Combined experimental and in silico elucidation of KP27 endolysin reveals a phage derived antibacterial with pH and thermal robustness

Introduction

Phage endolysins are highly specific peptidoglycan hydrolases that are encoded by bacteriophages at the end of the phage lytic cycle to split critical bonds in the bacterial cell wall peptidoglycan, which results in quick bacterial lysis and the release of newly formed phage progeny^1,2. The identification of endolysins comes from the observation that phage lysates also have the ability to lyse and kill bacteria. Thus, to find a lysing agent, scientists have discovered and characterized proteins known as endolysins. These proteins have appeared to be attractive alternatives and have become a prominent focus of attention in bacterial control research³. The peptidoglycan layer in the bacterial cell wall is a supramolecular assembly critical for safeguarding bacteria. Biochemically, it consists of repeating units of the disaccharides N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), which are linked by peptide bridges, creating a strong network². Owing to their high specificity, mechanism of action, potential to be engineered and manipulated, lack of antibiotic resistance, rapid bactericidal activity, and potential to evade antibiotic resistance mechanisms, endolysins are being intensely researched as potential antibacterial agents for a range of applications, including human and animal health, food safety, and biocontrol⁴. The phage KP27 endolysin is a 131-amino acid protein with a molecular mass of 14.7 kDa. It belongs to the VanY domain-containing family, although its functional activity differs from that of preliminary bioinformatics predictions. While in silico analyses suggested D-alanyl-D-alanine carboxypeptidase activity, experimental validation revealed L-alanyl-D-glutamate endopeptidase specificity. This enzymatic action cleaves the peptide bond between L-alanine and D-glutamic acid in the peptidoglycan stem peptide, effectively lysing bacterial cell walls. The gene encoding the KP27 endolysin was extracted from the KP27 phage, which infects Klebsiella pneumoniae, and its recombinant protein was produced in 2017⁵. Among the phages that infect gram-negative bacteria, the phages KP15 and KP27 have the most advanced lytic system. This system consists of four proteins, namely, holin, antholin, spanin, and endolysin, with over 99% similarity between the proteins of the two phages. For example, in the KP27 endolysin, glutamine replaces the glutamate found in the KP15 endolysin^5,6.

One of the main factors in the application of proteins and enzymes is their stability during production and storage, including their thermal stability, pH stability, and remaining catalytic activity⁷. To our knowledge, the KP27 endolysin is highly stable under various environmental conditions, such as high temperature and pH. This makes it a promising candidate for further investigation, especially given the limited research conducted on this particular endolysin compared with other endolysins. Solving the 3D structure of a protein provides a blueprint for its function, role in disease, and engineering for therapeutic or biotechnological applications. It is a foundation of modern biology and medicine⁸. However, according to the literature review, little structural information is available, especially concerning the 3D structure of KP27, which is currently available for use as an endolysin. Protein structure prediction can be either homology-based or de novo modeling. AlphaFold2, on the other hand uses de novo modeling through deep learning and multiple sequence alignment to predict protein structures without explicit use of templates and can model accurately even novel folds⁹. The current study aims to describe the antimicrobial activity of the bacteriophage-derived endolysin KP27 as a new therapeutic option, with an emphasis on its lytic activity against both gram-negative (E. coli) and gram-positive (S. aureus) pathogens across physiologically relevant pH and temperature gradients. To complement these functional assays, we also utilized in silico structural prediction via comparative modeling to determine the tertiary structure of KP27, followed by molecular docking simulations to determine its binding thermodynamics and substrate interaction motifs (Fig. 1).

Results

Cloning, expression, and purification of recombinant endolysin KP27

To enable recombinant production of endolysin KP27, the KP27 gene was amplified and cloned and inserted into the pET28a(+) expression vector at the NcoI and XhoI restriction sites, which are downstream of the T7 promoter and ribosome binding site (Fig. 2a). After transformation into E. coli and protein expression induction, KP27 was purified from cell lysates via Ni²⁺-NTA affinity chromatography. The purity and molecular weight of the recombinant protein were examined via SDS‒PAGE (Fig. 2b). Intense bands were present in elution fractions 2, 3, and 4 at approximately 15 kDa, which is consistent with the predicted protein size of KP27, indicating both efficient expression and effective purification of KP27 with minimal contaminating proteins.

MIC, MBC, and Disc Diffusion Analyses

The MIC is the lowest concentration of KP27 that shows visible inhibition of bacterial growth following incubation, and the MBC is the lowest concentration that results in a ≥ 99.9% decrease in viable bacterial cells, indicating bactericidal activity. As shown in Table 1, KP27 displayed an MIC of 8 μg/mL against S. aureus, indicating effective inhibition of growth at relatively low doses. The MBC was 32 μg/mL, implying that a fourfold-fold greater concentration is needed to cause complete killing of bacteria. For E. coli, a relatively high MIC value of 16 μg/mL was observed, indicating relatively low susceptibility to KP27. The MBC against E. coli was 64 μg/mL, which was four times greater than the MIC, implying that higher concentrations are needed to exert bactericidal activity against this gram-negative strain.

Table 1 Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and inhibition zone diameters of endolysin KP27 for S. aureus and E. coli. The MIC and MBC values are in μg/mL, and the inhibition zone diameters are in millimeters (mm). The data revealed greater antimicrobial activity of KP27 against gram-positive S. aureus than gram-negative E. coli.

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The antimicrobial properties of the endolysin were also tested via a disc diffusion assay against S. aureus and E. coli on 100 mm agar plates. As presented in Table 1, for both bacterial species, the diameter of the zone of inhibition around the disc was measured to estimate the lytic effect. The S. aureus disc yielded a clear and well-defined inhibition zone that measured 20 mm in diameter, reflecting strong antibacterial activity against this gram-positive bacterium. For E. coli, however, the zone of inhibition around the disc was significantly smaller, measuring 14 mm in diameter, reflecting relatively decreased susceptibility.

Antibacterial activity of endolysin KP27

The antibacterial properties of endolysin KP27 were tested against both gram-negative (E. coli) and gram-positive (S. aureus) bacteria over a concentration range of 0–8 μg/mL. As shown in Fig. 3A, endolysin KP27 displayed concentration-dependent increases in bactericidal activity against both bacteria. Moderate bactericidal activities, with activity levels of approximately 20% to 60% for both E. coli and S. aureus, were noted at low concentrations (0–2 μg/mL). However, a significant difference in susceptibility emerged at high endolysin concentrations. Whereas E. coli exhibited a gradual increase in bactericidal activity to a maximum of approximately 80% at 8 μg/mL, S. aureus showed an abrupt increase in susceptibility such that its bactericidal activity surpassed 100% at 4 μg/mL and was sustained at this level at 8 μg/mL. These data reveal that the endolysin KP27 is potently active against both gram-positive and gram-negative bacteria but more so against S. aureus. The disparity in maximal activity presumably stems from differences in the structure of the two bacterial cell envelopes, with the outer membrane of E. coli serving as a partial barrier to endolysin-induced lysis.

Turbidity reduction assay

The lytic activity of endolysin KP27 against S. aureus and E. coli was examined quantitatively by measuring the decrease in turbidity of the bacterial suspensions over a period of time (Fig. 3B). Both strains were treated with KP27 at 2 × MIC, 16 μg/mL for S. aureus and 32 μg/mL for E. coli, with the addition of 0.5 mM EDTA for E. coli to permeabilize the outer membrane. The results are plotted as the percentage of the initial OD600 with respect to untreated controls. As shown in Fig. 3B, a marked and rapid reduction in turbidity was noted for both bacterial species upon endolysin treatment. Within the initial 10 min, the OD600 of the S. aureus and E. coli suspensions decreased to approximately 60% and 70% of the initial value, respectively, reflecting significant cell lysis. This lytic activity was sustained over the subsequent 20 min, with both strains falling below 25% of their original turbidity after 30 min. The decline in turbidity reached a plateau from this point on, with no significant further decrease noted up to 60 min, indicating that maximal lysis was attained within the first 30 min of exposure. Statistical analysis indicated that the decreases in turbidity at each time point were highly significant compared with those of the control (p < 0.05 to p < 0.001).

Peptidoglycan degradation by endolysin KP27

Figure 4 shows the time course of the lytic activity of endolysin KP27 on cell wall peptidoglycan, which was measured as changes in optical density at 600 nm (OD600) over a 150-min incubation. In the control group (untreated, squares), the OD600 values were consistent throughout the experiment, with no appreciable lysis or decrease in cell wall integrity. In contrast, KP27-treated samples (circles) presented a pronounced, progressive decrease in the OD600, indicating effective peptidoglycan degradation and resulting in cell lysis. The decrease in the OD600 was noticeable as early as 15 min after treatment, with a statistically significant difference from that of the control (p < 0.05). The lytic effect increased in severity over time, with the KP27 group displaying a strong and sustained decrease in turbidity, which was lowest at 150 min (p < 0.01). The steady and significant decrease in the OD600 in the presence of KP27 compared with the untreated control highlights the strong enzymatic activity of KP27 against bacterial cell wall peptidoglycan.

pH stability profile of endolysin KP27

The pH-dependent lytic activity of endolysin KP27 was evaluated by measuring the residual turbidity of E. coli and S. aureus suspensions after preincubation of KP27 at 2 × MIC at a pH range of 3–11 (Fig. 5a). The results are expressed as percentages of the initial OD600, with lower values corresponding to greater lytic efficacy. Both bacterial species presented high residual turbidity at acidic pH values (3–5), reflecting the low lytic activity of KP27 under these conditions. As the pH approached neutrality, a dramatic reduction in turbidity occurred, corresponding to increased enzymatic activity. The lowest turbidity values, and hence maximal lytic activity, were obtained at pH 8 for S. aureus (44% of initial OD600) and at pH 8–9 for E. coli (59–60% of initial OD600). These data point to the wide pH tolerance of KP27, with optimal lytic activity under neutral to mildly alkaline conditions. At more alkaline pH values (10–11), partial activity loss was detected for both strains, as reflected by increased turbidity. Interestingly, S. aureus showed a more abrupt decrease in turbidity at neutral and mildly alkaline pH values than did E. coli, revealing a somewhat greater susceptibility to KP27 under these conditions. Statistical analysis revealed that the turbidity reduction at pH values of 6–9 was highly significant compared with that under acidic and strongly alkaline conditions (p < 0.01, p < 0.001). These data illustrate that KP27 retains strong and broad-spectrum lytic activity over a wide pH range, with high activity under conditions relevant to physiological and environmental applications.

Thermal stability profile of endolysin KP27

The thermal stability of the endolysin KP27 was assessed through measurement of the residual turbidity of E. coli and S. aureus suspensions upon treatment with KP27 at 2 × MIC after preincubation of the enzyme at temperatures ranging from 40 to 80 °C. Turbidity was quantified at OD600 and expressed as a percentage of the initial value; hence, lower turbidity is indicative of greater lytic activity of KP27. As presented in Fig. 5B, KP27 displayed high lytic activity after incubation at 40 °C and 50 °C, as demonstrated by the comparatively low turbidity values (~ 50–60% of the initial OD600) for both bacteria. This implies that the enzyme remains very active at these temperatures. With increasing incubation temperature to 60 °C and 70 °C, there was a gradual rise in turbidity, indicating a moderate decline in enzymatic activity; nevertheless, KP27 still preserved significant lytic activity, with turbidity values below 90% of the initial OD600. At 80 °C, a further increase in turbidity was observed for E. coli and S. aureus (close to 90% of the initial OD600), indicating an appreciable loss of lytic activity at this high temperature. Even so, the enzyme retained partial activity under these conditions as well. These observations indicate that KP27 manifests strong thermal stability, with high lytic activity up to 50 °C and moderate activity at temperatures as high as 70 °C. The thermal robustness underscores the potential of KP27 for applications where enzyme stability at elevated temperatures is needed.

Hemolytic activity of KP27 on red blood cells

The hemolytic activity of KP27 was assessed by incubating sheep red blood cells (RBCs) with increasing concentrations of endolysin (0–100 μg/mL). The degree of hemolysis was measured spectrophotometrically and quantified as a percentage, providing insight into the cytotoxic capacity of KP27 for human cells. The hemolytic capacity of endolysin KP27 was assessed by incubating sheep red blood cells (RBCs) with increasing concentrations of KP27 (0–100 μg/mL) and measuring hemolysis as a percentage of total lysis (Fig. 6). At all the concentrations tested, KP27 caused minimal hemolysis, with values consistently below 5%, even at the highest test concentration (100 μg/mL). Representative images of the assay supernatants also demonstrate the lack of visible hemolysis.

In silico characterization of KP27

AlphaFold-based prediction of the KP27 protein structure

To assess the structural characteristics of KP27, five high-confidence structural models were generated via AlphaFold (Fig. 7a). AlphaFold modeling of endolysin KP27 revealed a highly conserved core domain (residues 10–70) with excellent multiple sequence alignment (MSA) coverage (40–60% identity) and high per-residue confidence (pLDDT > 98%). Conversely, lower coverage at the N- and C-termini suggests more variable and less conserved regions (Fig. 7b). The per-residue confidence score (pLDDT) is consistently high across the core, peaking at approximately 98%, which indicates reliable structural predictions.

Structural superimposition and active site mapping of modeled KP27 with 2MXZ and 5OPZ

The AlphaFold structural model was superimposed on the experimentally determined structures 2MXZ, and 5OPZ. In Fig. 8, the predicted model is colored blue, its active site residues are red, the DALI-identified templates are green, and their active site residues are cyan. In the first superimposition (Fig. 8a), the AlphaFold model was aligned with 2MXZ. This identified the conserved residues His62, Asp69, and His127, reflecting the structural conservation of the active site in the AlphaFold prediction. The alignment of the AlphaFold model with 5OPZ (Fig. 8b) confirmed the correspondence of His62, Asp69, and His127 (AlphaFold) with His68, Asp75, and His123 (5OPZ). The residue Asp120 present in 5OPZ lacked a structural equivalent in the AlphaFold model and was hence excluded from consideration during downstream docking. On the whole, these structural superimpositions allow the precise identification of functionally important residues in the predicted models and attest to their reliability for use in docking studies and functional annotation.

Comparative molecular docking scores of KP27 models and reference peptidoglycan hydrolases against peptidoglycan-derived ligands

Molecular docking of two predicted endolysins, KP27, with the top-ranked analogs 2MXZ and 5OPZ, which are annotated as L-alanyl-D-glutamate peptidases, was carried out to determine the structural and functional similarity of KP27 with these characterized enzymes. The binding energy values calculated by AutoDock Vina are presented in Table 2. Among all the ligands screened, NAM-L-alanyl-D-isoglutamine showed the most favorable binding energies for all protein models, with values of -7.37 kcal/mol for AlphaFold-modeled KP27, -6.106 kcal/mol for 2MXZ, and -7.128 kcal/mol for 5OPZ. These consistently low energies point toward its potential as the most stable and specific ligand in these structural frameworks.

Table 2 Results of molecular docking (binding affinities in kcal/mol) of KP27 protein models and reference endolysins (2MXZ, 5OPZ) with diverse peptidoglycan-related ligands. For the KP27 model, NAM-L-alanyl-D-isoglutamine exhibited the most favorable binding energy (-7.37 kcal/mol), suggesting the strongest interaction. Similarly, 2MXZ and 5OPZ also displayed the best binding with NAM-L-alanyl-D-isoglutamine, with scores of -6.106 kcal/mol and -7.128 kcal/mol, respectively. Docking scores are presented in kcal/mol, where more negative values indicate stronger predicted ligand‒protein interactions. Essentially, NAM-L-alanyl-D-isoglutamine proved to be the best-performing ligand on several proteins. As 2mxz and 5opz are peptidoglycan hydrolases that act on certain peptide bonds of bacterial cell wall structures, the positive docking interactions indicate that the predicted KP27 also has a high level of structural homology in its active site and binding pockets with these enzymes.

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Moreover, PLIP analysis revealed that the interaction of the modeled endolysin KP27 with its cell wall substrate NAM-L-alanyl-D-isoglutamine is characterized by 11 important hydrogen bonds, which are mainly established by residues such as ARG41, GLN46, and TYR71, which play major roles in ligand binding via polar interactions. Furthermore, 2 hydrophobic interactions (TRP110, PHE122) and 1 salt bridge (HIS127 with a carboxylate) stabilize the ligand‒receptor complex, indicating a tight and specific binding mode (Fig. 9).

Circular dichroism analysis of the KP27 protein secondary structure

To characterize the secondary structure alteration of KP27 under various pH conditions, a circular dichroism (CD) experiment was performed. As shown in Fig. 10, at pH 8, the CD spectrum shows pronounced negative ellipticity peaks at approximately 208 nm and 222 nm, indicative of a well-preserved alpha-helical secondary structure. At pH 6.0 and pH 10.0, the spectra revealed a reduced ellipticity intensity, reflecting partial disruption of the secondary structure of endolysin KP27.

To provide a clear illustration, the CD spectral data were converted into percentages of the secondary structure and are summarized in Table 3.

Table 3 Effects of pH on the secondary structure composition of the KP27 protein. The percentage composition of secondary structure elements—alpha-helix, beta-sheet, and random coil/irregular—of the KP27 protein measured at different pH values (6.0, 8.0, and 10.0). At pH 8.0, the alpha-helix content reached its highest value of 55%, indicating an increased helical structure compared with those at pH 6.0 and 10.0. The beta-sheet content modestly increased from 14% at pH 6.0 to 18% at pH 8.0 and slightly decreased to 17% at pH 10.0. Moreover, the percentage of random coil or irregular structures is lower at pH 8.0 (27%) than at pH 6.0 (49%) and pH 10.0 (44%), suggesting a more ordered secondary structure at neutral to slightly basic pH values. These changes highlight how the structural conformation of KP27 is influenced by the environmental pH. The values are expressed as the mean percentages with their corresponding standard deviations.

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Assessment of 3D conformational dynamics in endolysin KP27 by fluorescence

In addition to evaluating the secondary structural changes, detailed fluorescence analyses were carried out to thoroughly examine the modifications and dynamics occurring within the tertiary structure of KP27, providing deeper insight into how its overall three-dimensional conformation responds to varying environmental conditions. The findings from the intrinsic and ANS fluorescence analyses are shown in Figs. 11 and 12, respectively. As shown in Fig. 11, intrinsic fluorescence emission spectra of endolysin KP27 revealed pH-dependent conformational changes. The maximal fluorescence intensity at pH 8.0 indicates a stable, native protein structure with buried tryptophan residues. The reduced fluorescence at pH values above or below 8.0 suggests partial unfolding and increased tryptophan exposure to the solvent, reflecting diminished structural stability.

The ANS fluorescence emission spectra of endolysin KP27 demonstrated pH-dependent changes in response to hydrophobic surface exposure. The highest fluorescence intensity at pH 6.0 indicates maximal exposure of hydrophobic patches, likely due to partial unfolding or loosening of the tertiary structure under acidic conditions. Intermediate fluorescence at pH 9.0 and 10.0 suggests moderate hydrophobic exposure linked to conformational changes in alkaline environments. The lowest intensities at pH 7.0 and 8.0 reflect a compact, native-like conformation with buried hydrophobic cores and minimal ANS binding (Fig. 12). These results highlight pH-sensitive structural alterations in KP27, with optimal folding near neutral to slightly alkaline pH and destabilization under acidic and strongly alkaline conditions.

Discussion

The present work provides a comprehensive characterization of recombinant KP27 endolysin, with an emphasis on its potential as a broad-spectrum antimicrobial agent against both gram-positive S. aureus and gram-negative E. coli. In addition to functional studies, the structural attributes of KP27 were also examined through molecular modeling and docking against peptidoglycan ligands, with important implications for its molecular function. For functional assays, KP27 was produced as a recombinant protein. As inferred from the MIC, MBC, and disc diffusion test data (Table 1), KP27 exhibited strong antimicrobial activity against both S. aureus and E. coli, with increased potency against S. aureus. The lower MIC and MBC values and the larger inhibition zone obtained for S. aureus reveal that KP27 inhibits and kills this gram-positive bacterium more effectively. Conversely, the higher MIC and MBC and smaller inhibition zone for E. coli reflect comparatively lower susceptibility, which is consistent with the established barrier function of the gram-negative outer membrane in restricting the access of lytic enzymes. The MIC and MBC values of the KP27 endolysins fall within their typical ranges for effective endolysins against these bacteria. Its activity against both gram-positive and gram-negative organisms unequivocally differentiates it from many phage lysins, which typically show strong activity against gram-positive species only. In comparison, highly active gram-positive endolysins such as PlyC exhibit MICs as low as 0.014–0.03 μg/mL against Streptococcus pyogenes¹⁰, and LysGH15, another well-characterized phage endolysin, has MICs of 8–32 μg/mL against several staphylococcal species, including S. aureus, S. epidermidis, S. hemolyticus, and S. hominis. Therefore, the MIC range of LysGH15 is similar to that of KP27 against S. aureus, and its antibacterial potency against staphylococci appears to be similar¹¹. Although the activity of LysGH15 is mostly against various staphylococci, KP27, with broader activity encompassing gram-negative bacteria, is an added advantage in its therapeutic potential. Nevertheless, like KP27, most of these enzymes typically lack inherent activity against gram-negative bacteria without assistance from membrane-permeabilizing agents. Moreover, the MIC value of KP27 is typically comparable to that reported for various natural and engineered endolysins against these bacterial species. For example, ZAM-CS, a new chimeric endolysin with activity against methicillin-resistant S. aureus (MRSA), has similar MIC values, approximately 10 µg/mL, and causes rapid killing of bacteria within 10 min, similar to the bactericidal speed of KP27¹². Evaluation of the bactericidal effect revealed that the KP27 endolysin had specific dose-dependent killing activity, with complete lysis of S. aureus and E. coli at concentrations ≥ 4 μg/mL. This activity was rapid, with a decrease in bacterial turbidity of > 50% within 10 min of exposure to twice the minimum inhibitory concentration (2 × MIC) for both microorganisms. KP27’s fast lysis kinetics, with > 50% turbidity reduction within 10 min at 2 × MIC for both bacteria, indicate highly effective enzymatic activity, comparable to that of advanced engineered molecules such as ZAM-CS, which also exhibit rapid killing times.

Furthermore, the KP27 endolysin is a peptidoglycan hydrolase that displays enzymatic specificity as an endopeptidase, targeting the peptide stem of bacterial peptidoglycan by specifically cleaving the bond between L-alanine and D-glutamic acid residues. This was evidenced by turbidimetric assays of the extracted peptidoglycan layer. The time-dependent mode of lysis shows that KP27 effectively hydrolyzes peptidoglycan, causing cell wall disruption and eventual bacterial lysis. The pH stability of the endolysin KP27 was also determined by measuring its residual lytic activity against E. coli and S. aureus over a pH range from 3 to 11 via a turbidity reduction assay. It displayed a pH stability profile with optimal lytic activity at neutral to slightly alkaline pH values (approximately pH 7–9) against both E. coli and S. aureus. Under such conditions, KP27 shows high lytic efficiency and causes a drastic decrease in bacterial turbidity (OD600). On the other hand, under extremely acidic (pH 3–4) and alkaline (pH 10–11) conditions, KP27 lytic activity is diminished, indicative of reduced killing of bacteria. Compared with other endolysins, many of which also exhibit optimal activity close to neutral pH, the profile of KP27 more closely matches those enzymes that are optimally active under physiological conditions. For example, LysMP, an endolysin with activity against lactic acid bacteria, exhibits optimal lytic activity at pH 6 and stability from pH 4 to 8 for prolonged times (up to 48 h)¹³. Similarly, certain gram-negative-targeting endolysins, improved by fusion with antimicrobial peptides, have stable enzymatic activity in the pH range of 5–10. Endolysins employed for decontamination of Salmonella typhimurium, E. coli, and Listeria monocytogenes also remain active in the pH range of 4–10¹⁴. This profile indicates that although KP27 is extremely effective under physiological conditions, its effectiveness may be compromised in highly acidic or alkaline environments, which is important for the design of therapeutic applications. Thermal stability was assessed by preincubating KP27 at a range of temperatures (40 to 80 °C) and subsequently conducting a turbidity assay against both bacterial species. KP27 endolysin exhibits considerable thermal stability, maintaining greater than 50% of its original lytic activity against both E. coli and S. aureus at 50 °C, with activity increasing further and approaching original levels at higher temperatures between 70 and 80 °C. This high level of thermal tolerance is statistically significant and demonstrates strong enzymatic functionality under heat stress conditions. Compared with other well-studied endolysins, the thermal stability profile of KP27 is competitive but differs within the diverse range exhibited among phage lysins. For example, the chimeric endolysin ZAM-MSC retains nearly complete activity from 4 to 42 °C for up to 24 h and demonstrates appreciable thermostability within this range but is not reported at temperatures above 42 °C¹⁵. The streptococcal multimeric endolysin PlyC exhibits poor thermal stability; its PlyCA catalytic subunit loses activity precipitously at elevated temperatures, although its PlyCB binding subunit itself remains stable up to approximately 90 °C, reflecting a partially thermolabile character that limits overall thermostability¹⁶. Conversely, extremophilic endolysins such as Ts2631 from Thermus scotoductus exhibit remarkable thermostability, with activity after 2 h at 95 °C and a melting temperature (Tm) of approximately 99.8 °C, indicating excellent heat resistance¹⁷. Likewise, the PhiKo endolysin from Thermus thermophilus has a melting temperature of approximately 91.7 °C and retains activity against extremophiles, indicating robust thermal resilience¹⁸. Other gram-negative endolysins, such as PlyB221 and PlyP32, exhibit stable activity up to temperatures of 50 °C and 45 °C, respectively, which is within the moderate thermal stability range that is common for many lysins applied in biocontrol scenarios¹⁹. Endolysins fused with antimicrobial peptides tend to remain stable from 4 to 65 °C, indicating a wide range of usability, although their thermostability is sometimes lower than that of extremophilic enzymes²⁰. In summary, the capacity of KP27 to retain and enhance activity at temperatures as high as 70–80 °C is among the most thermally resilient enzymes. Although it does not achieve the ultrahigh thermostability of specialist thermophilic endolysins such as Ts2631 or PhiKo, it outperforms endolysins with narrower thermal ranges such as the ZAM-MSC and PlyC holoenzymes. applications.

To determine the potential cytotoxicity and hemolytic ability of KP27, its impact on red blood cells (RBCs) was investigated as part of the safety analysis. KP27 displayed negligible hemolytic activity across all concentrations. The assay revealed no notable increase in hemolysis with increasing concentrations of KP27, demonstrating that KP27 does not jeopardize red blood cell membrane integrity even at the highest concentration tested.

Recent developments in computational protein structure prediction, such as AlphaFold, have allowed very accurate, high-resolution modeling of protein conformations⁹. In the absence of experimentally determined structural information available in the Protein Data Bank (PDB) and in vitro structure determination via techniques such as nuclear magnetic resonance (NMR) or X-ray crystallography for KP27, we used AlphaFold and then compared the predicted structure with high-resolution experimental structures of homologous proteins (PDB IDs: 2MXZ and 5OPZ). Molecular docking analyses with the cell wall substrate NAM-L-alanyl-D-isoglutamine were also carried out to investigate substrate recognition mechanisms and define the shape of the active site. The AlphaFold model consists of approximately 38% alpha helices, 23% beta sheets, and 39% random coil or loop regions, indicative of a well-defined and structured fold with a high content of ordered secondary elements (Fig. 7a). For definitive assignment of the secondary structure of KP27 endolysin, however, nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectroscopy are standard experimental approaches. Examples of numerous endolysins whose 3D structures have been modeled or experimentally determined via various platforms demonstrate the range of approaches successfully utilized. The Ts2631 endolysin from Thermus scotoductus has a mixed α/β fold with a buried active site made of histidine and cysteine residues, which is resolved by crystallography and modeling to identify key surface charge characteristics for peptidoglycan binding. SU57e endolysin was structurally predicted via platforms such as Jpred, Phyre2, and I-TASSER, which revealed conserved domain organization and catalytic motifs. The LysB mycobacteriophage endolysin, modeled through I-TASSER coupled with molecular dynamics, provides insight into its unique catalytic site and the dynamics of flexible loops, informing mutagenesis to improve lytic efficiency²¹ LysK endolysin, modeled through AlphaFold2, showed a confident fold enabling substrate binding and catalytic residue identification²². Finally, chimeric endolysins such as Cly2v were modeled through AlphaFold-based tools coupled with experimental verification, resulting in hybrid computational‒experimental pipelines²³. In addition, research on EcLys and LysB also underscores the role of flexible loops and peripheral parts in controlling substrate access and enzyme dynamics. This adds to the inherent structural divergence in the loop regions of modeled KP27, highlighting the functional importance of conformational flexibility for endolysin enzymatic activity and specificity. Comparative analysis with previously characterized phage endolysins, including PlyC, LysK, EcLys, and LysB, further validated the predicted domain organization and underscored the role of loop flexibility in regulating substrate accessibility and catalytic efficiency²⁴.

Molecular docking with a peptidoglycan substrate fragment (NAM-L-alanyl-D-isoglutamine) further revealed substrate recognition mechanisms and an active site configuration. The reference structure 2MXZ contains the catalytically relevant residues His66, Asp73, and His133. On the basis of crystallographic data reported in the literature, the homologous structure 5OPZ, derived from the Chix protein, share conserved active site residues: His68, Asp75, His123, and Asp120⁹. Owing to their high sequence and structural similarity, findings from docking studies may be extrapolated across these structures. The structural superimposition of the AlphaFold-predicted model with the experimentally determined reference structures 2MXZ and 5OPZ illustrates that both computational methods can generate models closely resembling experimentally resolved conformations (Fig. 8).

The results of molecular docking indicate that the AlphaFold-generated KP27 model has the most favorable predicted binding affinity for the peptidoglycan substrate on the basis of its binding energy of -7.37 kcal/mol. Comparatively, the experimental reference structures 2MXZ and 5OPZ exhibit lower binding energies closely approximating those of the AlphaFold prediction. This convergence between the AlphaFold model and the experimental 5OPZ structure highlights the validity of the predicted binding interactions and supports the possibility that AlphaFold might better capture major active site features supporting substrate interactions. The fact that the modeled KP27 affinities are comparable or modestly better than the experimental structures implies that the models are sufficiently adequate to depict the enzyme‒substrate interaction. The binding site was defined on the basis of conserved catalytic motifs typical of endolysins. Docking pose analysis indicated that key residues coordinate interactions with both the N-acetylmuramic acid moiety and the peptide stem, which are stabilized by hydrogen bonds and hydrophobic contacts (Fig. 9b).

Circular dichroism (CD) spectroscopy and intrinsic/ANS fluorescence data provide a comprehensive understanding of the pH-dependent conformational dynamics of endolysin KP27, linking its secondary and tertiary structural changes to enzymatic activity. CD spectra in the far-UV region (190–250 nm) are used to specifically monitor the secondary structure content of proteins. For endolysin KP27, at pH 8.0, the optimum enzymatic activity condition, the CD spectrum shows strong negative ellipticity peaks near 208 nm and 222 nm, characteristic of well-formed alpha-helices²⁵. This indicates a stable and native-like secondary structure with a high alpha-helical content (~ 55%). In contrast, at pH values of 6.0 and 10.0, these alpha-helical signals are significantly diminished, reflecting partial loss of helicity and increased random coil or disordered elements (~ 49% at pH 6.0 and ~ 44% at pH 10.0). These decreases suggest that the secondary structure is partially destabilized at acidic and alkaline pH values, which disrupts the regular folding patterns crucial for protein function. Intrinsic fluorescence, dominated by tryptophan residues, involves the local environment within the folded protein. At pH 8.0, the highest intrinsic fluorescence intensity indicates that tryptophans are buried in a nonpolar, hydrophobic core, reflecting a compact and well-folded tertiary structure. The lower fluorescence at pH values of 6.0 and 10.0 corresponds to partial unfolding, exposing tryptophans to the solvent and causing quenching. The surface of the ANS fluorescent probes was subjected to hydrophobic exposure. The lowest ANS fluorescence at pH 8.0 confirms the minimal number of exposed hydrophobic patches, which is consistent with a tightly folded tertiary structure. Elevated ANS signals at pH 6.0 and 10.0 reveal that acidic and alkaline conditions expose hydrophobic residues normally hidden inside the protein, indicating loosening or partial unfolding of the tertiary fold. The preservation of the alpha-helical secondary structure at pH 8.0 aligns with the maintenance of a stable tertiary fold, as reflected by intrinsic and ANS fluorescence data, creating the ideal conformation for catalytic function. Disruptions in the secondary structure at pH values of 6.0 and 10.0—due to protonation/deprotonation events affecting ionic and hydrogen bonding networks—also destabilize the tertiary structure. This unfolding or conformational loosening exposes hydrophobic regions and tryptophan residues to the solvent, perturbing the active site geometry and dynamics necessary for substrate binding and catalysis. Therefore, the enzymatic activity of endolysin KP27 strongly correlates with the integrity of both its secondary and tertiary structures, which are optimally preserved at approximately pH 8.0. Deviations toward acidic or alkaline pH cause conformational rearrangements at both structural levels, leading to a decrease in enzymatic efficiency.

Conclusion

The KP27 endolysin displays strong and broad-range antibacterial activity against both gram-positive (S. aureus) and gram-negative (E. coli) bacteria, with significantly greater efficacy against S. aureus. Its bactericidal action is dose-dependent and swift, resulting in considerable bacterial lysis at low doses within minutes. Structurally, KP27 functions as an endopeptidase that targets bacterial peptidoglycan, with molecular modeling confirming that the active site configuration and substrate binding are consistent with those of known endolysins. The enzyme maintains optimal activity and stable secondary and tertiary structures around neutral to slightly alkaline pH, with diminished function under extreme pH conditions due to structural changes. KP27 also exhibits considerable thermal stability, retains activity at temperatures up to 80 °C, and shows negligible hemolytic activity, indicating a favorable safety profile. These features position KP27 as a promising candidate for further development in therapeutic and biotechnological applications.

Materials and methods

Chemicals

The KP27 gene coding sequence was obtained from NCBI under accession number YP_007348788 and synthesized by Bioneer Corp. NcoI, XhoI restriction enzymes, and T4 DNA ligases were purchased from NEB, and the DNA markers, agarose, and plasmid extraction kits and gel extraction kits were from Sinaclon. The pET-28a(+) plasmid and bacteria E. coli DH5α and E. coli BL21 (DE3) pLysS were obtained from the Pasteur Institute of Iran. The Ni²⁺-NTA chromatography columns were from ARG Biotech, Tabriz. The SDS‒PAGE gel electrophoresis kits and protein concentration determination kits were from Arsam Farazist, Urmia. The bacteria E. coli and S. aureus subsp. aureus were gifts from Dr. Nima Shaykh-Baygloo, Department of Biology, Urmia University.

Cloning and verification of the KP27 gene

To construct the pET28a(+)-KP27 expression vector, the steps and guidelines set in previous research were followed [15]. In brief, the pGEM-B1 plasmid carrying the synthetic KP27 gene was initially digested via the two restriction enzymes NcoI and XhoI. Next, to isolate the KP27 fragment, the digestion product was subjected to agarose gel electrophoresis and extraction. Following this, the ligation reaction was carried out by mixing a ratio of 5:1 of the KP27 gene and the pET28a(+) plasmid, which had been digested at 24 °C for two hours. Finally, the developed plasmid was transformed into competent E. coli DH5α cells via heat shock and plated onto LB agar plates supplemented with 50 µg/mL kanamycin. To verify the cloning of the KP27 gene at the expected location between the two abovementioned restriction enzymes, after plasmid extraction, PCR was initially conducted. After the gene was confirmed to be present in the pET28a(+) plasmid, it was sequenced for final verification.

Expression and purification

After verifying the correctness of the cloning, for the expression and purification of KP27 endolysin, 2 mL of an overnight culture of BL21 bacteria harboring the plasmid with the KP27 gene was transferred to 200 mL of LB culture medium supplemented with the antibiotic kanamycin in a shaking incubator at 180 rpm and 37 °C. When the OD of the culture medium reached a turbidity of 0.6 to 0.8, lactose at a final concentration of 10 mM was added to induce the expression of endolysin, and the bacteria were incubated overnight in a shaking incubator at 180 rpm and 37 °C. The next day, the culture medium containing the bacteria was centrifuged for 20 min at 3000 rpm, and the bacterial pellet was collected. To reach the cytosolic content, bacteria containing endolysin protein were dissolved in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole), and the walls of the bacteria were disrupted by sonication on ice. The sonicated sample was transferred to a 1.5 mL microtube on ice and centrifuged for 15 min at 14,000 rpm. After the samples were centrifuged, the supernatants of all microtubes were collected in a Falcon tube for transfer to an affinity chromatography column.

To purify KP27 endolysin in a single step, a Ni²⁺-NTA column was utilized. Prior to sample loading, the column was washed and equilibrated with 5 column volumes of distilled water and lysis buffer. Then, the protein mixture was loaded onto the column. In the subsequent step, wash buffer was passed through the column for washing and removing materials and proteins that did not bind to the column. Finally, elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole) was used to separate the KP27 endolysin bound to the column. The endolysin eluate from the column was collected in 2 mL microtubes in 1.5 mL volumes, and prior to determining antibacterial activity, salts, imidazole and other small molecules were removed via a dialysis bag with 1200 Da pores (Sigma D2272).

Endolysin concentration determination and electrophoresis

The KP27 endolysin concentration was measured via the Bradford assay, which takes advantage of the binding of the Coomassie Brilliant Blue G-250 dye to protein, which was measured at an absorbance of 595 nm via a spectrophotometer. Purity was determined by SDS‒PAGE analysis under denaturing conditions: samples were denatured by boiling for 10 min in lysis buffer (which included SDS and β-mercaptoethanol) and electrophoresed through a 15% polyacrylamide resolving gel with a molecular weight marker. The gels were stained with Coomassie Brilliant Blue R-250, destained, and imaged to verify protein purity and molecular weight. Collectively, these findings ensured proper quantitation and verification before functional assays were performed.

MIC determination and MBC determination

To conduct this test, the required dilutions of bacteria and KP27 endolysin were prepared in MHB medium in a sterile 96-well microplate. To each well of the microplate, 30 µL of each endolysin dilution, 160 µL of MHB medium, and 10 µL of bacteria in the logarithmic phase at a concentration of 1 × 106 CFU/mL were added and mixed gently. In the control well, bacteria were mixed with MHB medium, while the blank well contained only MHB medium. The microplates were incubated at 37 °C for 18 h in the incubator. EDTA was used to permeabilize the membrane of the gram-negative bacterium E. coli at a concentration of 0.5 mM. MBC is the minimum concentration of an antibacterial agent that works to kill the total population of bacteria compared with the control group. To determine the MBC, the minimum inhibitory concentration (MIC) was first determined visually, after which 10 µL of the mixture from each well at concentrations above the MIC were cultured on agar plates. The MBC was determined as the lowest concentration of endolysin at which there were no bacterial colonies.

Disk diffusion test

Assessment of antibiotic resistance is important for controlling and minimizing the spread of resistant bacteria. Antibiotic sensitivity testing, analysis and interpretation of associated data, and antibiotic drug research and development are highly important. For this purpose, the bacteria S. aureus and E. coli were grown overnight, and the solution was diluted to 0.5 McFarland standard. A total of 10 µl of the solution was cultured on sterile MHA media. Antibiotic disks against E. coli include ampicillin (10 µg), ceftriaxone (30 µg), and gentamicin (10 µg), and those against S. aureus include gentamicin (10 µg) and erythromycin (15 µg). These disks are applied under a hood using sterile forceps, 20 mm from the plate edge and 25 mm away from one another on the medium. The samples were kept in an incubator at 37 °C for 18 h, and the halo diameter was measured in millimeters via a ruler. To examine the effects of the KP27 endolysin and measure the halo diameter, the bacteria E. coli and S. aureus were grown. To remove the outer membrane of the bacterium E. coli and increase the access of endolysin to the cell wall, the bacteria were treated with EDTA (0.5 mM) and incubated for 20 min. Both bacterial species are diluted to a 0.5 McFarland standard, and then 10 µl of each of the diluted bacterial species is cultured on a sterile MHA plate. Disks containing 72 µg of KP27 were applied to the agar medium via sterile forceps and incubated in an incubator at 37 °C for 18 h, after which the diameter of the halo formed was measured with a ruler.

Antibacterial activity assay of endolysin

A colony of the cultured standard strains of E. coli and S. aureus bacteria was picked from solid agar culture medium, added to tubes with 5 mL of liquid broth culture medium and incubated overnight at 37 °C with shaking at 180 rpm. The bacteria grown in liquid culture medium were diluted 1:30. To prepare the dilutions of KP27 endolysin, a two-fold serial dilution series was performed in a 96-well plate to create the following final well concentrations: 8, 4, 2, 1, 0.5 and 0.25 µg/mL. To exclude the potential effect of the endolysin buffer, the highest concentration of PBS was used as a negative control in one well instead of the protein, and a well with only culture medium and bacteria was prepared as a positive control. Finally, the microbial suspension of the two-hour bacterial culture was diluted 1:30, and then a 0.5 mM EDTA solution was inoculated into all test wells at the final volume to increase the permeability of the outer membrane of the gram-negative bacteria. Finally, the lid of the plate was closed, and the plate was placed in a 37 °C incubator for 24 h. To determine the effect of endolysin, after 24 h, the OD600 of the wells was read by an ELISA reader, and the percentage of killing was calculated via the formula [(OD_control-OD_test)/OD_control] × 100.

Turbidity reduction assay

The lytic activity of endolysin KP27 against S. aureus and E. coli was assessed by measuring the reduction in turbidity of bacterial suspensions with a spectrophotometer using a 400 µL cuvette. Overnight cultures of both strains were grown in Mueller‒Hinton broth at 37 °C with shaking until the medium reached the midexponential phase (OD600 ≈ 0.4–0.6). The cells were pelleted via centrifugation at 5,000×g for 5 min, washed twice with phosphate-buffered saline (PBS, pH 7.4), and resuspended to an OD600 of approximately 0.8. For S. aureus, 360 µL of bacterial suspension was mixed with 40 µL of endolysin KP27 solution at 16 μg/mL (2 × MIC) in a cuvette. For E. coli, the bacterial suspension was pretreated with 0.5 mM EDTA for 15 min at room temperature to permeabilize the outer membrane. Then, 360 µL of the EDTA-treated E. coli suspension was mixed with 40 µL of the endolysin KP27 solution at 32 μg/mL (2 × MIC). The control samples for both strains received 40 µL of buffer instead of endolysin. The optical density at 600 nm (OD600) was measured at 37 °C at 5-min intervals for 60 min with a spectrophotometer. The lytic activity was reported as the percentage decrease in the OD600 compared with the initial value. All the experiments were performed in triplicate, and the data are presented as the means ± standard deviations.

Cell wall peptidoglycan purification and assaying of KP27 endolysin activity

Purified peptidoglycan was used as a substrate to analyze the enzymatic activity of the KP27 endolysin. Thus, to analyze the impact of KP27 endolysin on the degradation of the S. aureus cell wall, the turbidity of the bacterial suspension was measured. The S. aureus strain was cultivated in 500 mL of MHB at 37 °C until it reached the middle of the logarithmic growth phase and then washed three times with PBS. The bacterial pellet was suspended in 14 mL of 4% SDS and boiled for 30 min. The cell wall was separated through centrifugation at 10,000×g for 15 min, washed four times with distilled water, and incubated in 10 mL of buffer (20 mM Tris-HCl, pH 6.8, 0.1 mM CaCl2 containing 0.5 mg/mL trypsin) at 37 °C for 18 h. After being washed four additional times with distilled water, the sample was ultimately dissolved in 2 mL of 10% trichloroacetic acid and incubated at 4 °C for 6 h. Finally, after four additional washes with distilled water, the cell wall was dissolved in PBS and incubated with KP27 endolysin at 37 °C.

pH and temperature stability assays of endolysin KP27

The pH and temperature stability of endolysin KP27 were tested by assaying its residual bacteriolytic activity through a turbidity reduction assay. For the pH stability assay, purified KP27 was incubated at 37 °C for 1 h in a range of universal buffers (40 mM boric acid and 40 mM phosphoric acid) adjusted to pH 3.0 to 11.0 (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0) with NaOH. After incubation, the enzyme samples were subjected to activity assays immediately via a turbidity reduction assay. For the thermal stability assay, individual aliquots of KP27 were incubated in phosphate-buffered saline (PBS, pH 7.4) at 40, 50, 60, 70, and 80 °C for 15 and 60 min. After heat treatment, the samples were cooled to room temperature before their activity was measured via a turbidity reduction assay. The residual enzymatic activity was calculated as the percentage reduction in OD600 compared with the initial reading and normalized against the untreated KP27 controls incubated at optimal conditions (pH 7.0 and 4 °C, respectively). All the experiments were conducted in triplicate.

Hemolysis assays of KP27 endolysin

Hemolytic activity was evaluated via a standardized spectrophotometric assay using sheep red blood cells (RBCs). Purified KP27 (0–100 μg/mL) was incubated with washed RBCs (1.6% v/v suspension in PBS) at 37 °C for 60 min, together with PBS-negative and 1% Triton X-100-positive controls. After centrifugation (1,500×g, 5 min), the hemoglobin release from the supernatant was measured at 541 nm, and the hemolysis percentage was calculated as [(OD _Sample – OD _PBS)/ (OD _Triton – OD _PBS)] × 100.

In silico structural characterization of endolysin KP27

With the increasing availability and robustness of computational modeling tools, in silico methods have become an essential adjunct to structural biology in cases where experimental data are scarce or missing. The mature amino acid sequences of KP27 (NCBI Accession: YP_007348788) and two structurally similar proteins, 2MXZ and 5OPZ, were submitted to the servers for comparison.

Predicting the 3D structure of proteins with AlphaFold

Because the three-dimensional structure of the endolysin KP27 protein has not been elucidated in vitro using methods such as nuclear magnetic resonance (NMR) and because there are no specific data on this protein in the Protein Data Bank (PDB), we aimed to obtain the nearest and most similar three-dimensional (3D) structure to the subject protein using bioinformatics strategies and tools, such as AlphaFold. The AlphaFold v2 pipeline was used to predict and examine the 3D structure of the protein, starting with the collection of the target amino acid sequence in FASTA format. Homologous sequences were found through the UniRef90 and MGnify databases for constructing multiple sequence alignments (MSAs) to provide a strong evolutionary context for structure prediction. AlphaFold, accessed through the UCSF ChimeraX interface, produced five ranked structural models. Each structure had per-residue confidence metrics, including predicted local distance difference test (pLDDT) scores (from 0 to 100), Predicted Aligned Error (PAE) matrices (in Ångströms), and sequence coverage statistics, which were all exported in JSON and image files. The top-ranked structure was examined in UCSF ChimeraX through visualization of pLDDT-colored cartoon representations (blue = high confidence, red = low confidence), PAE heatmaps for estimating interresidue error, and surface depictions to confirm structural validity. A quantitative evaluation was carried out using median pLDDT scores, maximum PAE values, and MSA coverage distributions, ensuring a transparent and reproducible assessment of model accuracy.

Comparison of the endolysin KP27 with existing endolysin structures 2MXZ and 5OPZ

This comparison was performed using advanced bioinformatics software and by querying major protein databases such as the Protein Data Bank (PDB), which provides access to experimentally determined 3D structures of proteins. Through this comparison, we can detect structural similarities and differences, which can provide important insights into the functional characteristics and enzymatic activity of KP27, particularly its ability to degrade bacterial cell walls. The structural comparison revealed an alignment between the 3D structures of KP27 and the selected endolysins. Features within the PDB platform, along with molecular modeling and docking programs such as AutoDock Vina, were used to evaluate how the binding interactions and active sites compare among these proteins. This structural‒functional comparison is essential for determining whether KP27 possesses critical features found in other endolysins, such as conserved domains, substrate specificity, and catalytic efficiency, that are important for its potential application in biotechnology and therapeutic applications.

Preparation of peptidoglycan components

The structures shown in Fig. 1 are important ligands and peptidoglycan-derived motifs selected for molecular docking experiments with endolysin KP27. To obtain the three-dimensional structural details of the ligands, essential constituents were either downloaded from the PubChem database (e.g., N-acetylglucosamine, N-acetylmuramic acid, and pentaglycine) or built manually using ChemDraw to represent complicated peptidoglycan fragments. All the structures were energy-minimized and geometrically optimized for proper protonation states and compatibility with molecular docking or MD simulations. This strategy combines experimentally validated compounds and tailor-made motifs, facilitating systematic modeling of bacterial cell wall interactions.

Molecular docking of KP27 with bacterial cell wall elements

Molecular docking simulations using AutoDock Vina were carried out to predict the binding interactions of KP27 with these bacterial cell wall ligands. In the absence of experimentally determined binding sites, blind docking was undertaken to investigate possible interaction sites over the entire KP27 surface. This computational method simulates the first, atomic-level step of bacterial lysis, specific substrate binding in the active site, yielding important information about the structural basis of endolysin function. The predicted structure was utilized to prepare the system for molecular docking simulations. This consisted of preparing the protein structure file, defining the simulation box and solvent environment, adding counterions for system neutralization, performing energy minimization to eliminate steric clashes, and finally running the docking calculations. The docking protocol consisted of exhaustive sampling of ligand conformations and orientations, followed by scoring according to the predicted binding affinities to determine the most favorable binding poses. The docking results were examined for binding energies and important intermolecular interactions to infer the possible molecular recognition mechanisms of KP27 with bacterial cell wall components.

Interaction analysis and binding energy calculation of KP27-ligand complexes

To obtain in-depth information on the molecular interactions between ligands and proteins, the PDB files of the docked complexes were submitted to the Protein–Ligand Interaction Profiler (PLIP) web server (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index). PLIP automatically detects and classifies the noncovalent interactions in a complex, including hydrogen bonds, hydrophobic contacts, salt bridges, π–π stacking, and π–cation interactions. This furnished a complete interaction fingerprint of the ligand binding mode. In parallel, binding affinities and energy terms, including total binding free energy, Van der Waals, hydrogen bonding, desolvation, and electrostatic contributions, were obtained from AutoDock Vina docking logs to quantify the energetic basis of ligand binding. The docking simulations were run with an exhaustiveness set to achieve extensive sampling of binding conformations. This integrated strategy allowed extensive mapping of the interaction landscape and energetic determinants of KP27-ligand complexes, making it straightforward to identify key residues involved in binding and informing follow-up experimental validation and functional studies.

Characterization of the secondary structure of KP27 via circular dichroism

Circular dichroism (CD) spectroscopy is a biophysical method used to study the structural properties of chiral molecules, most commonly proteins. CD measures the differential absorption of left- and right-handed circularly polarized light, which arises from the chiral nature of protein secondary structures, such as alpha-helices, beta-sheets, and random coils. Changes in the CD spectra at these wavelengths reveal alterations in secondary structure, allowing researchers to monitor folding, unfolding, or conformational changes under varying conditions, such as pH or temperature. Purified endolysin KP27 was prepared at a concentration of 0.2 mg/mL in buffer solutions adjusted to pH values of 6.0, 8.0, and 10.0. Far-UV circular dichroism (CD) spectra were recorded at room temperature using a spectropolarimeter equipped with a quartz cuvette with a 0.2 cm path length. Spectral data were collected over the wavelength range of 190–250 nm, with instrument settings optimized for resolution and sensitivity, including a 1 nm bandwidth and a scanning speed of 50 nm/min. Baseline correction was performed by subtracting the respective buffer spectra at each pH. The raw ellipticity data were converted to molar ellipticity ([θ], degrees cm² dmol^-1) considering protein concentration and path length, enabling a comparative analysis of secondary structure content. The spectral features characteristic of alpha helices, such as negative bands near 208 and 222 nm, were analyzed to assess the structural stability and conformational changes of KP27 under various pH conditions. Secondary structure estimations were optionally carried out via deconvolution software to quantify changes associated with pH-dependent protein folding.

Evaluation of the 3D structural dynamics of KP27

Intrinsic fluorescence generally refers to the natural emission of light by aromatic amino acid residues—primarily tryptophan—found within proteins when they are excited by ultraviolet (UV) light, typically at approximately 280 nm. It allows researchers to monitor structural transitions and conformational dynamics in proteins in real time without the need for external probes or labeling. Moreover, ANS is a hydrophobic dye that fluoresces more intensely when bound to exposed hydrophobic regions of proteins, serving as a probe for detecting normally buried patches. Therefore, we explored the 3D structural dynamics of KP27 by employing both intrinsic and ANS fluorescence spectroscopy, which together offered complementary information on the conformational stability of the protein and its local microenvironment under different conditions.

The intrinsic fluorescence emission spectra of endolysin KP27 (50 µg/mL) were measured at different pH values (6.0–10.0) by preparing protein samples in corresponding buffer solutions, equilibrating them at room temperature, and recording fluorescence emission between 305 and 410 nm with excitation at 280 nm via a fluorescence spectrometer and quartz cuvettes. The buffer background fluorescence was subtracted from each spectrum. This method allows the assessment of changes in tryptophan fluorescence intensity and emission profile as a sensitive probe of the protein’s 3D structural stability and local microenvironment in response to pH variations.

The ANS fluorescence emission spectra of endolysin KP27 were recorded to assess changes in surface hydrophobicity and conformational state under various pH conditions. Protein samples were prepared by diluting purified endolysin KP27 to 50 µg/mL in universal buffer solutions adjusted to pH values of 6.0, 7.0, 8.0, 9.0, and 10.0. The samples were incubated at room temperature for 15–30 min to ensure equilibration. ANS dye was added to each protein mixture at a fixed molar ratio, and the mixtures were incubated briefly to allow binding. Fluorescence emission spectra were then collected via a fluorescence spectrophotometer over the wavelength range of approximately 400–600 nm with an excitation wavelength set at 390 nm.

Statistical analysis

Statistical analysis and graphing were performed with GraphPad Prism 9 software. Two groups were compared via the unpaired t test, and one-way ANOVA to determine significant differences among groups. When a significant overall effect was detected, Dunnett’s post hoc test was applied to compare each treatment group with the untreated control with suitable post hoc tests was used for comparing multiple groups. A p value of less than 0.05 was regarded as statistically significant. Statistical significance is indicated in the figures as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Introduction

Results

Cloning, expression, and purification of recombinant endolysin KP27

MIC, MBC, and Disc Diffusion Analyses

Antibacterial activity of endolysin KP27

Turbidity reduction assay

Peptidoglycan degradation by endolysin KP27

pH stability profile of endolysin KP27

Thermal stability profile of endolysin KP27

Hemolytic activity of KP27 on red blood cells

In silico characterization of KP27

AlphaFold-based prediction of the KP27 protein structure

Structural superimposition and active site mapping of modeled KP27 with 2MXZ and 5OPZ

Comparative molecular docking scores of KP27 models and reference peptidoglycan hydrolases against peptidoglycan-derived ligands

Circular dichroism analysis of the KP27 protein secondary structure

Assessment of 3D conformational dynamics in endolysin KP27 by fluorescence

Discussion

Conclusion

Materials and methods

Chemicals

Cloning and verification of the KP27 gene

Expression and purification

Endolysin concentration determination and electrophoresis

MIC determination and MBC determination

Disk diffusion test

Antibacterial activity assay of endolysin

Turbidity reduction assay

Cell wall peptidoglycan purification and assaying of KP27 endolysin activity

pH and temperature stability assays of endolysin KP27

Hemolysis assays of KP27 endolysin

In silico structural characterization of endolysin KP27

Predicting the 3D structure of proteins with AlphaFold

Comparison of the endolysin KP27 with existing endolysin structures 2MXZ and 5OPZ

Preparation of peptidoglycan components

Molecular docking of KP27 with bacterial cell wall elements

Interaction analysis and binding energy calculation of KP27-ligand complexes

Characterization of the secondary structure of KP27 via circular dichroism

Evaluation of the 3D structural dynamics of KP27

Statistical analysis

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