Description

Endolysins are hydrolytic enzymes produced by bacteriophages during the lytic cycle (phage lysins). In practice, they become "enzybiotics" when used therapeutically against bacteria. Despite these advantages, applying endolysins to Gram-negative bacteria and delivering them in patients remains challenging. As protein therapeutics, they also carry familiar liabilities: protease degradation, instability at extreme pH or in serum, poor solubility during recombinant production, and poor oral delivery due to gastric conditions.

In this paper, the authors use genetic code expansion to introduce azido non-canonical amino acids—p-azido-L-phenylalanine (pAzF) and azidonorleucine (AnzL)—at solvent-exposed positions. The swap yields enhanced bacteriolytic activity, superior thermal and storage stability and preserved structural integrity.

Citation: Qi et al., 2025

Endolysins (phage-derived enzymes that lyse bacteria) are promising alternatives to antibiotics, but face the same challenges as other protein therapeutics: protease degradation, thermal instability, and poor serum persistence. These limitations are particularly acute for Gram-negative bacterial targets, where the outer membrane adds another barrier.

Incorporating azido ncAAs (pAzF and AnzL) at solvent-exposed positions improved all three parameters simultaneously. The modified endolysins showed enhanced bacteriolytic activity, superior thermal and storage stability, and preserved structural integrity compared to the wild-type enzyme (Qi et al., 2025).

This multi-parameter improvement from ncAA substitution is notable because conventional protein engineering often faces trade-offs, where optimising one property degrades another. ncAAs, by introducing chemical properties outside the natural amino acid repertoire, can break these trade-offs. The same approach applies to any therapeutic protein where activity, stability, and structural integrity must be maintained simultaneously.

Source publication