ИСТИНА

Staphylococcus aureus is one of the most dangerous bacterium for humans and animals. It is a cause of human organs and tissues infections. The treatment of staphylococcal infections is difficult because of 90% of its strains are resistant to antibiotics. Lytic enzymes of bacteriophages can be considered as promising alternative to conventional antibiotic therapy. The stability (residual activity dependence on the incubation time) of the lysins of phages 8161, K, and chimeric enzyme K-L (LysK CHAP endopeptidase and amidase domains, and the entire mature lysostaphin protein) was investigated at the physiologically relevant conditions (37°С, human serum, enzyme concentration of 0,2-0,4 mg/mL). Activity of the enzymes was measured in a turbidity reduction assay from time-dependent turbidity changes in a suspension of S. aureus cells (A600 = 0,6, 37°С, СNaCl 137 mM, CKCl 2,7 mM, CNa2HPO4 10 mM, CKH2PO4 1,76 mM, pH 7,4). The stability of lysin of phage 8161 depends on the enzyme concentration. Increase of the enzyme concentration from 0.2 to 0.4 mg/mL leads to the enzyme stability decrease. It can be concluded that intermolecular interactions (aggregation) is likely the main reason of the enzyme inactivation. At high concentration of phage 8161 lysin (0,4 mg/mL) two linear parts are presented on the dependence of residual activity logarithm versus incubation time. Such dependence corresponds to two different inactivation stages. The rate of chimeric enzyme K-L inactivation does not depend on its concentration and is described by the first order equation. Both inactivation constant and half-inactivation time were calculated (k ~ 3,33×10-5 s-1 τ1/2 ~ 347 min). The inactivation rate of phage K lysin depends on its concentration. Increase of the enzyme concentration from 0.2 to 0.4 mg/mL (inactivation curve with fracture) leads to the enzyme stability increase. Such dependences correspond to dissociative mechanism of the enzyme inactivation. It can be concluded that 8161 lysin inactivation has aggregative mechanism, chimeric enzyme inactivation has denaturation mechanism and phage K lysin inactivation has dissociative mechanism.