Optimization of the Structure of the Staphylococcus aureus Phage K Endolysin CHAP Domain to Increase Lytic Activity

In order to increase the lytic activity of the enzyme, structures of the recombinant modified CHAP domain of the phage K endolysin to Staphylococcus aureus have been developed, including (starting from the N-terminus) a sequence of various cationic peptides HB(X) fused with the sequence of the CHAP domain through the GSG4S linker region. Genetic engineering constructs encoding the new HB(X)-CHAP were obtained, which were cloned and expressed in the recipient strain E. coli BL21(DE3). All variants of HB(X)-CHAP, as well as the control variant of the CHAP domain, were isolated and purified using a single technique involving a combination of cation and anion exchange chromatography. The lytic activity of the obtained enzymes was studied by the turbidimetric method using an autoclavable culture of S. aureus. For the two most promising HB(X)-CHAP variants from the point of view of further use, the main physicochemical characteristics are determined. It was shown that the presence of the GSG4S linker site in the structure of the molecule led to at least a twofold increase in the activity in the lysis of S. aureus cells, while the cationic peptides did not have a positive effect on the lytic activity of endolysin against Staphylococcus aureus. The obtained data may allow a rational approach to the issue of choosing an approach to optimizing the structure in the future and will expand the possibilities for designing endolysins in order to create an effective drug.