Efficient production of a highly active lysozyme from European flat oyster Ostrea edulis

Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to improve its catalytic activity and secretory expression still need to be studied. In the present study, twelve lysozymes from different origins were heterologously expressed using the Komagataella phaffii expression system. Among them, the lysozyme from the European flat oyster Ostrea edulis (oeLYZ) showed the highest activity. Via a semi-rational approach to reduce the structural free energy, the double mutant Y15A/S39R (oeLYZ_dm) with the catalytic activity 1.8-fold greater than that of the wild type was generated. Subsequently, different N-terminal fusion tags were employed to enhance oeLYZ_dm expression. The fusion with peptide tag 6×Glu resulted in a remarkable increase in the recombinant oeLYZdm expression, from 2.81 × 10³ U mL^-1 to 2.11 × 10⁴ U mL^-1 in shake flask culture, and eventually reaching 2.05 × 10⁵ U mL^-1 in a 3-L fermenter. The work produced the greatest amount of heterologous oeLYZ expression in microbial systems that are known to exist. Reducing the structural free energy and employing the N-terminal fusion tags are effective strategies to improve the catalytic activity and secretory expression of lysozyme.