# Enhancing stability and safety of chimeric peptidoglycan hydrolases by linker engineering

## TL;DR

This paper improves the stability and safety of chimeric enzymes that kill harmful bacteria without harming beneficial microflora.

## Contribution

The study introduces linker engineering to enhance enzyme stability and confirms their safety and low resistance risk.

## Key findings

- Engineered linkers significantly improved enzyme stability and storage conditions.
- Chimeric enzymes showed no cytotoxic effects on eukaryotic cells or model organisms.
- Selected chimeras demonstrated minimal risk of resistance development.

## Abstract

Spread of antimicrobial resistance and lack of new antibiotics have brought attention to alternative strategies of combating pathogenic bacteria. One of these strategies takes advantage of the bacteriolytic activity of peptidoglycan hydrolases. The enzymes allow efficient elimination of pathogenic bacteria while preserving the natural microflora. Such enzymes must meet specific criteria of activity, stability, and safety to become efficient enzybiotics. In our previous work (10.1128/spectrum.03546-23), we have created three chimeric enzymes and demonstrated their high efficacy in the elimination of Enterococcus faecalis and Staphylococcus aureus. In this work, we investigated and addressed issues related to the stability and safety of these enzymes. To improve the stability, we engineered the linkers and optimized storage conditions. Moreover, we demonstrated that such enzymes do not have any cytotoxic effects on eukaryotic cells, Danio rerio or Galleria mellonella. We also investigated the prevalence of resistance development, a particularly important feature for new antimicrobials. In conclusion, we here propose efficient, safe, and stable chimeric enzybiotics to eliminate E. faecalis and S. aureus.

• Optimized linker design enhances enzyme stability.

• Generated chimeric lysins do not display cytotoxicity.

• Chimeras with minimal risk of resistance development were selected.

The online version contains supplementary material available at 10.1007/s00253-025-13651-7.