhanced SPR intensity and peak shifts, supporting the concentration-dependent modulation of nanoparticle optical properties. As shown in Fig. 2c, under the optimized synthesis conditions (1.0 mg/mL AgNPs and 1.0 mg/mL ampicillin), the Amp–AgNPs conjugate exhibited a distinct SPR peak at 340.5 nm. This characteristic peak confirms the formation of the novel hybrid nanostructure. The conjugate remained stable over 3 months at room temperature, as evidenced by unchanged UV–visible spectra, indicating preserved structural integrity and colloidal stability throughout the storage period. Functionalization of AgNPs with biomolecules enhances their stability and provides properties such as target specificity, fluorescence, and antimicrobial activity, supporting their broad applicability in biomedical nanotechnology 30 . The AgNPs used for ampicillin functionalization were biosynthesized by T. funiculosus and characterized by our previously study El deeb et al. 8 as spherical crystalline, stable (6 months), and mono-dispersed (PDI: 0.007), exhibiting SPR at 422.5 nm, average diameter of 34.32 nm, and Zeta potential of -18.41 mV. The pronounced 82 nm shift in Amp–AgNPs UV–visible spectra reflects significant change in the electronic environment and surface characteristics of the nanoparticles, supporting successful functionalization. Such molecular interactions promoted charge transfer and altered the electronic structure, particularly through transitions from non-bonding (n) orbitals to antibonding pi (π*) orbitals, ultimately leading to a blue shift in the SPR band, as described in the study by Onyangore et al. 54 . The noteworthy 82 nm shift sharply contrasts with the more minor shifts observed in previous studies, such as the shift from 408 nm to 427 nm upon ampicillin functionalization 55 , minimal shifts from 438 nm to the range of 421 – 426 nm noted with cephalosporin conjugates 56 , and the shift from 438 nm to 477 nm occurring after conjugation with ciprofloxacin 57 . Other reports have shown SPR bands for silver-drug conjugates generally remaining within the 400–450 nm range 58 , while ampicillin-functionalized AgNPs displayed an SPR at 390 nm 59 . In the direct synthesis of AgNPs using ampicillin as reducing and stabilizing agent, Khatoon et al. 60 reported an SPR peak at 406 nm, confirming ampicillin capability in nanoparticle synthesis however the peak remained within the expected range for AgNPs. The consistent SPR band at 340.5 nm for 3 months further confirms the long-term stability of the Amp–AgNPs, demonstrating their suitability for potential therapeutic applications. Similar stability results were reported by Brown et al. 61 . DLS analysis of synthesized Amp–AgNPs displayed a size range of 22.2 to 39.46 nm, with an average diameter of 27.26 nm, while Zeta potential measurements indicated a negative surface charge of –24.9 mV (Fig. 2d). The reduction in hydrodynamic size from 34.32 (AgNPs) to 27.26 nm following ampicillin conjugation is attributed to the active functional groups of ampicillin, which interact with and reorganize the nanoparticle surface, increase surface charge, and compress the stabilizing sheath. These modifications result in a smaller and more stable particle size, as evidenced by the increase in negative Zeta potential from –18.6 to –24.9 mV. Similar findings were reported by Lopez-Carrizales et al. 62 where the conjugation of ampicillin led to a reduction in AgNPs size from 8.57 to 4.01 nm. This decrease was attributed to ampicillin favoring the homogeneous dispersion of the nanoparticles and enhancing colloidal stability, as reflected by an increase in negative Zeta potential from –40.01 to –51.00 mV. Comparable effects have also been observed with tetracycline, where surface modification influences nanoparticle dispersion, surface charge, and overall colloidal stability 63 . In a study by Rogowska et al. 32 functionalization of AgNPs with ampicillin resulted in an increase in particle size and a shift in zeta potential toward less negative values, indicating that the impact on colloidal stability may vary depending on the capping agents and synthesis conditions. In contrast, Adil et al. 56 observed larger hydrodynamic diameters in antibiotic-functionalized AgNPs compared to AgNPs, attributing the increase in size to the binding of antibiotic molecules to the nanoparticle surface. FTIR spectroscopy further validated the conjugation between AgNPs and ampicillin (Fig. 2e). In the FTIR spectrum of pure ampicillin a prominent band appeared at 1736 cm⁻¹, corresponding to the carbonyl (C=O) stretching vibration. The disappearance of this band in the Amp–AgNPs spectrum suggests that the carbonyl group of ampicillin is involved in the interaction with AgNPs. The bands observed at 1457 cm⁻¹ and 1530 cm⁻¹, assigned to β-lactam ring vibrations in pure ampicillin, were not detected in the Amp–AgNPs spectrum. The disappearance of these functional group signals supports the binding of the β-lactam group during the conjugation process. Previous studies have reported only shifts in hydroxyl and amide group regions upon antibiotic conjugation, supporting the role of these groups in stabilization and surface binding 58,64 . The FTIR spectrum of AgNPs synthesized directly using ampicillin showed a shift of the amine peak from 1605 to 1625 cm⁻¹ while the β-lactam ring and other characteristic bands remained unchanged compared to pure ampicillin 60 . Also, Murei et al. 34 observed no new functional groups upon conjugation, suggesting that the synthetic route significantly influences the surface chemistry and degree of interaction between AgNPs and antibiotics. In contrast, the presented data reveal distinct spectral features indicative of direct bonding that enhanced conjugation efficiency. HR-TEM imaging provided direct visual evidence of the morphology and structural organization of the Amp–AgNPs conjugate (Fig. 3a-c). The TEM images revealed that the AgNPs exhibited a well-defined spherical morphology surrounded by ampicillin molecules. This distinctive structural arrangement suggests effective adsorption or potential covalent interaction between AgNPs and ampicillin, which may contribute to enhanced functionality for diverse biomedical applications. No aggregation of AgNPs was observed and the calculated PDI of 0.037 indicates excellent stability and a highly mono-disperse distribution. The SAED pattern confirmed the retention of the crystalline nature of AgNPs, with no detectable alterations upon conjugation with ampicillin (Fig. 3d). These findings are consistent with previous reports 59,61,65 . The PDI of the conjugate was 0.037, indicating it is highly mono-disperse and confirming its excellent suitability for biomedical applications 66 . Although the PDI increased slightly from 0.007 for the AgNPs to 0.037 after conjugation, this minor rise is attributed to surface functionalization with ampicillin. Stability of Amp–AgNPs under variable pH and temperature conditions UV–Vis spectroscopic analysis demonstrated a pH-dependent variation in the optical properties of Amp–AgNPs conjugate across a pH range of 1 to 13 (Fig. 4a). A prominent absorption peak at 340.5 nm was observed from highly acidic (pH 1) to moderately alkaline (pH 9) conditions, indicating a broad tolerance for pH variations. Elevated alkaline pH values (11–13) caused significantly diminished or poorly resolved this peak. The dissociation of the Amp–AgNPs complex at higher pH values was further supported by the appearance of distinct absorbance bands corresponding to free ampicillin (281.5 nm) and AgNPs (422.5 nm). Visual inspection revealed slightly aggregation or precipitation under strong acidic conditions after 4 hours of incubation (Fig. 4a). After 48 hours, the reaction mixture maintained the characteristic 340.5 nm peak, with reduced intensity, indicating time-dependent stability and some preservation of the conjugate even under extreme pH conditions. The Amp–AgNPs displayed impressive resilience across a broad pH spectrum, particularly maintaining stability at pH 5.0. This finding is consistent with research on the stability of AgNPs biosynthesized by T. funiculosus , which also demonstrated enhanced stability at pH 5.5 8 . Ampicillin, on the other hand, shows optimal stability at pH 7.5, experiencing over 70% degradation at pH 3.4 after 12 hours, underscoring the importance of a near-neutral pH for preserving its efficacy 67,68 . Therefore, the observed stability of the conjugates at various pH levels indicates that the conjugation with AgNPs aids in maintaining the structural integrity of ampicillin, even at the extremes of its natural stability range. The UV–Vis spectroscopy analysis of the thermal stability of Amp–AgNPs was conducted across a temperature range of 5 °C to 55 °C (Fig. 4b). A consistent absorption peak at 340.5 nm was observed throughout the temperature gradient. The stability of this peak suggests that the bonding interaction between ampicillin and the AgNPs surface remains intact even at elevated temperatures. Visual inspection of the samples (inset) further supported the spectrophotometric results, showing no visible aggregation or precipitation across the temperature range (Fig. 4b). This evidence implies that Amp–AgNPs maintain colloidal stability and structural integrity up to 55 °C. Regarding thermal tolerance, biosynthesized AgNPs showed optimal stability at elevated temperatures, particularly around 60 °C 8 . Correspondingly, the Amp–AgNPs maintained their structural integrity at temperatures up to 55 °C. This stands in contrast to free ampicillin, which maintains over 90 % of its initial concentration for 48 hours only under refrigerated conditions (8 ± 2 °C), and for up to 24 hours at 25 ± 2 °C and 30 ± 2 °C. However, at 37 ± 2 °C, its stability falls below the 90% threshold within 12 hours, making it less suitable for use in warmer environments 69,70 . The enhanced thermal stability of the Amp–AgNPs indicates that nanoparticle conjugation significantly improves the physicochemical resilience of ampicillin, potentially extending its efficacy in challenging storage or treatment conditions. Similarly, the colloidal stability of biogenic synthesized AgNPs formed stable complexes with ampicillin within a pH range of 6 to 8, with optimal stability observed at pH 7–8. Additionally, enhanced adsorption efficiency was reported at elevated incubation temperatures ranging from 4 to 42 °C 32 . The imipenem-AgNPs conjugate exhibited excellent stability, with no changes in absorbance peaks, color, visible aggregation, or clarity observed over three months of storage at 4 °C and 25 °C 71 . Also, the colloidal stability of biogenic AgNPs–nisin conjugate was confirmed at pH 4 and 8, as no shift in the SPR band or decrease in its intensity was observed, indicating that the nanoparticles remained stable and did not undergo aggregation under the tested pH conditions 72 . Exploring the binding mechanism of ampicillin to nitrate reductase via in silico approaches The 3D structure of nitrate reductase was built using homology modeling based on the obtained homologous templates. The obtained 3D structure of nitrate reductase is shown in Fig. 5. The model consists of the molybdenum cofactor (MoCo) domain that binds the Moco where nitrate reduction occurs and this is the catalytic site center where nitrate binds and is reduced. Flavin adenine dinucleotide (FAD)-Binding domain which binds FAD for NAD(P)H-mediated electron transfer, where FAD accepts electrons from NADPH and transfers them to the MoCo center. Nitrate-binding pocket near the MoCo center, with conserved residues (e.g., Arg, His, or Ser) aiding in substrate orientation. Electron transfer pathways involving conserved cysteine residues coordinating with the MoCo centers. Potential phosphorylation sites (as seen in other fungal NRs) that regulate enzyme activity in response to nitrogen availability. Ramachandran plot (Fig. 6) showed that almost all β-sheets, right-handed α-helices and left-handed α-helices residues are found in the core regions (Favored Regions), with some residues in loops or flexible regions. too few residues (Gly, and Pro) appear in the disallowed regions (and this is acceptable due to large flexible loops of the model). To find out the most active pocket grid analysis implemented in DOCK 6 was performed and the most active pocket was used for docking. Pocket-Cavity Search within this structure showed that it has a large area shown as iso-surface in Fig. 7. Largest cavity, consistent with the molybdenum cofactor (MoCo) binding site where nitrate reduction occurs (pocket 1 1089 ų - Catalytic MoCo Active Site, consists of His84, Arg89, likely stabilize the MoCo center (analogous to Aspergillus NR), Asp213, Asp221acts as proton donors for catalysis, Cys237, Trp238, which are redox-active residues for electron transfer, aromatic/hydrophobic residues (Phe18, Tyr230, Phe236) may position nitrate substrates, and disulfide potential (Cys237) suggests redox regulation. Pocket 2 (511 ų - FAD/NADPH-Binding Domain), this pocket consists of a positively charged residues (Arg2, Lys3, Lys5, Arg124) – Binds NADPH phosphate groups, aromatic amino acids (Trp117, Tyr121) which Stack with FAD’s isoalloxazine ring and Asp122 that may hydrogen-bond to NADPH. Pocket 3 (173 ų - Allosteric Regulation Site) which has charged cluster (Arg192, Glu194, Lys199) - Potential phosphorylation site (e.g., by fungal kinases) and Trp203 mediates protein-protein interactions. Pocket 4 (154 ų - Proton Relay/Substrate Channel), composed of acidic residues (Asp164, Glu165, Asp254) and His163 act as proton transfer during catalysis and proton shuttle, respectively. Pocket 5 (142 ų - Redox-Sensing Disulfide) with Cys41 and Tyr52 work as potential disulfide bond formation under oxidative stress and electron transfer roles. Pocket 6 (129 ų - Substrate Access Channel), with charged (Asp46, Lys47) and aromatic (Tyr53, Trp117) residues guides nitrate into Pocket 1. Pocket 7 (122 ų - Heme-Binding Interface), Arg146, Asp151 and Tyr147, Tyr150 which are Heme propionate coordination and stabilization. Pocket 8 (118 ų - Solvent-Exposed Electrostatic Patch), basic residues (Arg25, Lys29, Arg35) may anchor NR to membranes or partner proteins. The 2D structure of the lowest energy docked structure was shown in Fig. 8, where ampicillin was fitted in the active site pocket of the modeled nitrate reductase model. It is obvious from this model that ampicillin formed HB network with residues His84, Arg89, Val92, and Gln95 where His84 and Arg89 are likely near the catalytic site (common in nitrate reductases for redox reactions). Hydrogen bonds stabilize the β-lactam ring of ampicillin, potentially interfering with substrate binding. In addition, it had electrostatic and hydrophobic interaction with residues Val14, Serl5, Thr16, Val9l, Pro93, Ser100, Leu152, Phe236, Cys237, Trp238. This may lead to disruption of the enzyme’s charge balance near the active site. The hydrophobic cluster (Phe236, Trp238, Leu152) suggests ampicillin binds in a partially buried pocket, shielding it from solvent. The docking score was found to be -8.4 kcal/mol. His84 & Arg89 are likely near the catalytic site, which is critical for nitrate reduction (common in NADH-dependent nitrate reductase). Ampicillin’s β-lactam ring is stabilized by these H-bonds Competitive or mixed-type inhibition, where ampicillin directly competes with nitrate or induces conformational changes. Arg89 (positively charged) may interact with ampicillin’s anionic carboxylate group. Ser15, Thr16 (polar) could stabilize the drug’s polar groups. Phe236, Trp238, Leu152, Val91, Pro93 form a hydrophobic pocket, burying ampicillin’s nonpolar regions (e.g., phenyl group). Cys237 may contribute to binding via weak S/π interactions with the β-lactam ring. The hydrophobic cluster shields ampicillin from solvent, increasing residence time in the binding site. Disruption of the charge balance near the active site (due to Arg89 interaction) may impair redox chemistry. A score of -8.4 kcal/mol suggests strong binding (typical high-affinity inhibitors range from -7 to -12 kcal/mol). The docking results confirmed the interaction between ampicillin and the enzyme-capped AgNPs, indicating the formation of a stable hybrid conjugate. This discovery opens new avenues for exploring the potential of drugs to conjugate with silver nanoparticles through nitrate reductase, presenting a promising strategy for improved drug delivery. Antibacterial activity and ultra-structural disruption of β-Lactamase-producing bacteria induced by Amp–AgNPs Amp–AgNPs demonstrated significant antibacterial activity against β-lactamase-producing bacterial strains (Fig. 9 and Table 1). At low concentrations (5–10 µg/mL), Amp–AgNPs were produced measurable inhibition zones ranging from 7.7 to 10.7 mm. At a concentration of 50 µg/mL, Amp–AgNPs resulted in inhibition zones measuring 27.3 ± 0.3 mm for E. coli , 25.0 ± 1.0 mm for E. faecalis , and 26.3 ± 0.3 mm for S. aureus . These measurements significantly exceeded those recorded for AgNPs (17.7 ± 0.3, 15.7 ± 0.3, and 16.7 ± 0.3 mm, respectively) and the positive control (15.3 ± 0.3, 13.3 ± 0.3, and 16.3 ± 0.3 mm, respectively). Ampicillin was ineffective across all tested concentrations. The MIC values for Amp–AgNPs conjugate were significantly lower than AgNPs, ampicillin, and the positive control (Table 1). Amp–AgNPs MICs were 3.3 ± 0.3, 4.7 ± 0.3, and 4.3 ± 0.3 µg/mL for E. coli , E. faecalis , and S. aureus , respectively. However, AgNPs MIC values were 8.7 ± 0.3, 7.7 ± 0.3, and 8.3 ± 0.3 µg/mL, respectively while for the positive control were 17.3 ± 0.3, 15.7 ± 0.3, and 9.3 ± 0.3 µg/mL, respectively. Ampicillin demonstrated no inhibitory effect even at concentrations of 100 µg/mL. These results suggest that ampicillin conjugation with AgNPs presenting a promising strategy for combating β-lactamase-producing pathogens. Bacterial strains Escherichia coli Enterococcus faecalis Staphylococcus aureus Concentration ( µg/mL) Amp AgNPs Positive control Amp–AgNPs Amp AgNPs Positive control Amp–AgNPs Amp AgNPs Positive control Amp–AgNPs 5 Inhibition zone diameter (mm) - - - 8.3±0.3 - - - 7.7±0.3 - - - 7.7±0.3 10 - 7.3 ±0.3 - 10.7±0.3 - 7.0±0.0 - 11.7±0.3 - 8.3±0.3 7.7±0.3 10.3±0.3 20 - 8.3±0.3 7.0±0.0 14.3±0.3 - 8.7±0.3 7.0±0.0 14.3±0.3 - 12.3±0.3 12.3±0.3 13.7±0.3 30 - 11.7±0.3 9.7±0.3 18.3±0.3 - 12.3±0.3 8.3±0.3 16.3±0.3 - 13.7±0.3 13.3±0.3 17.7±0.3 40 - 14.7±0.3 12.3±0.3 22.7±0.3 - 13.3±0.3 10.3±0.3 19.7±1.0 - 15.7±0.3 14.7±0.3 21.0±0.3 50 - 17.7±0.3 15.3±0.3 27.3±0.3 - 15.3±0.3 13.7±0.3 25.0±1.0 - 16.7±0.3 16.3±0.3 26.3±0.3 MIC ( µg/mL) - 8.7±0.3 17.3±0.3 3.3±0.3 - 7.7±0.3 15.7±0.3 4.7±0.3 - 8.3±0.3 9.3±0.3 4.3±0.3 Table 1 . Inhibitory zone diameter and MIC of of Amp–AgNPs against β-lactamase-producing bacteria ( p < 0.05). Scanning electron microscopy provided compelling evidence of structural damage of various β-lactamase-producing E. coli , E. faecalis , and S. aureus (Fig. 10). The untreated control cells displayed well-preserved morphology with smooth, intact surfaces, showing healthy cellular integrity. Cells treated with ampicillin maintained their architecture, exhibiting minimal morphological changes, which confirmed their resistance to β-lactam antibiotics. Treatment with AgNPs alone resulted in moderate membrane disruption, pore formation, and increased surface roughness. In contrast, the positive control caused partial structural damage, including membrane wrinkling and deformation. Cells exposed to the Amp–AgNPs conjugate showed extensive morphological alteration, characterized by pore formation, membrane rupture, collapse of cellular structures, and leakage of intracellular contents. This level of damage was consistent across all tested strains. These results indicated that conjugating ampicillin with AgNPs effectively overcomes β-lactamase-mediated resistance by combining targeted membrane disruption with intracellular delivery of the antibiotic. Amp–AgNPs synthesized in this study exhibited significantly enhanced antibacterial activity against β-lactamase-producing E. coli , E. faecalis , and S. aureus . This synergistic enhancement underscores the potential of AgNPs as effective carriers for conventional antibiotics, particularly in combating resistant bacterial strains. Similar results have been documented in the previous studies. For instance, Alfahad et al. 73 documented inhibition zones of 14.17 mm for Amp–AgNPs against Salmonella typhi using 3% aqueous AgNPs. Similarly, Rogowska et al. 32 reported inhibition zones for Ag-CGG-Ampicillin conjugates synthesized using Actinomycetes CGG 11n, with zones ranging from 5.0 ± 0 mm to 12 ± 0 mm across different pathogens. Brown et al. 61 showed that Amp–AgNPs achieved complete eradication of resistant E. coli and Pseudomonas aeruginosa within 4–6 hours, compared to 6–8 hours for AgNPs alone, highlighting the conjugate’s enhanced bactericidal kinetics and its potential to prevent biofilm formation. Ibraheem et al. 57 reported that a conjugate of AgNPs, polyethylene glycol, and ciprofloxacin produced remarkably larger inhibition zones of 36 mm for Acinetobacter baumannii , 39 mm for S. aureus , and 40 mm for Serratia marcescens compared to the individual components. Also, Jalil et al. 64 demonstrated that AgNPs conjugated with amoxicillin exhibited superior antimicrobial activity against Streptococcus pneumoniae , S. aureus , P. aeruginosa , and methicillin-resistant S. aureus , reinforcing the value of nanoparticle-mediated drug delivery systems. The results of the MIC assays further substantiate the superior antibacterial efficacy of the Amp–AgNPs conjugate. The results highlight a synergistic interaction between ampicillin and AgNPs, enabling effective bacterial inhibition at significantly lower concentrations. Similarly, Khatoon et al. 60 documented MICs of 18.75 µg/mL and 9.375 µg/mL for Amp–AgNPs against ampicillin-sensitive E. coli and S. aureus , respectively and MICs of 10 µg/mL and 3 µg/mL for ampicillin-resistant counterparts. The MIC values were lower than the MICs reported for chemically synthesized AgNPs, which range from 280 to 720 µg/mL. Also, Rogowska et al. 32 demonstrated enhanced antibacterial efficacy using AgNPs functionalized with ampicillin, with MICs of 3.125 µg/mL for P. aeruginosa , 25 µg/mL for E. coli , and 6.25 µg/mL for K. pneumoniae . Antibiotic–AgNPs conjugates enhance antimicrobial efficacy with lower MICs and offering a more effective therapeutic strategy 34 . The effectiveness of the conjugation strategy is demonstrated by imipenem–AgNP conjugate, which exhibited markedly lower MICs (2–16 µg/mL) against 200 P. aeruginosa isolates compared to imipenem alone (64 to >512 µg/mL) and AgNPs (4–32 µg/mL), highlighting their enhanced potency 74 . AgNPs functionalized with glucosamine demonstrated enhanced activity, with MICs as low as 8 µg/mL against methicillin-resistant Staphylococcus aureus 75 . These findings corroborate our current results and consistently support that conjugation of AgNPs with conventional antibiotics markedly reduces MIC values even in resistant strains. The enhanced antibacterial efficacy of Amp–AgNPs conjugates can be attributed to multiple synergistic mechanisms involving the physicochemical properties of AgNPs and the biological activity of the antibiotics. Conjugation significantly enhances antibacterial activity against Gram-positive and Gram-negative bacteria, including MDR strains by enabling bioactive molecules to retain and even amplify their function when bound to the surface of AgNPs 76 . This enhanced potency likely results from the hydroxyl and amide groups of ampicillin and the AgNPs surface, which leads to improved cellular uptake and intracellular retention. Scanning electron microscopy analysis in the current study revealed pronounced morphological disruptions in E. coli , E. faecalis , and S. aureus treated with Amp–AgNPs, including membrane rupture, cytoplasmic leakage, and complete structural collapse, effects that were not observed with either ampicillin or AgNPs alone. AgNPs enhance antibiotic efficacy by increasing bacterial membrane permeability and facilitating greater intracellular drug uptake 33 . AgNPs disrupt bacterial membranes by binding to sulfur-containing proteins, compromising structural and enzymatic integrity, while also interfering with protein synthesis and DNA replication, ultimately causing irreversible cellular damage and death 57 . Conjugation enhances adhesion and membrane penetration through van der Waals and electrostatic interactions, disrupts DNA and metabolic processes, generates reactive oxygen species, and impairs electron transport, leading to bacterial cell death 77 . By integrating membrane disruption, intracellular interference, and enhanced delivery, the conjugates present a robust strategy for next-generation antimicrobial therapies, highlighting the need for further validation through in vivo studies. Effect of Amp–AgNPs conjugate on β-lactamases activity in clinical bacterial strains All tested strains ( E. coli , E. faecalis , and S. aureus ) were confirmed as β-lactamase producers, indicated by a distinct color change from blue to colorless in the iodometric assay, reflecting the enzymatic hydrolysis of free ampicillin (Fig. 11). In contrast, AgNPs and Amp–AgNPs maintained the blue color, demonstrating the absence of β-lactam ring hydrolysis. The positive control displayed a pale blue color, consistent with β-lactamase activity. The sustained blue coloration observed in the Amp–AgNPs treatment confirmed the stability of the conjugated form, suggesting that the β-lactam ring is shielded from enzymatic degradation. The iodometric assay results revealed clear evidence of β-lactamase activity in E. coli , E. faecalis , and S. aureus , as indicated by the color change from blue to colorless upon ampicillin treatment. This confirms the susceptibility of conventional β-lactam antibiotics to enzymatic hydrolysis, a major mechanism underlying bacterial resistance 78,79 . In contrast, samples treated with Amp–AgNPs retained the blue coloration, indicating that this complex prevent β-lactamase activity and maintained ampicillin structural integrity. Moreover, Amp–AgNPs demonstrated superior stability compared to the positive control, suggesting a protective role conferred by nanoparticle conjugation 80 . A plausible explanation lies in the alteration of steric and electronic characteristics of ampicillin upon conjugation with AgNPs. These modifications may prevent optimal substrate alignment within the β-lactamase active site, impairing catalytic function through steric hindrance or electronic redistribution 81 . This inhibition mechanism resembles competitive inhibition or substrate mimicry, a concept well-documented in the context of β-lactamase inhibitors 82 . Unlike classical inhibitors such as clavulanic acid, sulbactam, and tazobactam, which act through covalent acylation and irreversible enzyme inactivation 83 . This aligns with emerging efforts to develop non-β-lactam inhibitors capable of resisting enzymatic degradation across a broad spectrum of β-lactamase classes 84,85 . By preserving the β-lactam core and conferring enzymatic protection, Amp–AgNPs address the structural and functional limitations of existing β-lactamase inhibitors 86 . Due to the increasing prevalence of β-lactamase-mediated resistance and the decreasing effectiveness of current inhibitors 87 , hybrid nano-antibiotic platforms offer a promising new approach for antimicrobial drug design, providing renewed hope to combat resistance and enhance antibiotic effectiveness. Materials and Methods Functional SDS-PAGE profiling and microplate assay confirmation of nitrate reductase involved in the biosynthesis and capping of AgNPs by T. funiculosus The isolation and characterization of the proteins present in T. funiculosus filtrate and the corresponding biosynthesized AgNPs were conducted using SDS-PAGE as described by Laemmli 88 . Three distinct fungal filtrates were prepared from various patches of T. funiculosus , each utilized for synthesizing AgNPs as outlined by El deeb et al. 8 . SDS-PAGE analysis was performed on each fungal filtrate and its respective biosynthesized AgNPs to confirm protein presence and ensure the reproducibility of the results. Electrophoresis was conducted using a 12% SDS-polyacrylamide resolving gel within an OmniPAGE Mini Vertical Protein Electrophoresis System, UK, at a constant voltage of 100 V. After electrophoresis, the gel was stained with Coomassie Brilliant Blue dye and visualized using a gel imaging system (GelLITE, Cambridge, UK). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), with a molecular weight of 38 kDa, was run alongside the samples as an internal reference to validate the accuracy and reliability of molecular weight estimation. Also, a standard molecular weight marker (Promega, ADV8491, USA) was included to facilitate precise protein size determination. To confirm the identity of the protein band detected by SDS-PAGE as nitrate reductase for the fungal filtrate and the biosynthesized AgNPs, a colorimetric microplate-based nitrate reductase assay was performed using a commercial kit (Nitrate Reductase Microplate Assay Kit, Cat. No. ORB219870, Biorbyt, USA). The assay quantified nitrate reductase activity based on the enzymatic reduction of nitrate to nitrite by chromogenic azo-compound detection under standard assay conditions. One unit of nitrate reductase activity was defined as the amount of enzyme required to generate 1 μmol of nitrite per hour. The assay was conducted in triplicate according to the manufacturer's instructions, and enzyme activity was expressed as units per gram (U/g) of fungal biomass. Absorbance was measured at 540 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Finland), and the data were analyzed using SkanIt Software for Microplate Readers RE, version 6.0.1.6. The detection of enzymatic activity corresponding to the 58 kDa protein band confirmed the functional presence of nitrate reductase in the fungal filtrate and the AgNPs, indicating its catalytic role in the biosynthesis and capping of AgNPs. Preparation and physicochemical characterization of Amp–AgNPs conjugate Ampicillin solutions (0.2, 0.5, 1.0, 2.0, 3.0, and 4.0 mg/mL) were prepared in distilled water and mixed with biosynthesized AgNPs (1.0 mg/mL) to form Amp–AgNPs conjugate. Conversely, varying concentrations of AgNPs (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) were mixed with ampicillin (1.0 mg/mL) to identify the optimal nanoparticle concentration for conjugation. The optimal concentration of ampicillin and AgNPs for functionalization was determined using UV–visible spectroscopy. The reaction mixtures were incubated at room temperature for 24 hours and repeated in triplicate for reproducibility. The successful formation of the Amp–AgNPs conjugates was confirmed using multiple characterization techniques. To monitor the surface Plasmon resonance (SPR) shifts, UV–visible spectra were conducted using a UV–visible spectrophotometer (JAS.CO V-770, Japan) within the wavelength range of 200 – 800 nm. Deionized water was used as a blank to adjust the baseline. Measurements were repeated after six months of preservation at room temperature to evaluate the long-term stability of the Amp–AgNPs. The size distribution and average hydrodynamic diameter of Amp–AgNPs were assessed by DLS, while Zeta potential (surface charge) was measured with a NANOTRAC WAVE II, Germany to evaluate colloidal stability. FTIR spectroscopy was conducted to explore the functional groups and potential interactions involved in the formation of the Amp–AgNPs conjugate. The spectra of pure ampicillin, biosynthesized AgNPs, and the conjugated complex were recorded using a Platinum-ATR FTIR spectrometer (Bruker Alpha, Germany) in the range of 4000 – 400 cm⁻¹. This spectral range facilitated the identification of characteristic functional groups and the assessment of possible chemical bonding or interactions between ampicillin and AgNPs. High-resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) analyses were performed to investigate the morphology, interactions, and crystalline structure of the Amp–AgNPs conjugate. These analyses were conducted using a JEOL JSM 100CX TEM instrument (JEOL Ltd., Japan) at the Electron Microscope Unit of Cairo University, Egypt. TEM imaging was used to evaluate the shape, size distribution, and dispersion of the nanoparticles, as well as to provide insight into the surface interaction and conjugation of ampicillin with AgNPs. The polydispersity index (PDI) was calculated from the measured particle sizes to assess the uniformity and dispersity of the nanoparticles 66 . SAED was utilized to confirm the crystalline nature of the nanoparticles and to evaluate any potential structural changes or aggregation that may have arisen during the conjugation process. Stability assessment of Amp–AgNPs under physiological pH and temperature conditions The stability of the Amp–AgNPs conjugate was evaluated under varying pH and temperature conditions to assess its physicochemical robustness following the method described by Rogowska et al. 32 . For pH stability, aliquots were adjusted to pH values of 1, 3, 5, 7, 9, 11, and 13 using 0.1 M HCl or 0.1 M NaOH and incubated at room temperature for 48 hours. Thermal stability was assessed by incubating separate aliquots at 5, 15, 25, 35, 45, and 55 °C for 48 hours in a controlled water bath. UV–Vis absorbance spectra (200 – 800 nm) were recorded to monitor changes in the characteristic peak at 340.5 nm, indicative of stable Amp–AgNPs conjugation. Shifts or reductions in peak intensity were used to evaluate conjugate integrity. The test was conducted in triplicates, and visual inspections were performed to detect signs of aggregation, turbidity, or precipitation providing complementary evidence of colloidal stability. Computational investigation of ampicillin interaction with nitrate reductase enzyme capped AgNPs Nitrate reductase enzyme’s sequence of T. funiculosus was obtained from the UniProt database (ID: F1CF56). The obtained sequence was used to search for a homology models using BLAST database, highly similar sequences with known crystal structures were used for multiple alignment and threading of the query enzyme (PDB IDs: 1SOX, 2A99, 2A9B, 2A9D, 2BIH, 2BII, 3HBG, 3HBP, 3HC2, 3R18, 3R19). The 3D model of the enzyme was then designed using SWISSPdb 89 , where the template models were fitted together before threading the query sequence. This process enhances the modeling and decreases the clashes of the new model. The obtained model was validated using Ramachandran plot 90 . Furthermore, energy minimization was performed using GROMOS96 force-field 91 . The obtained model was compared with the AI generator of Aphafold2, where the RMS value was found to be 1.55 Å. The obtained 3D model was used as a receptor for docking ampicillin substrate which was obtained from the PubChem database. Docking protocols were created based on prior studies 92 using USCF DOCK6 93 . The AM1-BBC method was used in DOCK6 to add charge to the protein and ampicillin. Utilizing a probe radius of 1 point 4 Å, the molecular surface was created. Using spheres within 9 Å of the native ligand, which is encircled by a box with a margin of 5 Å, the active site of a target was identified. Polar hydrogens were added to the targets in Vina, and then Kollman charges were added. In the meantime, ligand preparation involved the use of Gestaiger charges. To generate the center coordinate, a grid box was applied at a spacing of 1 Å, centered on the native ligand position. After comparing the ten conformations produced, docked structures were selected from the obtained conformations based on the binding energy (kcal/mol) of these conformations. LIGPLOT + (version 2.2) was used to visualize and determine the interactions between ligand and receptor's amino acids. It examines the intricate receptor-ligand structure's 2D hydrogen bond (HB) interaction. It provides a graphical representation of HB, hydrophobic bonds, and their bond lengths at the ideal docking position 94 . Integrated evaluation of antibacterial activity and morphological disruption by Amp–AgNPs conjugate against β-lactamase-producing bacteria The antibacterial susceptibility test and the minimum inhibitory concentration (MIC) determinations were conducted as described by El deeb et al. 8 . The assays were performed against Gram-negative and Gram-positive β-lactamase-producing bacteria, including Escherichia coli (SUMCC 22014), Enterococcus faecalis (SUMCC 22015), and Staphylococcus aureus (SUMCC 22016). All strains were isolated from clinical samples obtained from the Sohag University Microbial Culture Collection, Egypt. Five concentrations (5, 10, 20, 30, 40, and 50 µg/mL) were tested for: ampicillin, biosynthesized AgNPs, ampicillin/sulbactam (2:1 ratio), and the Amp–AgNPs conjugate. All experiments were performed in triplicate to ensure accuracy. The antibacterial activity was evaluated using the disk diffusion method on Mueller–Hinton agar (MHA). Sterile filter paper disks were loaded with the designated concentrations of each tested compound. The selected concentration range for Amp–AgNPs was based on the typical daily intake of silver from natural sources approximately 0.4 – 27 µg/day 95 , thus ensuring relevance to environmentally realistic exposure levels. Ampicillin served as the negative control to confirm bacterial resistance to ampicillin, while ampicillin/sulbactam was used as the positive control. The formed plates were incubated at 37 °C for 24 hrs followed by measuring the formed inhibition zone in millimeter (mm). The MIC for ampicillin, AgNPs, positive control, and Amp–AgNPs were determined for the tested pathogens by broth microdilution method using 96-well microtiter plates according to the principles described by Kowalska-Krochmal & Dudek-Wicher 96 . Concentrations ranging from 1 to 100 μg/mL were prepared to facilitate MIC determination. Resazurin, a blue dye that becomes pink upon reduction by metabolically active cells, was employed as an indicator of bacterial viability. From the above assay, an inoculum was taken from each well that showed no visual growth and spotted on MHA plates to validate the MIC assay. To evaluate the morphological alterations and potential molecular interactions induced by Amp–AgNPs, SEM analysis was performed on the tested bacterial cells before and after 6 hours of exposure to the MIC of each tested compound. For ampicillin, a concentration of 100 μg/mL was used. The analysis included untreated control cells, cells treated with ampicillin, AgNPs, positive control, and the Amp–AgNPs conjugate. Morphological analysis was performed according to the protocol described by Singh et al. 97 using a JEOL JSM-5400LV SEM (JEOL Ltd., Japan) operated at 15 – 25 kV. Impact of Amp–AgNPs conjugate on β-lactamases enzymatic activity The iodometric method was employed to evaluate β-lactamase activity, following the protocols described by Sharma et al. 98 and Aliyu et al. 99 . A loopful of a dense 24-hour culture of E. coli , E. faecalis , and S. aureus from MHA was mixed with 1.0 mL of a 10000 µg/mL solution of ampicillin, AgNPs, ampicillin/sulbactam (β-lactamase inhibitor used as a positive control), and the Amp–AgNPs conjugate. The samples were incubated at room temperature for 30 minutes with gentle agitation at 15-minute intervals. After the incubation period, two drops of a 1 % soluble starch solution and one drop of iodine solution were added to the mixture and gently shaken. A change in color from blue to colorless indicated positive β-lactamase activity, whereas the persistence of the blue color signified a negative result. Results were recorded within 10 minutes, and all tests were performed in triplicate for confirmation. Statistical analysis All experiments were carried out in triplicate and the results were presented as mean ± standard deviation. The data were statistically analyzed by one-way analysis of variance (ANOVA) using XLSTAT version 2023.2.0 software 100 . Differences at P < 0.05 were regarded as statistically significant. Conclusion This study successfully demonstrated the pivotal role of nitrate reductase in the biosynthesis and stabilization of AgNPs by T. funiculosus , with the enzyme retained and catalytically active on the nanoparticle surface. The formation of the Amp–AgNPs conjugate was confirmed through spectroscopic and microscopic analyses, demonstrating strong interactions between ampicillin and AgNPs that enhance physicochemical stability. The conjugate exhibited broad pH and thermal tolerance that is essential for biomedical applications. In silico modeling of nitrate reductase revealed its active site architecture and demonstrated strong binding affinity with ampicillin. Amp–AgNPs significantly exceeded ampicillin and AgNPs in inhibiting β-lactamase-producing bacterial strains. Also, Amp–AgNPs protects the antibiotic’s β-lactam ring from enzymatic degradation and causing pronounced bacterial ultra-structural damage. SEM analysis indicated extensive damage to the membranes of β-lactamase-producing bacteria, confirming the enhanced bactericidal efficacy through a synergistic mechanism involving membrane disruption and antibiotic delivery. The obtained results confirm the potential of NR-mediated AgNPs as nano-carriers for antibiotic delivery, offering a novel approach to overcome multidrug-resistant infections and advance antimicrobial nanobiotechnology. Future studies should explore in vivo efficacy, ensure biosafety, and extend this biomolecular strategy to a broader range of drug delivery applications. Declarations Data availability All data generated or analyzed during this study are included in this published article. Nitrate reductase enzyme’s sequence of Talaromyces funiculosus was obtained from the UniProt database (ID: F1CF56). The other datasets used and/or analyzed during the current study are available from the corresponding author upon request. Acknowledgements The authors thank Dr. Mohamed A. Morsy (Al-Azhar Virology Research Center, Faculty of Medicine, Al-Azhar University, Cairo, Egypt) for his valuable assistance in protein detection. Author contributions All authors registered have made fundamental, direct, and intellectual contributions to the work discussed in this publication. Conceptualization: B.A.E and G.G.F.; methodology: M.S.B., G.G.F. and M.I.; data collection: G.G.F. and M.I.; data curation: B.A.E, M.S.B., G.G.F., and M.I.; manuscript preparation, review, and editing: B.A.E, G.G.F., M.S.B., and M.I. All authors have read and agreed to the published version of the manuscript. Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Competing interests The authors declare no competing interests. Additional information Supplementary information file for this paper is available online. Correspondence and requests for materials should be addressed to B.A.E. References Siddiqi, K. S., Husen, A. & Rao, R. A. K. A review on biosynthesis of silver nanoparticles and their biocidal properties. J. Nanobiotechnol. 16 , 14. https://doi.org/10.1186/s12951-018-0334-5 (2018). Choudhary, S. et al. Phyco-synthesis of silver nanoparticles by environmentally safe approach and their applications. Sci. Rep. 14 , 9568. https://doi.org/10.1038/s41598-024-60195-3 (2024). Rafique, M., Sadaf, I., Rafique, M. S. & Tahir, M. B. A review on green synthesis of silver nanoparticles and their applications. Artif. Cells Nanomed. 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(2024). https://www.xlstat.com/en Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7042236","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":485340845,"identity":"e224669a-26a1-470f-844a-42f30b0efdb8","order_by":0,"name":"Gerges Gad Faheem","email":"","orcid":"","institution":"Sohag University","correspondingAuthor":false,"prefix":"","firstName":"Gerges","middleName":"Gad","lastName":"Faheem","suffix":""},{"id":485340846,"identity":"3affbda6-d62f-448c-ae4f-5dce185f893d","order_by":1,"name":"Bahig A. 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marker, (\u003cstrong\u003eLanes 1–3\u003c/strong\u003e) Proteins from \u003cem\u003eT. funiculosus \u003c/em\u003efiltrate, (\u003cstrong\u003eLanes 4–6\u003c/strong\u003e) Protein capped AgNPs, (\u003cstrong\u003eGAPDH\u003c/strong\u003e) Internal protein reference (38 kDa), (\u003cstrong\u003eNR\u003c/strong\u003e) Nitrate reductase band at 58 kDa.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/81ea4df74a17bffb22f7296d.jpeg"},{"id":86862207,"identity":"eddbbffa-9f88-42d5-a165-de3579fa04eb","added_by":"auto","created_at":"2025-07-16 12:26:54","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":732209,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of Amp–AgNPs:\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea-c\u003c/strong\u003e) UV-visible absorption spectra of Amp–AgNPs synthesized using varying concentrations of ampicillin, using varying concentrations of AgNPs, and at optimal concentration of ampicillin and AgNPs, respectively, (\u003cstrong\u003ed\u003c/strong\u003e) DLS analysis, (\u003cstrong\u003ee\u003c/strong\u003e) FTIR spectra of ampicillin, AgNPs, and Amp–AgNPs.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/6ff28921d573a44fa7406496.jpeg"},{"id":86861366,"identity":"8219434b-79c4-4019-a345-abe00a4ca1d0","added_by":"auto","created_at":"2025-07-16 12:18:54","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":439351,"visible":true,"origin":"","legend":"\u003cp\u003eHR-TEM images of Amp–AgNPs show AgNPs surrounded by ampicillin molecules: (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e) Amp–AgNPs at different scale bars, (\u003cstrong\u003ed\u003c/strong\u003e) SAED pattern.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/dd015a15a6729f1383df2586.jpeg"},{"id":86861371,"identity":"7e11631e-f9ec-4c3b-9e3d-43e0ae62dcba","added_by":"auto","created_at":"2025-07-16 12:18:54","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":327528,"visible":true,"origin":"","legend":"\u003cp\u003eStability of Amp–AgNPs at different:\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003epH values.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eTemperatures.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/089f936c4c10427a1a5f8f52.jpeg"},{"id":86861380,"identity":"35fbde19-dc5a-4dc0-af33-ef60f52e1eaa","added_by":"auto","created_at":"2025-07-16 12:18:55","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":349670,"visible":true,"origin":"","legend":"\u003cp\u003e3D model of nitrate reductase\u003cem\u003e \u003c/em\u003ebuilt by homology modeling, helixes are magenta, β-sheets are yellow and coils are light and dark blue.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/facb6f2027ebc262af67f890.jpeg"},{"id":86861382,"identity":"1f96463c-a48a-439e-99e0-6a5f95a73bc7","added_by":"auto","created_at":"2025-07-16 12:18:55","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":350558,"visible":true,"origin":"","legend":"\u003cp\u003e2D Ramachandran plot of the homology model of nitrate reductase.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/232ec6c5fd9af9d084d4dc03.jpeg"},{"id":86861370,"identity":"9ff01721-f2ca-4b4b-a9e2-49d0b4bf18dc","added_by":"auto","created_at":"2025-07-16 12:18:54","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":478773,"visible":true,"origin":"","legend":"\u003cp\u003eIsosurface representation of the pockets obtained by the cavity search tool.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/d8d6901f006bc07b7addd389.jpeg"},{"id":86861368,"identity":"114b19cc-4d48-4b3a-86c0-12530127f842","added_by":"auto","created_at":"2025-07-16 12:18:54","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":567906,"visible":true,"origin":"","legend":"\u003cp\u003e2D represents the ampicillin interaction within the active pocket of nitrate reductase enzyme. HB are shown in green line, hydrophobic and electrostatic attraction are shown in red.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/83c59e9a3a44b4cb34369de6.jpeg"},{"id":86861378,"identity":"6486e09f-a624-4c46-acb3-1b28211b8507","added_by":"auto","created_at":"2025-07-16 12:18:55","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2367319,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activities of Amp–AgNPs against β-lactamase-producing bacteria. Images show the effect on: \u003cem\u003eE. coli\u003c/em\u003e (\u003cstrong\u003ea\u003c/strong\u003e–\u003cstrong\u003ed\u003c/strong\u003e), \u003cem\u003eE. faecalis\u003c/em\u003e (\u003cstrong\u003ee\u003c/strong\u003e–\u003cstrong\u003eh\u003c/strong\u003e), and \u003cem\u003eS. aureus\u003c/em\u003e (\u003cstrong\u003ei\u003c/strong\u003e–\u003cstrong\u003el\u003c/strong\u003e) treated with: ampicillin (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e i\u003c/strong\u003e), AgNPs (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e), positive control (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003e k\u003c/strong\u003e), and Amp–AgNPs conjugate (\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e l\u003c/strong\u003e), respectively, Numerals indicate applied concentrations in µg/mL.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/9774141bc3b23f64374283ab.jpeg"},{"id":86861372,"identity":"25e9f9b2-0b17-4ac1-8f33-ca20e3943e00","added_by":"auto","created_at":"2025-07-16 12:18:54","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2813760,"visible":true,"origin":"","legend":"\u003cp\u003eSEM analysis revealing ultra-structural disruption of β-lactamase-producing bacteria. Micrographs show morphological alterations in \u003cem\u003eE. coli \u003c/em\u003e(\u003cstrong\u003ea\u003c/strong\u003e–\u003cstrong\u003ee\u003c/strong\u003e), \u003cem\u003eE. faecalis \u003c/em\u003e(\u003cstrong\u003ef\u003c/strong\u003e–\u003cstrong\u003ej\u003c/strong\u003e), and \u003cem\u003eS. aureus\u003c/em\u003e (\u003cstrong\u003ek\u003c/strong\u003e–\u003cstrong\u003eo\u003c/strong\u003e) subjected to: untreated cells (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e), ampicillin (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e), AgNPs (\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e h\u003c/strong\u003e,\u003cstrong\u003e m\u003c/strong\u003e), positive control (\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003e n\u003c/strong\u003e), and Amp–AgNPs conjugate (\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e j\u003c/strong\u003e,\u003cstrong\u003e o\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/69e66c9da61757e691578204.jpeg"},{"id":86861388,"identity":"0a63f43a-4e6e-4e81-b5a8-adc1e2f38b4a","added_by":"auto","created_at":"2025-07-16 12:18:55","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":400116,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Amp–AgNPs on β-lactamase activity in bacterial strains assessed by iodometric assay: (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eE. coli\u003c/em\u003e, (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eE. faecalis\u003c/em\u003e, (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/3c9474c3dbd25499ba298a33.jpeg"},{"id":99545382,"identity":"716a9352-e7ad-45e9-94af-8d1c1da40d8f","added_by":"auto","created_at":"2026-01-05 16:06:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10803372,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/86505970-7c55-41f8-9a07-c0b25dc9a2e0.pdf"},{"id":86862442,"identity":"72384f5a-2394-43ad-9527-155c53f8ff10","added_by":"auto","created_at":"2025-07-16 12:34:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":143162,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7042236/v1/11da9ee9374ee24dbf73fe19.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biomolecular strategy for designing antibiotic–silver nanoparticles conjugate via nitrate reductase mediated β-lactamase inhibition with molecular docking insights","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanoparticles have become transformative tools in medicine, biotechnology, and environmental applications due to their unique physicochemical properties at the nanoscale\u003cstrong\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e. Their synthesis can be achieved through physical, chemical, or green methods categorized into top-down and bottom-up approaches based on the formation mechanism\u003cstrong\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e. Top-down methods involve breaking down bulk materials into nano-scale particles using mechanical or thermal forces, producing uniform nanoparticles but requiring complex equipment, additional energy inputs, and lacking stabilizers to prevent agglomeration\u003cstrong\u003e\u003csup\u003e3,4\u003c/sup\u003e\u003c/strong\u003e. The bottom-up approach assembles nanoparticles from molecular precursors through nucleation and growth, typically using chemical or biological synthesis pathways involving metal salts\u003cstrong\u003e\u003csup\u003e5\u003c/sup\u003e\u003c/strong\u003e. While chemical synthesis allows for rapid production, it may involve hazardous substances, which can restrict medical applications\u003cstrong\u003e\u003csup\u003e6\u003c/sup\u003e\u003c/strong\u003e. In contrast, biological synthesis offers an environmentally friendly alternative, utilizing molecules derived from plants and microorganisms\u003cstrong\u003e\u003csup\u003e7,8\u003c/sup\u003e\u003c/strong\u003e. Fungi, particularly endophytic fungi, are efficient biological agents for nanoparticle synthesis due to their ease of cultivation and high secretion of enzymes and proteins that enhance nanoparticle stability \u003cstrong\u003e\u003csup\u003e9,10\u003c/sup\u003e\u003c/strong\u003e. Endophytes produce diverse bioactive compounds and extracellular enzymes with industrial and therapeutic relevance\u003cstrong\u003e\u003csup\u003e11-13\u003c/sup\u003e\u003c/strong\u003e. Among these, nitrate reductase (NR) is an enzyme implicated in the extracellular synthesis of AgNPs, facilitating the reduction of metal ions\u003cstrong\u003e\u003csup\u003e14-16\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eNitrate reductase, a molybdo-flavoprotein, catalyzes the nitrate reduction to nitrite using pyridine nucleotides as electron donors\u003cstrong\u003e\u003csup\u003e17\u003c/sup\u003e\u003c/strong\u003e. Nitrate reductase is categorized based on its coenzyme specificity: NADH-specific in higher plants, NADH/NADPH in algae, and NADPH-specific in fungi\u003cstrong\u003e\u003csup\u003e18,19\u003c/sup\u003e\u003c/strong\u003e. It is a multidomain enzyme comprising the prosthetic groups molybdopterin, Fe-heme, and FAD (flavin adenine dinucleotide) in a 1:1:1 stoichiometry that mediates an electron transfer from NAD(P)H to nitrate\u003cstrong\u003e\u003csup\u003e20\u003c/sup\u003e\u003c/strong\u003e. The biosynthesis of AgNPs is proposed to involve NADH as an electron donor and NADH-dependent nitrate reductase as a catalytic mediator in the reduction of silver ions to metallic silver, a function consistent with the enzyme’s electron transfer capabilities and structural composition\u003cstrong\u003e\u003csup\u003e21,22\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCapping agents are molecules that encase and stabilize AgNPs, preventing agglomeration, and ensuring their stability\u003cstrong\u003e\u003csup\u003e23\u003c/sup\u003e\u003c/strong\u003e. Proteins function effectively as green capping agents in the synthesis of nanoparticles due to their capacity to bind to metal surfaces through functional groups like amine, carboxyl, and thiol residues\u003cstrong\u003e\u003csup\u003e24\u003c/sup\u003e\u003c/strong\u003e. These biomolecules not only stabilize AgNPs by curtailing agglomeration but also play a crucial role in influencing their surface charge (Zeta potential), size, and shape\u003cstrong\u003e\u003csup\u003e25\u003c/sup\u003e\u003c/strong\u003e. Protein-capped nanoparticles enhance biocompatibility, offering a biodegradable, non-toxic alternative to synthetic stabilizers, and enable surface modifications for various medical applications\u003cstrong\u003e\u003csup\u003e26\u003c/sup\u003e\u003c/strong\u003e. The stabilization of nanoparticles by capping agents is mediated through various mechanisms, including steric hindrance, depletion stabilization, electrostatic interactions, hydration forces, and van der Waals forces\u003cstrong\u003e\u003csup\u003e27\u003c/sup\u003e\u003c/strong\u003e. Nitrate reductase primarily facilitates the bio-reduction of silver ions while associated proteins possibly including the enzyme itself may adsorb onto the nanoparticle surface functioning as capping agents that contribute to their stabilization\u003cstrong\u003e\u003csup\u003e28,29\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFunctionalization of AgNPs involves the modification of the nanoparticle surface with specific ligands, functional groups, or biomolecules to improve physicochemical properties and biological interactions\u003cstrong\u003e\u003csup\u003e30\u003c/sup\u003e\u003c/strong\u003e. This strategy improves stability, enables targeted delivery, enhances stability, and facilitates controlled drug release\u003cstrong\u003e\u003csup\u003e31\u003c/sup\u003e\u003c/strong\u003e. The synthesis of AgNPs functionalized with antibiotics holds potential medical applications, making the elucidation of the binding mechanisms on their surface crucial for further study\u003cstrong\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/strong\u003e. Conjugates of antibiotics and AgNPs may be considered an alternative treatment to resistant bacterial strains because combined formulations of antibiotics-nanoparticles not only reduce the dose of medicine but also minimize the chances of toxicity\u003cstrong\u003e\u003csup\u003e33\u003c/sup\u003e\u003c/strong\u003e. Multidrug-resistant (MDR) microorganisms are being targeted through the functionalized nanoparticles with less potent antibiotics to enhance their antimicrobial efficacy\u003cstrong\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/strong\u003e. β-lactamase-producing bacteria pose a significant challenge in modern medicine, as they produce enzymes that hydrolyze the β-lactam ring of antibiotics, thus rendering them ineffective\u003cstrong\u003e\u003csup\u003e35\u003c/sup\u003e\u003c/strong\u003e. These enzymes are characteristic of MDR strains, which severely reduce the efficacy of β-lactam antibiotics, including penicillin, ampicillin, and cephalosporin\u003cstrong\u003e\u003csup\u003e36\u003c/sup\u003e\u003c/strong\u003e. Addressing β-lactam resistance is essential for improving the therapeutic potential of antibiotic-conjugated nanoparticles\u003cstrong\u003e\u003csup\u003e37\u003c/sup\u003e\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn silico molecular docking has emerged as a pivotal tool in fundamental and applied biological research, enabling the rational investigation of molecular interactions\u003cstrong\u003e\u003csup\u003e38\u003c/sup\u003e\u003c/strong\u003e. These computational studies facilitate the elucidation of interaction mechanisms between functionalized nanoparticles and biomolecular targets, providing structural and energetic insights that complement experimental observations\u003cstrong\u003e\u003csup\u003e39\u003c/sup\u003e\u003c/strong\u003e. Advanced bioinformatics platforms allow for the prediction of binding affinities, identification of active site preferences, and assessment of structural compatibility among AgNPs capping proteins and antibiotics, thereby uncovering potential modes of action and synergistic effects\u003cstrong\u003e\u003csup\u003e40\u003c/sup\u003e\u003c/strong\u003e. Computational approaches provide in-depth insights into nanoparticle behavior and interactions within complex biological systems\u003cstrong\u003e\u003csup\u003e41\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThis study presents an integrated experimental and computational approach to investigate the role of fungal nitrate reductase in the green synthesis, capping, and functionalization of AgNPs produced by \u003cem\u003eTalaromyces funiculosus\u003c/em\u003e (SUMCC 22011). The novelty of this research lies in recognizing nitrate reductase not only as a biocatalyst for the biosynthesis of AgNPs but also as a capping agent. Ampicillin (Amp) was conjugated with the biosynthesized AgNPs in a defined stoichiometric ratio, resulting in a stable nano-antibiotic conjugate (Amp–AgNPs). Comprehensive characterization techniques, including ultraviolet–visible spectroscopy (UV–Vis), dynamic light scattering (DLS), Zeta potential analysis, Fourier transform infrared spectroscopy (FTIR), and high-resolution transmission electron microscopy (HR-TEM) confirmed the successful formation and structural integrity of ampicillin–AgNPs conjugate (Amp–AgNPs). The physicochemical stability of Amp–AgNPs was assessed under varying pH and temperature conditions. Molecular docking was utilized to model the active site architecture of nitrate reductase and its interactions with ampicillin, providing mechanistic insights into the formation of Amp–AgNPs conjugate. The antibacterial activity of Amp–AgNPs was assessed, and the associated morphological alterations in bacterial cells were confirmed by scanning electron microscopy (SEM). The β-lactamase inhibition ability of Amp–AgNPs was evaluated using the iodometric assay. This integrative experimental and computational approach provides a sustainable and innovative platform for AgNPs biosynthesis and functionalization for biomedical applications.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eFunctional verification of nitrate reductase in the biosynthesis and capping of AgNPs \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTalaromyces funiculosus \u003c/em\u003e(SUMCC 22011) is an endophytic fungus isolated from the wild medicinal plant\u003cem\u003e Euphorbia hirta \u003c/em\u003eand identified based on morphological characteristics and phylogenetic analysis, as reported in our previous study El deeb et al.\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of \u003cem\u003eT. funiculosus\u003c/em\u003e filtrates and the corresponding biosynthesized AgNPs revealed distinct protein profiles (Fig. 1). Fungal filtrates showed protein bands at 58, 75, and 100 kDa. A prominent band consistently observed at 58 kDa across all fungal filtrates and AgNPs derived from three independent fungal patches indicates the presence of a protein involved in the synthesis and capping of AgNPs. A colorimetric microplate-based assay for NR activity was performed to confirm the identity of the observed protein band. The results validated the enzymatic activity associated with nitrate reduction in the fungal filtrate and the AgNPs-capped protein. The NR activity of the fungal filtrates was 1.856 \u0026plusmn; 0.004 U/g biomass, and the activity increased to 2.407 \u0026plusmn; 0.006 U/g for the AgNPs, indicating 29.68 % enhancement following biosynthesis. The detection of enzymatic activity at the same molecular weight position in the SDS-PAGE further supports the presence of functionally active NR in both fractions. The results confirm that NR activity is preserved during AgNPs biosynthesis and remains catalytically active when associated with the nanoparticle surface.\u003c/p\u003e\n\u003cp\u003eSeveral studies have highlighted the involvement of proteins in AgNPs biosynthesis. For instance, \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e exhibited an 85 kDa protein band, identified as a stabilizing agent for AgNPs\u003cstrong\u003e\u003csup\u003e42\u003c/sup\u003e\u003c/strong\u003e. Similarly, extracellular proteins with molecular masses of 45 kDa were reported in \u003cem\u003eAspergillus niger\u003c/em\u003e cell filtrates\u003cstrong\u003e\u003csup\u003e28\u003c/sup\u003e\u003c/strong\u003e, while \u003cem\u003eAspergillus flavus\u003c/em\u003e secreted proteins of 32 and 35 kDa, in the culture filtrate and bound to AgNPs\u003cstrong\u003e\u003csup\u003e43\u003c/sup\u003e\u003c/strong\u003e. \u003cem\u003eTrichoderma asperellum\u003c/em\u003e was shown to produce protein bands at 70 and 55 kDa, which were directly linked to nitrate reductase activity and AgNPs biosynthesis\u003cstrong\u003e\u003csup\u003e44\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFor further confirmation that the 58 kDa band belongs to NR, A colorimetric assay was employed for the fungal filtrates and the AgNPs after drying at room temperature and suspended in ionized distilled water. To the best of our knowledge, this is the first quantitative study to report a significant enhancement in NR activity following the biosynthesis of AgNPs in a fungal system. Nitrate reductase is an enzyme produced by various microorganisms, plays a crucial role in the extracellular biosynthesis of AgNPs by catalyzing the reduction of silver ions to elemental silver via an NADH-dependent electron transfer mechanism\u003cstrong\u003e\u003csup\u003e45\u003c/sup\u003e\u003c/strong\u003e. While it typically facilitates the conversion of nitrate to nitrite, the capacity of the enzyme to mediate electron transfer is effectively utilized in AgNPs biosynthesis to reduce silver ions, leading to their nucleation and subsequent formation\u003cstrong\u003e\u003csup\u003e46\u003c/sup\u003e\u003c/strong\u003e. This mechanism is conserved across a diverse range of microbial taxa including fungi, yeasts, and bacteria\u003cstrong\u003e\u003csup\u003e14,47,48\u003c/sup\u003e\u003c/strong\u003e. Among filamentous fungi, \u003cem\u003eFusarium oxysporum\u003c/em\u003e has been extensively characterized for its NR-mediated nanoparticle synthesis, with reported enzymatic rates up to 220 nmol/h/mL\u003cstrong\u003e\u003csup\u003e49\u003c/sup\u003e\u003c/strong\u003e. Similarly, \u003cem\u003eAspergillus oryzae\u003c/em\u003e and \u003cem\u003eTrichoderma reesei\u003c/em\u003e have been shown to utilize nitrate reductase effectively in AgNPs biosynthesis\u003cstrong\u003e\u003csup\u003e15,50\u003c/sup\u003e\u003c/strong\u003e. Intra-genus variability in NR activity has also been documented among \u003cem\u003eAspergillus\u003c/em\u003e species, with \u003cem\u003eA. fumigatus\u003c/em\u003e exhibiting the highest activity, followed by \u003cem\u003eA. clavatus\u003c/em\u003e and \u003cem\u003eA. niger\u003c/em\u003e, while \u003cem\u003eA. flavus\u003c/em\u003e demonstrated comparatively lower enzymatic performance\u003cstrong\u003e\u003csup\u003e51\u003c/sup\u003e\u003c/strong\u003e. Other fungal genera have also displayed considerable NR activity. For instance, \u003cem\u003ePenicillium\u003c/em\u003e spp. exhibited significant enzymatic activity quantified at 270 nmol/h/mL\u003cstrong\u003e\u003csup\u003e52\u003c/sup\u003e\u003c/strong\u003e. Among yeasts, \u003cem\u003eCryptococcus laurentii\u003c/em\u003e and \u003cem\u003eRhodotorula glutinis\u003c/em\u003e showed NR activities of 266.56 nmol/h/mL and 216.85 nmol/h/mL, respectively\u003cstrong\u003e\u003csup\u003e48\u003c/sup\u003e\u003c/strong\u003e. In bacterial systems, \u003cem\u003eBacillus subtilis\u003c/em\u003e demonstrated nitrate reductase activity of 152 nmol/h/mL\u003cstrong\u003e\u003csup\u003e47\u003c/sup\u003e\u003c/strong\u003e, while crude metabolite extracts from \u003cem\u003eEscherichia coli\u003c/em\u003e displayed activity levels equivalent to 2.18 U/mL\u003cstrong\u003e\u003csup\u003e53\u003c/sup\u003e\u003c/strong\u003e. The significant increase in nitrate reductase activity in the AgNPs-associated fraction of \u003cem\u003eT. funiculosus\u003c/em\u003e highlights a functional integration between the enzyme and the nanoparticle matrix. This interaction enhances catalytic efficiency, suggesting a stable nano\u0026ndash;bio configuration with potential applications in developing high-performance bio-catalytic systems for therapeutic and industrial use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical and morphological validation of Amp\u0026ndash;AgNPs formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUV\u0026ndash;visible spectroscopy provided initial confirmation of the successful formation of Amp\u0026ndash;AgNPs conjugate. As shown in Fig. 2a, increasing the ampicillin concentration (0.2\u0026ndash;4.0 mg/mL) resulted in a gradual shift of the characteristic AgNPs SPR peak at 422.5 nm, along with the appearance of a secondary peak at 340.5 nm. These spectral changes indicate strong interactions between the \u0026beta;-lactam antibiotic and the nanoparticle surface. Similarly, varying the AgNPs concentration from 0.2 to 1.0 mg/mL (Fig. 2b) led to enhanced SPR intensity and peak shifts, supporting the concentration-dependent modulation of nanoparticle optical properties. As shown in Fig. 2c, under the optimized synthesis conditions (1.0 mg/mL AgNPs and 1.0 mg/mL ampicillin), the Amp\u0026ndash;AgNPs conjugate exhibited a distinct SPR peak at 340.5 nm. This characteristic peak confirms the formation of the novel hybrid nanostructure. The conjugate remained stable over 3 months at room temperature, as evidenced by unchanged UV\u0026ndash;visible spectra, indicating preserved structural integrity and colloidal stability throughout the storage period.\u003c/p\u003e\n\u003cp\u003eFunctionalization of AgNPs with biomolecules enhances their stability and provides properties such as target specificity, fluorescence, and antimicrobial activity, supporting their broad applicability in biomedical nanotechnology\u003cstrong\u003e\u003csup\u003e30\u003c/sup\u003e\u003c/strong\u003e. The AgNPs used for ampicillin functionalization were biosynthesized by \u003cem\u003eT. funiculosus\u003c/em\u003e and characterized by our previously study El deeb et al.\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e as spherical crystalline, stable (6 months), and mono-dispersed (PDI: 0.007), exhibiting SPR at 422.5 nm, average diameter of 34.32 nm, and Zeta potential of -18.41 mV. The pronounced 82 nm shift in Amp\u0026ndash;AgNPs UV\u0026ndash;visible spectra reflects significant change in the electronic environment and surface characteristics of the nanoparticles, supporting successful functionalization. Such molecular interactions promoted charge transfer and altered the electronic structure, particularly through transitions from non-bonding (n) orbitals to antibonding pi (\u0026pi;*) orbitals, ultimately leading to a blue shift in the SPR band, as described in the study by Onyangore et al.\u003cstrong\u003e\u003csup\u003e54\u003c/sup\u003e\u003c/strong\u003e. The noteworthy 82 nm shift sharply contrasts with the more minor shifts observed in previous studies, such as the shift from 408 nm to 427 nm upon ampicillin functionalization\u003cstrong\u003e\u003csup\u003e55\u003c/sup\u003e\u003c/strong\u003e, minimal shifts from 438 nm to the range of 421 \u0026ndash; 426 nm noted with cephalosporin conjugates\u003cstrong\u003e\u003csup\u003e56\u003c/sup\u003e\u003c/strong\u003e, and the shift from 438 nm to 477 nm occurring after conjugation with ciprofloxacin\u003cstrong\u003e\u003csup\u003e57\u003c/sup\u003e\u003c/strong\u003e. Other reports have shown SPR bands for silver-drug conjugates generally remaining within the 400\u0026ndash;450 nm range\u003cstrong\u003e\u003csup\u003e58\u003c/sup\u003e\u003c/strong\u003e, while ampicillin-functionalized AgNPs displayed an SPR at 390 nm\u003cstrong\u003e\u003csup\u003e59\u003c/sup\u003e\u003c/strong\u003e. In the direct synthesis of AgNPs using ampicillin as reducing and stabilizing agent, Khatoon et al.\u003cstrong\u003e\u003csup\u003e60\u003c/sup\u003e\u003c/strong\u003e reported an SPR peak at 406 nm, confirming ampicillin capability in nanoparticle synthesis however the peak remained within the expected range for AgNPs. The consistent SPR band at 340.5 nm for 3 months further confirms the long-term stability of the Amp\u0026ndash;AgNPs, demonstrating their suitability for potential therapeutic applications. Similar stability results were reported by Brown et al.\u003cstrong\u003e\u003csup\u003e61\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDLS analysis of synthesized Amp\u0026ndash;AgNPs displayed a size range of 22.2 to 39.46\u0026nbsp;nm, with an average diameter of 27.26 nm, while Zeta potential measurements indicated a negative surface charge of \u0026ndash;24.9 mV (Fig. 2d). The reduction in hydrodynamic size from 34.32 (AgNPs) to 27.26 nm following ampicillin conjugation is attributed to the active functional groups of ampicillin, which interact with and reorganize the nanoparticle surface, increase surface charge, and compress the stabilizing sheath. These modifications result in a smaller and more stable particle size, as evidenced by the increase in negative Zeta potential from \u0026ndash;18.6 to \u0026ndash;24.9 mV. Similar findings were reported by Lopez-Carrizales et al.\u003cstrong\u003e\u003csup\u003e62\u003c/sup\u003e\u003c/strong\u003e where the conjugation of ampicillin led to a reduction in AgNPs size from 8.57 to 4.01 nm. This decrease was attributed to ampicillin favoring the homogeneous dispersion of the nanoparticles and enhancing colloidal stability, as reflected by an increase in negative Zeta potential from \u0026ndash;40.01 to \u0026ndash;51.00 mV. Comparable effects have also been observed with tetracycline, where surface modification influences nanoparticle dispersion, surface charge, and overall colloidal stability\u003cstrong\u003e\u003csup\u003e63\u003c/sup\u003e\u003c/strong\u003e. In a study by Rogowska et al.\u003cstrong\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/strong\u003e functionalization of AgNPs with ampicillin resulted in an increase in particle size and a shift in zeta potential toward less negative values, indicating that the impact on colloidal stability may vary depending on the capping agents and synthesis conditions. In contrast, Adil et al.\u003cstrong\u003e\u003csup\u003e56\u003c/sup\u003e\u003c/strong\u003e observed larger hydrodynamic diameters in antibiotic-functionalized AgNPs compared to AgNPs, attributing the increase in size to the binding of antibiotic molecules to the nanoparticle surface.\u003c/p\u003e\n\u003cp\u003eFTIR spectroscopy further validated the conjugation between AgNPs and ampicillin (Fig. 2e). In the FTIR spectrum of pure ampicillin a prominent band appeared at 1736 cm⁻\u0026sup1;, corresponding to the carbonyl (C=O) stretching vibration. The disappearance of this band in the Amp\u0026ndash;AgNPs spectrum suggests that the carbonyl group of ampicillin is involved in the interaction with AgNPs. The bands observed at 1457 cm⁻\u0026sup1; and 1530 cm⁻\u0026sup1;, assigned to \u0026beta;-lactam ring vibrations in pure ampicillin, were not detected in the Amp\u0026ndash;AgNPs spectrum. The disappearance of these functional group signals supports the binding of the \u0026beta;-lactam group during the conjugation process. Previous studies have reported only shifts in hydroxyl and amide group regions upon antibiotic conjugation, supporting the role of these groups in stabilization and surface binding\u003cstrong\u003e\u003csup\u003e58,64\u003c/sup\u003e\u003c/strong\u003e. The FTIR spectrum of AgNPs synthesized directly using ampicillin showed a shift of the amine peak from 1605 to 1625 cm⁻\u0026sup1; while the \u0026beta;-lactam ring and other characteristic bands remained unchanged compared to pure ampicillin\u003cstrong\u003e\u003csup\u003e60\u003c/sup\u003e\u003c/strong\u003e. Also, Murei et al.\u003cstrong\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/strong\u003e observed no new functional groups upon conjugation, suggesting that the synthetic route significantly influences the surface chemistry and degree of interaction between AgNPs and antibiotics. In contrast, the presented data reveal distinct spectral features indicative of direct bonding that enhanced conjugation efficiency.\u003c/p\u003e\n\u003cp\u003eHR-TEM imaging provided direct visual evidence of the morphology and structural organization of the Amp\u0026ndash;AgNPs conjugate (Fig. 3a-c). The TEM images revealed that the AgNPs exhibited a well-defined spherical morphology surrounded by ampicillin molecules. This distinctive structural arrangement suggests effective adsorption or potential covalent interaction between AgNPs and ampicillin, which may contribute to enhanced functionality for diverse biomedical applications. No aggregation of AgNPs was observed and the calculated PDI of 0.037 indicates excellent stability and a highly mono-disperse distribution. The SAED pattern confirmed the retention of the crystalline nature of AgNPs, with no detectable alterations upon conjugation with ampicillin (Fig. 3d). These findings are consistent with previous reports\u003cstrong\u003e\u003csup\u003e59,61,65\u003c/sup\u003e\u003c/strong\u003e. The PDI of the conjugate was 0.037, indicating it is highly mono-disperse and confirming its excellent suitability for biomedical applications\u003cstrong\u003e\u003csup\u003e66\u003c/sup\u003e\u003c/strong\u003e. Although the PDI increased slightly from 0.007 for the AgNPs to 0.037 after conjugation, this minor rise is attributed to surface functionalization with ampicillin.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability of Amp\u0026ndash;AgNPs under variable pH and temperature conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUV\u0026ndash;Vis spectroscopic analysis demonstrated a pH-dependent variation in the optical properties of Amp\u0026ndash;AgNPs conjugate across a pH range of 1 to 13 (Fig. 4a). A prominent absorption peak at 340.5 nm was observed from highly acidic (pH 1) to moderately alkaline (pH 9) conditions, indicating a broad tolerance for pH variations. Elevated alkaline pH values (11\u0026ndash;13) caused significantly diminished or poorly resolved this peak. The dissociation of the Amp\u0026ndash;AgNPs complex at higher pH values was further supported by the appearance of distinct absorbance bands corresponding to free ampicillin (281.5 nm) and AgNPs (422.5 nm). Visual inspection revealed slightly aggregation or precipitation under strong acidic conditions after 4 hours of incubation (Fig. 4a). After 48 hours, the reaction mixture maintained the characteristic 340.5 nm peak, with reduced intensity, indicating time-dependent stability and some preservation of the conjugate even under extreme pH conditions. The Amp\u0026ndash;AgNPs displayed impressive resilience across a broad pH spectrum, particularly maintaining stability at pH 5.0. This finding is consistent with research on the stability of AgNPs biosynthesized by \u003cem\u003eT. funiculosus\u003c/em\u003e, which also demonstrated enhanced stability at pH 5.5\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e. Ampicillin, on the other hand, shows optimal stability at pH 7.5, experiencing over 70% degradation at pH 3.4 after 12 hours, underscoring the importance of a near-neutral pH for preserving its efficacy\u003cstrong\u003e\u003csup\u003e67,68\u003c/sup\u003e\u003c/strong\u003e. Therefore, the observed stability of the conjugates at various pH levels indicates that the conjugation with AgNPs aids in maintaining the structural integrity of ampicillin, even at the extremes of its natural stability range.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe UV\u0026ndash;Vis spectroscopy analysis of the thermal stability of Amp\u0026ndash;AgNPs was conducted across a temperature range of 5 \u0026deg;C to 55 \u0026deg;C (Fig. 4b). A consistent absorption peak at 340.5 nm was observed throughout the temperature gradient. The stability of this peak suggests that the bonding interaction between ampicillin and the AgNPs surface remains intact even at elevated temperatures. Visual inspection of the samples (inset) further supported the spectrophotometric results, showing no visible aggregation or precipitation across the temperature range (Fig. 4b). This evidence implies that Amp\u0026ndash;AgNPs maintain colloidal stability and structural integrity up to 55 \u0026deg;C. Regarding thermal tolerance, biosynthesized AgNPs showed optimal stability at elevated temperatures, particularly around 60 \u0026deg;C\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e. Correspondingly, the Amp\u0026ndash;AgNPs maintained their structural integrity at temperatures up to 55 \u0026deg;C. This stands in contrast to free ampicillin, which maintains over 90 % of its initial concentration for 48 hours only under refrigerated conditions (8 \u0026plusmn; 2 \u0026deg;C), and for up to 24 hours at 25 \u0026plusmn; 2 \u0026deg;C and 30 \u0026plusmn; 2 \u0026deg;C. However, at 37 \u0026plusmn; 2 \u0026deg;C, its stability falls below the 90% threshold within 12 hours, making it less suitable for use in warmer environments\u003cstrong\u003e\u003csup\u003e69,70\u003c/sup\u003e\u003c/strong\u003e. The enhanced thermal stability of the Amp\u0026ndash;AgNPs indicates that nanoparticle conjugation significantly improves the physicochemical resilience of ampicillin, potentially extending its efficacy in challenging storage or treatment conditions.\u003c/p\u003e\n\u003cp\u003eSimilarly, the colloidal stability of biogenic synthesized AgNPs formed stable complexes with ampicillin within a pH range of 6 to 8, with optimal stability observed at pH 7\u0026ndash;8. Additionally, enhanced adsorption efficiency was reported at elevated incubation temperatures ranging from 4 to 42 \u0026deg;C\u003cstrong\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/strong\u003e. The imipenem-AgNPs conjugate exhibited excellent stability, with no changes in absorbance peaks, color, visible aggregation, or clarity observed over three months of storage at 4 \u0026deg;C and 25 \u0026deg;C\u003cstrong\u003e\u003csup\u003e71\u003c/sup\u003e\u003c/strong\u003e. Also, the colloidal stability of biogenic AgNPs\u0026ndash;nisin conjugate was confirmed at pH 4 and 8, as no shift in the SPR band or decrease in its intensity was observed, indicating that the nanoparticles remained stable and did not undergo aggregation under the tested pH conditions\u003cstrong\u003e\u003csup\u003e72\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExploring the binding mechanism of ampicillin to nitrate reductase via in silico approaches\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3D structure of nitrate reductase was built using homology modeling based on the obtained homologous templates. The obtained 3D structure of nitrate reductase is shown in Fig. 5.\u003c/p\u003e\n\u003cp\u003eThe model consists of the molybdenum cofactor (MoCo) domain that binds the Moco where nitrate reduction occurs and this is the catalytic site center where nitrate binds and is reduced. Flavin adenine dinucleotide (FAD)-Binding domain which binds FAD for NAD(P)H-mediated electron transfer, where FAD accepts electrons from NADPH and transfers them to the MoCo center. Nitrate-binding pocket near the MoCo center, with conserved residues (e.g., Arg, His, or Ser) aiding in substrate orientation. Electron transfer pathways involving conserved cysteine residues coordinating with the MoCo centers. Potential phosphorylation sites (as seen in other fungal NRs) that regulate enzyme activity in response to nitrogen availability.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRamachandran plot (Fig. 6) showed that almost all \u0026beta;-sheets, right-handed \u0026alpha;-helices and left-handed \u0026alpha;-helices residues are found in the core regions (Favored Regions), with some residues in loops or flexible regions. too few residues (Gly, and Pro) appear in the disallowed regions (and this is acceptable due to large flexible loops of the model).\u003c/p\u003e\n\u003cp\u003eTo find out the most active pocket grid analysis implemented in DOCK 6 was performed and the most active pocket was used for docking. Pocket-Cavity Search within this structure showed that it has a large area shown as iso-surface in Fig. 7.\u003c/p\u003e\n\u003cp\u003eLargest cavity, consistent with the molybdenum cofactor (MoCo) binding site where nitrate reduction occurs (pocket 1 1089 \u0026Aring;\u0026sup3; - Catalytic MoCo Active Site, consists of His84, Arg89, likely stabilize the MoCo center (analogous to \u003cem\u003eAspergillus\u003c/em\u003e NR), Asp213, Asp221acts as proton donors for catalysis, Cys237, Trp238, which are redox-active residues for electron transfer, aromatic/hydrophobic residues (Phe18, Tyr230, Phe236) may position nitrate substrates, and disulfide potential (Cys237) suggests redox regulation. Pocket 2 (511 \u0026Aring;\u0026sup3; - FAD/NADPH-Binding Domain), this pocket consists of a positively charged residues (Arg2, Lys3, Lys5, Arg124) \u0026ndash; Binds NADPH phosphate groups, aromatic amino acids (Trp117, Tyr121) which Stack with FAD\u0026rsquo;s isoalloxazine ring and Asp122 that may hydrogen-bond to NADPH. Pocket 3 (173 \u0026Aring;\u0026sup3; - Allosteric Regulation Site) which has charged cluster (Arg192, Glu194, Lys199) - Potential phosphorylation site (e.g., by fungal kinases) and Trp203 mediates protein-protein interactions. Pocket 4 (154 \u0026Aring;\u0026sup3; - Proton Relay/Substrate Channel), composed of acidic residues (Asp164, Glu165, Asp254) and His163 act as proton transfer during catalysis and proton shuttle, respectively. Pocket 5 (142 \u0026Aring;\u0026sup3; - Redox-Sensing Disulfide) with Cys41 and\u0026nbsp; Tyr52 work as potential disulfide bond formation under oxidative stress and electron transfer roles. Pocket 6 (129 \u0026Aring;\u0026sup3; - Substrate Access Channel), with charged (Asp46, Lys47) and aromatic (Tyr53, Trp117) residues guides nitrate into Pocket 1. Pocket 7 (122 \u0026Aring;\u0026sup3; - Heme-Binding Interface), Arg146, Asp151 and Tyr147, Tyr150 which are Heme propionate coordination and stabilization. Pocket 8 (118 \u0026Aring;\u0026sup3; - Solvent-Exposed Electrostatic Patch), basic residues (Arg25, Lys29, Arg35) may anchor NR to membranes or partner proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 2D structure of the lowest energy docked structure was shown in Fig. 8, where ampicillin was fitted in the active site pocket of the modeled nitrate reductase model.\u003c/p\u003e\n\u003cp\u003eIt is obvious from this model that ampicillin formed HB network with residues His84, Arg89, Val92, and Gln95 where His84 and Arg89 are likely near the catalytic site (common in nitrate reductases for redox reactions). Hydrogen bonds stabilize the \u0026beta;-lactam ring of ampicillin, potentially interfering with substrate binding. In addition, it had electrostatic and hydrophobic interaction with residues Val14, Serl5, Thr16, Val9l, Pro93, Ser100, Leu152, Phe236, Cys237, Trp238.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis may lead to disruption of the enzyme\u0026rsquo;s charge balance near the active site. The hydrophobic cluster (Phe236, Trp238, Leu152) suggests ampicillin binds in a partially buried pocket, shielding it from solvent. The docking score was found to be -8.4 kcal/mol.\u003c/p\u003e\n\u003cp\u003eHis84 \u0026amp; Arg89 are likely near the catalytic site, which is critical for nitrate reduction (common in NADH-dependent nitrate reductase). Ampicillin\u0026rsquo;s \u0026beta;-lactam ring is stabilized by these H-bonds\u003c/p\u003e\n\u003cp\u003eCompetitive or mixed-type inhibition, where ampicillin directly competes with nitrate or induces conformational changes. Arg89 (positively charged) may interact with ampicillin\u0026rsquo;s anionic carboxylate group. Ser15, Thr16 (polar) could stabilize the drug\u0026rsquo;s polar groups. Phe236, Trp238, Leu152, Val91, Pro93 form a hydrophobic pocket, burying ampicillin\u0026rsquo;s nonpolar regions (e.g., phenyl group). Cys237 may contribute to binding via weak S/\u0026pi; interactions with the \u0026beta;-lactam ring. The hydrophobic cluster shields ampicillin from solvent, increasing residence time in the binding site. Disruption of the charge balance near the active site (due to Arg89 interaction) may impair redox chemistry.\u003c/p\u003e\n\u003cp\u003eA score of -8.4 kcal/mol suggests strong binding (typical high-affinity inhibitors range from -7 to -12 kcal/mol).\u003c/p\u003e\n\u003cp\u003eThe docking results confirmed the interaction between ampicillin and the enzyme-capped AgNPs, indicating the formation of a stable hybrid conjugate. This discovery opens new avenues for exploring the potential of drugs to conjugate with silver nanoparticles through nitrate reductase, presenting a promising strategy for improved drug delivery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial activity and ultra-structural disruption of \u0026beta;-Lactamase-producing bacteria induced by Amp\u0026ndash;AgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmp\u0026ndash;AgNPs demonstrated significant antibacterial activity against \u0026beta;-lactamase-producing bacterial strains (Fig. 9 and Table 1). At low concentrations (5\u0026ndash;10 \u0026micro;g/mL), Amp\u0026ndash;AgNPs were produced measurable inhibition zones ranging from 7.7 to 10.7 mm. At a concentration of 50 \u0026micro;g/mL, Amp\u0026ndash;AgNPs resulted in inhibition zones measuring 27.3 \u0026plusmn; 0.3 mm for \u003cem\u003eE. coli\u003c/em\u003e, 25.0 \u0026plusmn; 1.0 mm for \u003cem\u003eE. faecalis\u003c/em\u003e, and 26.3 \u0026plusmn; 0.3 mm for \u003cem\u003eS. aureus\u003c/em\u003e. These measurements significantly exceeded those recorded for AgNPs (17.7 \u0026plusmn; 0.3, 15.7 \u0026plusmn; 0.3, and 16.7 \u0026plusmn; 0.3 mm, respectively) and the positive control (15.3 \u0026plusmn; 0.3, 13.3 \u0026plusmn; 0.3, and 16.3 \u0026plusmn; 0.3 mm, respectively). Ampicillin was ineffective across all tested concentrations.\u003c/p\u003e\n\u003cp\u003eThe MIC values for Amp\u0026ndash;AgNPs conjugate were significantly lower than AgNPs, ampicillin, and the positive control (Table 1). Amp\u0026ndash;AgNPs MICs were 3.3 \u0026plusmn; 0.3, 4.7 \u0026plusmn; 0.3, and 4.3 \u0026plusmn; 0.3 \u0026micro;g/mL for \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eE. faecalis\u003c/em\u003e, and\u003cem\u003e S. aureus\u003c/em\u003e, respectively. However, AgNPs MIC values were 8.7 \u0026plusmn; 0.3, 7.7 \u0026plusmn; 0.3, and 8.3 \u0026plusmn; 0.3 \u0026micro;g/mL, respectively while for the positive control were 17.3 \u0026plusmn; 0.3, 15.7 \u0026plusmn; 0.3, and 9.3 \u0026plusmn; 0.3 \u0026micro;g/mL, respectively. Ampicillin demonstrated no inhibitory effect even at concentrations of 100 \u0026micro;g/mL. These results suggest that ampicillin conjugation with AgNPs presenting a promising strategy for combating \u0026beta;-lactamase-producing pathogens.\u003c/p\u003e\n\u003ctable width=\"763\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"106\"\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial strains\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"216\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEscherichia\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e \u003cem\u003ecoli\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"217\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"224\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"106\"\u003e\n\u003cp\u003e\u003cstrong\u003eConcentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003e\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e\u003cstrong\u003eAmp\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u003cstrong\u003eAgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u003cstrong\u003ePositive control\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmp\u0026ndash;AgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmp\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e\u003cstrong\u003eAgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e\u003cstrong\u003ePositive control\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmp\u0026ndash;AgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmp\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e\u003cstrong\u003eAgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e\u003cstrong\u003ePositive control\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e\u003cstrong\u003eAmp\u0026ndash;AgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e\u003cstrong\u003e5 \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"6\" width=\"43\"\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition zone diameter (mm)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e8.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e7.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e7.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e\u003cstrong\u003e10 \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e7.3 \u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e10.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e7.0\u0026plusmn;0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e11.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e8.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e7.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e10.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e\u003cstrong\u003e20 \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e8.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e7.0\u0026plusmn;0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e14.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e8.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e7.0\u0026plusmn;0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e14.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e12.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e12.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e13.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e\u003cstrong\u003e30 \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e11.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e9.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e18.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e12.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e8.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e16.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e13.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e13.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e17.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e\u003cstrong\u003e40 \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e14.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e12.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e22.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e13.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e10.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e19.7\u0026plusmn;1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e15.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e14.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e21.0\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e\u003cstrong\u003e50 \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e17.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e15.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e27.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e15.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e13.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e25.0\u0026plusmn;1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e16.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e16.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e26.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"106\"\u003e\n\u003cp\u003e\u003cstrong\u003eMIC \u003c/strong\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003e\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e8.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e17.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e3.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e7.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e15.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e4.7\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"39\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e8.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e9.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e4.3\u0026plusmn;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Inhibitory zone diameter and MIC of of Amp\u0026ndash;AgNPs against \u0026beta;-lactamase-producing bacteria (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy provided compelling evidence of structural damage of various \u0026beta;-lactamase-producing \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eE.\u003c/em\u003e \u003cem\u003efaecalis\u003c/em\u003e, and \u003cem\u003eS.\u003c/em\u003e \u003cem\u003eaureus\u003c/em\u003e (Fig. 10). The untreated control cells displayed well-preserved morphology with smooth, intact surfaces, showing healthy cellular integrity. Cells treated with ampicillin maintained their architecture, exhibiting minimal morphological changes, which confirmed their resistance to \u0026beta;-lactam antibiotics. Treatment with AgNPs alone resulted in moderate membrane disruption, pore formation, and increased surface roughness. In contrast, the positive control caused partial structural damage, including membrane wrinkling and deformation. Cells exposed to the Amp\u0026ndash;AgNPs conjugate showed extensive morphological alteration, characterized by pore formation, membrane rupture, collapse of cellular structures, and leakage of intracellular contents. This level of damage was consistent across all tested strains. These results indicated that conjugating ampicillin with AgNPs effectively overcomes \u0026beta;-lactamase-mediated resistance by combining targeted membrane disruption with intracellular delivery of the antibiotic.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmp\u0026ndash;AgNPs synthesized in this study exhibited significantly enhanced antibacterial activity against \u0026beta;-lactamase-producing \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eE. faecalis\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e. This synergistic enhancement underscores the potential of AgNPs as effective carriers for conventional antibiotics, particularly in combating resistant bacterial strains. Similar results have been documented in the previous studies. For instance, Alfahad et al.\u003cstrong\u003e\u003csup\u003e73\u003c/sup\u003e\u003c/strong\u003e documented inhibition zones of 14.17 mm for Amp\u0026ndash;AgNPs against \u003cem\u003eSalmonella typhi\u003c/em\u003e using 3% aqueous AgNPs. Similarly, Rogowska et al.\u003cstrong\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/strong\u003e reported inhibition zones for Ag-CGG-Ampicillin conjugates synthesized using \u003cem\u003eActinomycetes\u003c/em\u003e CGG 11n, with zones ranging from 5.0 \u0026plusmn; 0 mm to 12 \u0026plusmn; 0 mm across different pathogens. Brown et al.\u003cstrong\u003e\u003csup\u003e61\u003c/sup\u003e\u003c/strong\u003e showed that Amp\u0026ndash;AgNPs achieved complete eradication of resistant \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e within 4\u0026ndash;6 hours, compared to 6\u0026ndash;8 hours for AgNPs alone, highlighting the conjugate\u0026rsquo;s enhanced bactericidal kinetics and its potential to prevent biofilm formation. Ibraheem et al.\u003cstrong\u003e\u003csup\u003e57\u003c/sup\u003e\u003c/strong\u003e reported that a conjugate of AgNPs, polyethylene glycol, and ciprofloxacin produced remarkably larger inhibition zones of 36 mm for \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, 39 mm for \u003cem\u003eS. aureus\u003c/em\u003e, and 40 mm for \u003cem\u003eSerratia marcescens\u003c/em\u003e compared to the individual components. Also, Jalil et al.\u003cstrong\u003e\u003csup\u003e64\u003c/sup\u003e\u003c/strong\u003e demonstrated that AgNPs conjugated with amoxicillin exhibited superior antimicrobial activity against \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, and methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e, reinforcing the value of nanoparticle-mediated drug delivery systems.\u003c/p\u003e\n\u003cp\u003eThe results of the MIC assays further substantiate the superior antibacterial efficacy of the Amp\u0026ndash;AgNPs conjugate. The results highlight a synergistic interaction between ampicillin and AgNPs, enabling effective bacterial inhibition at significantly lower concentrations. Similarly, Khatoon et al.\u003cstrong\u003e\u003csup\u003e60\u003c/sup\u003e\u003c/strong\u003e documented MICs of 18.75 \u0026micro;g/mL and 9.375 \u0026micro;g/mL for Amp\u0026ndash;AgNPs against ampicillin-sensitive \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, respectively and MICs of 10 \u0026micro;g/mL and 3 \u0026micro;g/mL for ampicillin-resistant counterparts. The MIC values were lower than the MICs reported for chemically synthesized AgNPs, which range from 280 to 720 \u0026micro;g/mL. Also, Rogowska et al.\u003cstrong\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/strong\u003e demonstrated enhanced antibacterial efficacy using AgNPs functionalized with ampicillin, with MICs of 3.125 \u0026micro;g/mL for \u003cem\u003eP. aeruginosa\u003c/em\u003e, 25 \u0026micro;g/mL for \u003cem\u003eE. coli\u003c/em\u003e, and 6.25 \u0026micro;g/mL for \u003cem\u003eK. pneumoniae\u003c/em\u003e. Antibiotic\u0026ndash;AgNPs conjugates enhance antimicrobial efficacy with lower MICs and offering a more effective therapeutic strategy\u003cstrong\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/strong\u003e. The effectiveness of the conjugation strategy is demonstrated by imipenem\u0026ndash;AgNP conjugate, which exhibited markedly lower MICs (2\u0026ndash;16 \u0026micro;g/mL) against 200 \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates compared to imipenem alone (64 to \u0026gt;512 \u0026micro;g/mL) and AgNPs (4\u0026ndash;32 \u0026micro;g/mL), highlighting their enhanced potency\u003cstrong\u003e\u003csup\u003e74\u003c/sup\u003e\u003c/strong\u003e. AgNPs functionalized with glucosamine demonstrated enhanced activity, with MICs as low as 8 \u0026micro;g/mL against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003cstrong\u003e\u003csup\u003e75\u003c/sup\u003e\u003c/strong\u003e. These findings corroborate our current results and consistently support that conjugation of AgNPs with conventional antibiotics markedly reduces MIC values even in resistant strains.\u003c/p\u003e\n\u003cp\u003eThe enhanced antibacterial efficacy of Amp\u0026ndash;AgNPs conjugates can be attributed to multiple synergistic mechanisms involving the physicochemical properties of AgNPs and the biological activity of the antibiotics. Conjugation significantly enhances antibacterial activity against Gram-positive and Gram-negative bacteria, including MDR strains by enabling bioactive molecules to retain and even amplify their function when bound to the surface of AgNPs\u003cstrong\u003e\u003csup\u003e76\u003c/sup\u003e\u003c/strong\u003e. This enhanced potency likely results from the hydroxyl and amide groups of ampicillin and the AgNPs surface, which leads to improved cellular uptake and intracellular retention. Scanning electron microscopy analysis in the current study revealed pronounced morphological disruptions in \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eE. faecalis\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e treated with Amp\u0026ndash;AgNPs, including membrane rupture, cytoplasmic leakage, and complete structural collapse, effects that were not observed with either ampicillin or AgNPs alone. AgNPs enhance antibiotic efficacy by increasing bacterial membrane permeability and facilitating greater intracellular drug uptake\u003cstrong\u003e\u003csup\u003e33\u003c/sup\u003e\u003c/strong\u003e. AgNPs disrupt bacterial membranes by binding to sulfur-containing proteins, compromising structural and enzymatic integrity, while also interfering with protein synthesis and DNA replication, ultimately causing irreversible cellular damage and death\u003cstrong\u003e\u003csup\u003e57\u003c/sup\u003e\u003c/strong\u003e. Conjugation enhances adhesion and membrane penetration through van der Waals and electrostatic interactions, disrupts DNA and metabolic processes, generates reactive oxygen species, and impairs electron transport, leading to bacterial cell death\u003cstrong\u003e\u003csup\u003e77\u003c/sup\u003e\u003c/strong\u003e. By integrating membrane disruption, intracellular interference, and enhanced delivery, the conjugates present a robust strategy for next-generation antimicrobial therapies, highlighting the need for further validation through in vivo studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of Amp\u0026ndash;AgNPs conjugate on \u0026beta;-lactamases activity in clinical bacterial strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tested strains (\u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eE. faecalis\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e) were confirmed as \u0026beta;-lactamase producers, indicated by a distinct color change from blue to colorless in the iodometric assay, reflecting the enzymatic hydrolysis of free ampicillin (Fig. 11). In contrast, AgNPs and Amp\u0026ndash;AgNPs maintained the blue color, demonstrating the absence of \u0026beta;-lactam ring hydrolysis. The positive control displayed a pale blue color, consistent with \u0026beta;-lactamase activity. The sustained blue coloration observed in the Amp\u0026ndash;AgNPs treatment confirmed the stability of the conjugated form, suggesting that the \u0026beta;-lactam ring is shielded from enzymatic degradation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe iodometric assay results revealed clear evidence of \u0026beta;-lactamase activity in \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eE. faecalis\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e, as indicated by the color change from blue to colorless upon ampicillin treatment. This confirms the susceptibility of conventional \u0026beta;-lactam antibiotics to enzymatic hydrolysis, a major mechanism underlying bacterial resistance\u003cstrong\u003e\u003csup\u003e78,79\u003c/sup\u003e\u003c/strong\u003e. In contrast, samples treated with Amp\u0026ndash;AgNPs retained the blue coloration, indicating that this complex prevent \u0026beta;-lactamase activity and maintained ampicillin structural integrity. Moreover, Amp\u0026ndash;AgNPs demonstrated superior stability compared to the positive control, suggesting a protective role conferred by nanoparticle conjugation\u003cstrong\u003e\u003csup\u003e80\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eA plausible explanation lies in the alteration of steric and electronic characteristics of ampicillin upon conjugation with AgNPs. These modifications may prevent optimal substrate alignment within the \u0026beta;-lactamase active site, impairing catalytic function through steric hindrance or electronic redistribution\u003cstrong\u003e\u003csup\u003e81\u003c/sup\u003e\u003c/strong\u003e. This inhibition mechanism resembles competitive inhibition or substrate mimicry, a concept well-documented in the context of \u0026beta;-lactamase inhibitors\u003cstrong\u003e\u003csup\u003e82\u003c/sup\u003e\u003c/strong\u003e. Unlike classical inhibitors such as clavulanic acid, sulbactam, and tazobactam, which act through covalent acylation and irreversible enzyme inactivation\u003cstrong\u003e\u003csup\u003e83\u003c/sup\u003e\u003c/strong\u003e. This aligns with emerging efforts to develop non-\u0026beta;-lactam inhibitors capable of resisting enzymatic degradation across a broad spectrum of \u0026beta;-lactamase classes\u003cstrong\u003e\u003csup\u003e84,85\u003c/sup\u003e\u003c/strong\u003e. By preserving the \u0026beta;-lactam core and conferring enzymatic protection, Amp\u0026ndash;AgNPs address the structural and functional limitations of existing \u0026beta;-lactamase inhibitors\u003cstrong\u003e\u003csup\u003e86\u003c/sup\u003e\u003c/strong\u003e. Due to the increasing prevalence of \u0026beta;-lactamase-mediated resistance and the decreasing effectiveness of current inhibitors\u003cstrong\u003e\u003csup\u003e87\u003c/sup\u003e\u003c/strong\u003e, hybrid nano-antibiotic platforms offer a promising new approach for antimicrobial drug design, providing renewed hope to combat resistance and enhance antibiotic effectiveness.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eFunctional SDS-PAGE profiling and microplate assay confirmation of nitrate reductase involved in the biosynthesis and capping of AgNPs by \u003cem\u003eT. funiculosus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolation and characterization of the proteins present in \u003cem\u003eT. funiculosus\u003c/em\u003e filtrate and the corresponding biosynthesized AgNPs were conducted using SDS-PAGE as described by Laemmli\u003cstrong\u003e\u003csup\u003e88\u003c/sup\u003e\u003c/strong\u003e. Three distinct fungal filtrates were prepared from various patches of \u003cem\u003eT. funiculosus\u003c/em\u003e, each utilized for synthesizing AgNPs as outlined by El deeb et al.\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e. SDS-PAGE analysis was performed on each fungal filtrate and its respective biosynthesized AgNPs to confirm protein presence and ensure the reproducibility of the results. Electrophoresis was conducted using a 12% SDS-polyacrylamide resolving gel within an OmniPAGE Mini Vertical Protein Electrophoresis System, UK, at a constant voltage of 100 V. After electrophoresis, the gel was stained with Coomassie Brilliant Blue dye and visualized using a gel imaging system (GelLITE, Cambridge, UK). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), with a molecular weight of 38 kDa, was run alongside the samples as an internal reference to validate the accuracy and reliability of molecular weight estimation. Also, a standard molecular weight marker (Promega, ADV8491, USA) was included to facilitate precise protein size determination.\u003c/p\u003e\n\u003cp\u003eTo confirm the identity of the protein band detected by SDS-PAGE as nitrate reductase for the fungal filtrate and the biosynthesized AgNPs, a colorimetric microplate-based nitrate reductase assay was performed using a commercial kit (Nitrate Reductase Microplate Assay Kit, Cat. No. ORB219870, Biorbyt, USA). The assay quantified nitrate reductase activity based on the enzymatic reduction of nitrate to nitrite by chromogenic azo-compound detection under standard assay conditions. One unit of nitrate reductase activity was defined as the amount of enzyme required to generate 1 μmol of nitrite per hour. The assay was conducted in triplicate according to the manufacturer's instructions, and enzyme activity was expressed as units per gram (U/g) of fungal biomass. Absorbance was measured at 540 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Finland), and the data were analyzed using SkanIt Software for Microplate Readers RE, version 6.0.1.6. The detection of enzymatic activity corresponding to the 58 kDa protein band confirmed the functional presence of nitrate reductase in the fungal filtrate and the AgNPs, indicating its catalytic role in the biosynthesis and capping of AgNPs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation and physicochemical characterization of Amp–AgNPs conjugate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmpicillin solutions (0.2, 0.5, 1.0, 2.0, 3.0, and 4.0 mg/mL) were prepared in distilled water and mixed with biosynthesized AgNPs (1.0 mg/mL) to form Amp–AgNPs conjugate. Conversely, varying concentrations of AgNPs (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) were mixed with ampicillin (1.0 mg/mL) to identify the optimal nanoparticle concentration for conjugation. The optimal concentration of ampicillin and AgNPs for functionalization was determined using UV–visible spectroscopy. The reaction mixtures were incubated at room temperature for 24 hours and repeated in triplicate for reproducibility. The successful formation of the Amp–AgNPs conjugates was confirmed using multiple characterization techniques.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo monitor the surface Plasmon resonance (SPR) shifts, UV–visible spectra were conducted using a UV–visible spectrophotometer (JAS.CO V-770, Japan) within the wavelength range of 200 – 800 nm. Deionized water was used as a blank to adjust the baseline. Measurements were repeated after six months of preservation at room temperature to evaluate the long-term stability of the Amp–AgNPs.\u003c/p\u003e\n\u003cp\u003eThe size distribution and average hydrodynamic diameter of Amp–AgNPs were assessed by DLS, while Zeta potential (surface charge) was measured with a NANOTRAC WAVE II, Germany to evaluate colloidal stability.\u003c/p\u003e\n\u003cp\u003eFTIR spectroscopy was conducted to explore the functional groups and potential interactions involved in the formation of the Amp–AgNPs conjugate. The spectra of pure ampicillin, biosynthesized AgNPs, and the conjugated complex were recorded using a Platinum-ATR FTIR spectrometer (Bruker Alpha, Germany) in the range of 4000 – 400 cm⁻¹. This spectral range facilitated the identification of characteristic functional groups and the assessment of possible chemical bonding or interactions between ampicillin and AgNPs.\u003c/p\u003e\n\u003cp\u003eHigh-resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) analyses were performed to investigate the morphology, interactions, and crystalline structure of the Amp–AgNPs conjugate. These analyses were conducted using a JEOL JSM 100CX TEM instrument (JEOL Ltd., Japan) at the Electron Microscope Unit of Cairo University, Egypt. TEM imaging was used to evaluate the shape, size distribution, and dispersion of the nanoparticles, as well as to provide insight into the surface interaction and conjugation of ampicillin with AgNPs. The polydispersity index (PDI) was calculated from the measured particle sizes to assess the uniformity and dispersity of the nanoparticles\u003cstrong\u003e\u003csup\u003e66\u003c/sup\u003e\u003c/strong\u003e. SAED was utilized to confirm the crystalline nature of the nanoparticles and to evaluate any potential structural changes or aggregation that may have arisen during the conjugation process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability assessment of Amp–AgNPs under physiological pH and temperature conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stability of the Amp–AgNPs conjugate was evaluated under varying pH and temperature conditions to assess its physicochemical robustness following the method described by Rogowska et al.\u003cstrong\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/strong\u003e. For pH stability, aliquots were adjusted to pH values of 1, 3, 5, 7, 9, 11, and 13 using 0.1 M HCl or 0.1 M NaOH and incubated at room temperature for 48 hours. Thermal stability was assessed by incubating separate aliquots at 5, 15, 25, 35, 45, and 55 °C for 48 hours in a controlled water bath. UV–Vis absorbance spectra (200 – 800 nm) were recorded to monitor changes in the characteristic peak at 340.5 nm, indicative of stable Amp–AgNPs conjugation. Shifts or reductions in peak intensity were used to evaluate conjugate integrity. The test was conducted in triplicates, and visual inspections were performed to detect signs of aggregation, turbidity, or precipitation providing complementary evidence of colloidal stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComputational investigation of ampicillin interaction with nitrate reductase enzyme capped AgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNitrate reductase enzyme’s sequence of\u003cem\u003e\u0026nbsp;T. funiculosus\u0026nbsp;\u003c/em\u003ewas obtained from the UniProt database (ID: F1CF56). The obtained sequence was used to search for a homology models using BLAST database, highly similar sequences with known crystal structures were used for multiple alignment and threading of the query enzyme (PDB IDs: 1SOX, 2A99, 2A9B, 2A9D, 2BIH, 2BII, 3HBG, 3HBP, 3HC2, 3R18, 3R19).\u003c/p\u003e\n\u003cp\u003eThe 3D model of the enzyme was then designed using SWISSPdb\u003cstrong\u003e\u003csup\u003e89\u003c/sup\u003e\u003c/strong\u003e, where the template models were fitted together before threading the query sequence. This process enhances the modeling and decreases the clashes of the new model. The obtained model was validated using Ramachandran plot\u003cstrong\u003e\u003csup\u003e90\u003c/sup\u003e\u003c/strong\u003e. Furthermore, energy minimization was performed using GROMOS96 force-field\u003cstrong\u003e\u003csup\u003e91\u003c/sup\u003e\u003c/strong\u003e. The obtained model was compared with the AI generator of Aphafold2, where the RMS value was found to be 1.55 Å. The obtained 3D model was used as a receptor for docking ampicillin substrate which was obtained from the PubChem database.\u003c/p\u003e\n\u003cp\u003eDocking protocols were created based on prior studies\u003cstrong\u003e\u003csup\u003e92\u003c/sup\u003e\u003c/strong\u003e using USCF DOCK6\u003cstrong\u003e\u003csup\u003e93\u003c/sup\u003e\u003c/strong\u003e. The AM1-BBC method was used in DOCK6 to add charge to the protein and ampicillin. Utilizing a probe radius of 1 point 4 Å, the molecular surface was created. Using spheres within 9 Å of the native ligand, which is encircled by a box with a margin of 5 Å, the active site of a target was identified. Polar hydrogens were added to the targets in Vina, and then Kollman charges were added. In the meantime, ligand preparation involved the use of Gestaiger charges. To generate the center coordinate, a grid box was applied at a spacing of 1 Å, centered on the native ligand position. After comparing the ten conformations produced, docked structures were selected from the obtained conformations based on the binding energy (kcal/mol) of these conformations.\u003c/p\u003e\n\u003cp\u003eLIGPLOT\u003csup\u003e+\u003c/sup\u003e (version 2.2) was used to visualize and determine the interactions between ligand and receptor's amino acids. It examines the intricate receptor-ligand structure's 2D hydrogen bond (HB) interaction. It provides a graphical representation of HB, hydrophobic bonds, and their bond lengths at the ideal docking position\u003cstrong\u003e\u003csup\u003e94\u003c/sup\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntegrated evaluation of antibacterial activity and morphological disruption by Amp–AgNPs conjugate against β-lactamase-producing bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibacterial susceptibility test and the minimum inhibitory concentration (MIC) determinations were conducted as described by El deeb et al.\u003cstrong\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/strong\u003e. The assays were performed against Gram-negative and Gram-positive β-lactamase-producing bacteria, including \u003cem\u003eEscherichia coli\u003c/em\u003e (SUMCC 22014), \u003cem\u003eEnterococcus faecalis\u0026nbsp;\u003c/em\u003e(SUMCC 22015), and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (SUMCC 22016). All strains were isolated from clinical samples obtained from the Sohag University Microbial Culture Collection, Egypt. Five concentrations (5, 10, 20, 30, 40, and 50 µg/mL) were tested for: ampicillin, biosynthesized AgNPs, ampicillin/sulbactam (2:1 ratio), and the Amp–AgNPs conjugate. All experiments were performed in triplicate to ensure accuracy.\u003c/p\u003e\n\u003cp\u003eThe antibacterial activity was evaluated using the disk diffusion method on Mueller–Hinton agar (MHA). Sterile filter paper disks were loaded with the designated concentrations of each tested compound. The selected concentration range for Amp–AgNPs was based on the typical daily intake of silver from natural sources approximately 0.4 – 27 µg/day\u003cstrong\u003e\u003csup\u003e95\u003c/sup\u003e\u003c/strong\u003e, thus ensuring relevance to environmentally realistic exposure levels. Ampicillin served as the negative control to confirm bacterial resistance to ampicillin, while ampicillin/sulbactam was used as the positive control. The formed plates were incubated at 37 °C for 24 hrs followed by measuring the formed inhibition zone in millimeter (mm).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe MIC for ampicillin, AgNPs, positive control, and Amp–AgNPs were determined for the tested pathogens by broth microdilution method using 96-well microtiter plates according to the principles described by Kowalska-Krochmal \u0026amp; Dudek-Wicher\u003cstrong\u003e\u003csup\u003e96\u003c/sup\u003e\u003c/strong\u003e. Concentrations ranging from 1 to 100 μg/mL were prepared to facilitate MIC determination. Resazurin, a blue dye that becomes pink upon reduction by metabolically active cells, was employed as an indicator of bacterial viability. From the above assay, an inoculum was taken from each well that showed no visual growth and spotted on MHA plates to validate the MIC assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the morphological alterations and potential molecular interactions induced by Amp–AgNPs, SEM analysis was performed on the tested bacterial cells before and after 6 hours of exposure to the MIC of each tested compound. For ampicillin, a concentration of 100 μg/mL was used. The analysis included untreated control cells, cells treated with ampicillin, AgNPs, positive control, and the Amp–AgNPs conjugate. Morphological analysis was performed according to the protocol described by Singh et al.\u003cstrong\u003e\u003csup\u003e97\u003c/sup\u003e\u003c/strong\u003e using a JEOL JSM-5400LV SEM (JEOL Ltd., Japan) operated at 15 – 25 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of Amp–AgNPs conjugate on β-lactamases enzymatic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe iodometric method was employed to evaluate β-lactamase activity, following the protocols described by Sharma et al.\u003cstrong\u003e\u003csup\u003e98\u003c/sup\u003e\u003c/strong\u003e and Aliyu et al.\u003cstrong\u003e\u003csup\u003e99\u003c/sup\u003e\u003c/strong\u003e. A loopful of a dense 24-hour culture of \u003cem\u003eE.\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e, \u003cem\u003eE.\u003c/em\u003e \u003cem\u003efaecalis\u003c/em\u003e, and \u003cem\u003eS.\u003c/em\u003e \u003cem\u003eaureus\u003c/em\u003e from MHA was mixed with 1.0 mL of a 10000 µg/mL solution of ampicillin, AgNPs, ampicillin/sulbactam (β-lactamase inhibitor used as a positive control), and the Amp–AgNPs conjugate. The samples were incubated at room temperature for 30 minutes with gentle agitation at 15-minute intervals. After the incubation period, two drops of a 1 % soluble starch solution and one drop of iodine solution were added to the mixture and gently shaken. A change in color from blue to colorless indicated positive β-lactamase activity, whereas the persistence of the blue color signified a negative result. Results were recorded within 10 minutes, and all tests were performed in triplicate for confirmation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll experiments were carried out in triplicate and the results were presented as mean ± standard deviation. The data were statistically analyzed by one-way analysis of variance (ANOVA) using XLSTAT version 2023.2.0 software\u003cstrong\u003e\u003csup\u003e100\u003c/sup\u003e\u003c/strong\u003e. Differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 were regarded as statistically significant.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully demonstrated the pivotal role of nitrate reductase in the biosynthesis and stabilization of AgNPs by \u003cem\u003eT. funiculosus\u003c/em\u003e, with the enzyme retained and catalytically active on the nanoparticle surface. The formation of the Amp–AgNPs conjugate was confirmed through spectroscopic and microscopic analyses, demonstrating strong interactions between ampicillin and AgNPs that enhance physicochemical stability. The conjugate exhibited broad pH and thermal tolerance that is essential for biomedical applications. In silico modeling of nitrate reductase revealed its active site architecture and demonstrated strong binding affinity with ampicillin. Amp–AgNPs significantly exceeded ampicillin and AgNPs in inhibiting β-lactamase-producing bacterial strains. Also, Amp–AgNPs protects the antibiotic’s β-lactam ring from enzymatic degradation and causing pronounced bacterial ultra-structural damage. SEM analysis indicated extensive damage to the membranes of β-lactamase-producing bacteria, confirming the enhanced bactericidal efficacy through a synergistic mechanism involving membrane disruption and antibiotic delivery. The obtained results confirm the potential of NR-mediated AgNPs as nano-carriers for antibiotic delivery, offering a novel approach to overcome multidrug-resistant infections and advance antimicrobial nanobiotechnology. Future studies should explore in vivo efficacy, ensure biosafety, and extend this biomolecular strategy to a broader range of drug delivery applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. Nitrate reductase enzyme\u0026rsquo;s sequence of \u003cem\u003eTalaromyces funiculosus\u003c/em\u003e was obtained from the UniProt database (ID: F1CF56). The other datasets used and/or analyzed during the current study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr. Mohamed A. Morsy (Al-Azhar Virology Research Center, Faculty of Medicine, Al-Azhar University, Cairo, Egypt) for his valuable assistance in protein detection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors registered have made fundamental, direct, and intellectual contributions to the work discussed in this publication. Conceptualization: B.A.E and G.G.F.; methodology: M.S.B., G.G.F. and M.I.; data collection: G.G.F. and M.I.; data curation: B.A.E, M.S.B., G.G.F., and M.I.; manuscript preparation, review, and editing: B.A.E, G.G.F., M.S.B., and M.I. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpen access funding provided by The Science, Technology \u0026amp; Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupplementary information file for this paper is available online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to B.A.E.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiddiqi, K. 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(2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.xlstat.com/en\u003c/span\u003e\u003cspan address=\"https://www.xlstat.com/en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biosynthesis mechanism, Amp–AgNPs characterizations, In silico docking, Iodometric assay, β-lactamase inhibition, Drug delivery","lastPublishedDoi":"10.21203/rs.3.rs-7042236/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7042236/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, mechanistic analysis using SDS-PAGE identified a 58 kDa protein as the biomolecule responsible for AgNPs biosynthesis and capping. Colorimetric microplate-based assay confirmed the protein as nitrate reductase, with structural preservation evidenced by a 29.7% activity increase (1.856 to 2.407 U/g) following AgNPs synthesis. Functionalization of AgNPs with ampicillin was indicated by SPR shift from 422.5 to 340.5 nm and disappearance of the FTIR band at 1736 cm⁻¹. Amp–AgNPs conjugate was stable (3 months), spherical, mono-dispersed (PDI: 0.037), average diameter of 27.26 nm, Zeta potential of − 24.9 mV, and showed broad pH (1–9) and thermal (5–55°C) stability. Docking analysis revealed strong binding of ampicillin within the nitrate reductase catalytic pocket through hydrogen bonding, hydrophobic, and electrostatic interactions, confirming the conjugate stability. Amp–AgNPs (50 µg/mL) exhibited potent antibacterial activity against β-lactamase-producing bacteria with inhibition zones of 27.3 mm (Escherichia coli), 25.0 mm (Enterococcus faecalis), and 26.3 mm (Staphylococcus aureus), and MICs of 3.3, 4.7, and 4.3 µg/mL, respectively. SEM analysis revealed severe structural changes, indicating synergistic membrane disruption and antibiotic delivery. Amp–AgNPs showed potent β-lactamase inhibition in the iodometric assay, supporting their potential as alternative therapeutic agents. Future studies should focus on in vivo efficacy and expand this strategy to additional drug delivery applications.\u003c/p\u003e","manuscriptTitle":"Biomolecular strategy for designing antibiotic–silver nanoparticles conjugate via nitrate reductase mediated β-lactamase inhibition with molecular docking insights","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 12:18:49","doi":"10.21203/rs.3.rs-7042236/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-10T12:01:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-28T07:37:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204350395590865859535808692096158414980","date":"2025-08-25T09:27:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240409956798806583183022948931042318184","date":"2025-07-26T13:37:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-24T06:31:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313577122355654286525760044610871354694","date":"2025-07-24T06:19:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137139385312590709270093409994585947974","date":"2025-07-14T03:31:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T02:59:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-14T02:56:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-08T04:00:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-04T10:44:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-04T02:14:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b07704ba-427c-4a0a-bf4b-42207fe7d909","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51523131,"name":"Biological sciences/Biochemistry"},{"id":51523132,"name":"Biological sciences/Biotechnology"},{"id":51523133,"name":"Biological sciences/Drug discovery"},{"id":51523134,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-01-05T16:02:05+00:00","versionOfRecord":{"articleIdentity":"rs-7042236","link":"https://doi.org/10.1038/s41598-025-30539-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-03 15:58:21","publishedOnDateReadable":"January 3rd, 2026"},"versionCreatedAt":"2025-07-16 12:18:49","video":"","vorDoi":"10.1038/s41598-025-30539-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-30539-8","workflowStages":[]},"version":"v1","identity":"rs-7042236","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7042236","identity":"rs-7042236","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}