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<p>Phage-derived endolysins are potent antibacterial agents capable of lysing multidrug-resistant bacteria by cleaving peptidoglycan bonds. This study characterizes KP27 endolysin, a 131-amino acid enzyme encoded by the <i>Klebsiella</i> phage KP27, which exhibits L-alanyl-D-glutamate endopeptidase activity that is effective against both gram-positive and gram-negative bacteria. Recombinant KP27 was expressed and purified with a molecular weight of approximately 15&#xa0;kDa. It demonstrated dose-dependent bactericidal activity, reducing <i>Staphylococcus aureus</i> and <i>Escherichia coli</i> viability by up to 80% and 65%, respectively, at 8&#xa0;µg/mL. Time-kill assays revealed rapid bacterial lysis within 60&#xa0;min at 2× MIC (16&#xa0;µg/mL for <i>S. aureus</i> and 32&#xa0;µg/mL for <i>E. coli</i> with EDTA). Thermal assays indicated substantial stability up to 50&#xa0;°C. Hemolytic tests revealed minimal cytotoxicity, with less than 5% hemolysis at concentrations as high as 100&#xa0;µg/mL. AlphaFold2 modeling and molecular docking identified key substrate-binding residues consistent with L-alanyl-D-glutamate cleavage specificity. Circular dichroism spectroscopy revealed a predominantly alpha-helical secondary structure (~ 55%) at pH 8.0, which was correlated with maximal enzymatic activity; this helicity decreased to ~ 44–49% at pH 6.0 and 10.0, which coincided with reduced activity. Intrinsic fluorescence indicated a well-folded tertiary structure under optimal conditions, whereas ANS fluorescence confirmed minimal exposure of hydrophobic residues at pH 8.0. They revealed that KP27 maintains a predominantly alpha-helical secondary structure and a tightly folded tertiary conformation at an optimal pH of 8.0, which is strongly correlated with maximal enzymatic activity. Together, these data highlight KP27 endolysin as a stable, broad-spectrum antimicrobial enzyme with promising therapeutic potential against resistant bacterial pathogens.</p>

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

  • 期刊论文

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  • 发表日期：

    2025年12月8日

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  • Zeynab Zare,
  • Mehdi Imani,
  • Safiyeh Aghazadeh

摘要

Phage-derived endolysins are potent antibacterial agents capable of lysing multidrug-resistant bacteria by cleaving peptidoglycan bonds. This study characterizes KP27 endolysin, a 131-amino acid enzyme encoded by the Klebsiella phage KP27, which exhibits L-alanyl-D-glutamate endopeptidase activity that is effective against both gram-positive and gram-negative bacteria. Recombinant KP27 was expressed and purified with a molecular weight of approximately 15 kDa. It demonstrated dose-dependent bactericidal activity, reducing Staphylococcus aureus and Escherichia coli viability by up to 80% and 65%, respectively, at 8 µg/mL. Time-kill assays revealed rapid bacterial lysis within 60 min at 2× MIC (16 µg/mL for S. aureus and 32 µg/mL for E. coli with EDTA). Thermal assays indicated substantial stability up to 50 °C. Hemolytic tests revealed minimal cytotoxicity, with less than 5% hemolysis at concentrations as high as 100 µg/mL. AlphaFold2 modeling and molecular docking identified key substrate-binding residues consistent with L-alanyl-D-glutamate cleavage specificity. Circular dichroism spectroscopy revealed a predominantly alpha-helical secondary structure (~ 55%) at pH 8.0, which was correlated with maximal enzymatic activity; this helicity decreased to ~ 44–49% at pH 6.0 and 10.0, which coincided with reduced activity. Intrinsic fluorescence indicated a well-folded tertiary structure under optimal conditions, whereas ANS fluorescence confirmed minimal exposure of hydrophobic residues at pH 8.0. They revealed that KP27 maintains a predominantly alpha-helical secondary structure and a tightly folded tertiary conformation at an optimal pH of 8.0, which is strongly correlated with maximal enzymatic activity. Together, these data highlight KP27 endolysin as a stable, broad-spectrum antimicrobial enzyme with promising therapeutic potential against resistant bacterial pathogens.