ИСТИНА

A staphylolytic LysK-lysostaphin triple fusion enzyme possesses three lytic activities comprised of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To appreciate the power of possible therapeutic applications the kinetic properties (activity, stability) of the chimeric enzyme were investigated. To reduce the immunogenicity of the enzyme the biophysical effect of inclusion of LysK-lysostaphin triple fusion enzyme in anionic polymer matrices was tested. Triple fusion enzyme reveals a maximal lytic activity under the following conditions: pH 6.0-10.0, t 20-30°C, NaCl 400-800 mM. At 37°C (temperature of functioning) the chimeric enzyme is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7±3.0 hrs. At storage conditions (22°C, 4°C, pH 6.0-9.0, the enzyme concentration of 0.2-0.8 mg/mL) triple fusion enzyme is inactivated by a complex mechanism (combination of monomolecular and bimolecular mechanisms). The optimal storage conditions (enzyme retains 100% activity during 140 days) were appointed (4°C, the enzyme concentration of 0.8 mg/mL, pH 6.0/7.5). It can be concluded that no additional stabilization of the enzyme is needed at 4°C. Triple fusion enzyme was included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol (PGLU-PEG). Both activity and stability of the enzyme are retained. The inclusion of triple fusion enzyme in a polymer matrix may result in a reduced immunogenicity of biocatalyst which is a prerequisite for commercialization of a proteinaceous antimicrobial agent.