A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers.

Abstract

A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal (opt) conditions: pHopt 6.0-10.0, topt 20-30°C, NaClopt 400-800mM. At the working temperature of 37°C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0h. At storage temperatures of 22 and 4°C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100% activity after 140days of incubation (4°C, the enzyme concentration of 0.8mg/mL, pH6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.