Surface immobilization and properties optimization of phage hydrolase against Gram-negative bacteria

Immobilized hydrolase not only reduces the production of antibiotic-resistant bacteria, but also effectively improves the stability of hydrolase in external use. In this study, phage hydrolase LysSSE1 against Gram-negative bacteria were surface immobilized and optimized for their bactericidal activity. Different anti-pathogen surface materials were prepared, where LysSSE1 was immobilized on the glass surface with a silica-affinity peptide and into which different peptide linkers were introduced. Immobilized enzymes inserted into the natural amino acid peptide linker exhibited higher bactericidal activity, greater stability, and more consistent bactericidal performance compared to those without the peptide linker. Among these immobilized enzymes, LysSSE1-NL-SiAP1 exhibited the strongest bactericidal activity and the best repeatable bactericidal performance, which only reduced the original performance by about 5% after three bactericidal cycles. Modeling analysis suggested that the presence of peptide linker might increase the molecular flexibility of the proximal hydrolase domain to better interact with the bacterial substrate. Our surface immobilization strategy could be extended to other lytic proteins, providing support for the development of surface sterilization methods and materials.