Unlocking Lysostaphin’s Full Potential Through Linker Engineering

VI. International Enzyme and Bioprocess Days, Kocaeli, Türkiye, 27 - 29 Ağustos 2025, ss.71-72, (Özet Bildiri)

The rise of antibiotic-resistant Staphylococcus aureus, including biofilm-forming strains, requires

alternative antimicrobials. Lysostaphin targets the S. aureus cell wall by cleaving glycine-glycine bonds via

its catalytic domain (CAT) and binding pentaglycine bridges through its cell wall binding domain (CBD).

The linker connecting these domains is crucial for enzyme function, yet its alteration effects remain

underexplored. Here, we engineered four lysostaphin variants with linkers differing in length and amino

acid composition—rigid-short, rigid-long, flexible-short, and flexible-long—replacing the native flexible

linker. These were recombinantly expressed in E. coli BL21(DE3) under varying IPTG concentrations and

temperatures. To evaluate the linker effect on activity, the enzymatic activities of variant cell lysates were

compared with the wild-type enzyme using a turbidity assay against S. aureus strains ATCC 29213.

Although the study is ongoing and will be extended to include purified enzymes, the results indicate that

both linker length and composition significantly influence enzymatic activity and expression levels. The

short and flexible linker, due to its similarity to the natural linker, was observed to enhance both activity and

solubility and to shorten expression time. Despite activity decline after later hours, significant enzyme

expression and activity were observed early in induction (2h) for some variants. These preliminary findings

demonstrate that linker modifications impact activity and temporal expression, highlighting linker

engineering as a strategy to optimize lysostaphin’s antimicrobial feature and enhance the feasibility of

industrial production. This study provides insights into modular bacteriocin design and suggests that linker

tailoring can improve efficacy against resistant pathogens.