ИСТИНА

Phage lytic enzymes are widely applied in clinical medicine as an alternative to antibiotics, owing to the fact that near 90% strains of Staphylococci are resistant to antibiotics. The general aim is to find the optimum conditions for lysin activity, because it determines the ability of its practical application. The phage 8161 enzyme activity depends on its concentration, pH, the substrate concentration, salts content. The activity of phage 8161 enzyme grows when increasing the substrate content to A600 0.4 units of optical density, later it does not change (it reaches saturation). All the later studies of the environmental factors influence on the enzyme activity were carried out with «saturating» substrate concentration (A600 = 0.6 units of optical density). The concentration dependence of enzyme activity is linear in the range from 0 to 0.5 mkg/ml (рН 7.5, 37ºС), later it flattens out (2-12 mkg/ml). The cause of this phenomenon may be enzyme molecules aggregation. It was demonstrated that the effective hydrodynamic radius of the enzyme particles grows from 80 to 130 nm when increasing its concentration from 4 to 16 mkg/ml. The pH-dependence of enzyme activity looks like a bell-shaped curve with optimum pH 6-8.5, where physiological pH 7.5 lies. The dependences of the activity of the phage 8161 enzyme from ionic strength (KH2PO4 concentration) and the concentration of physiologically active NaCl have optima. The maximum activity was observed with KH2PO4 concentration 100 mM and in 200-400 mM NaCl. The influence of NaCl on the lysin activity may be caused by the change of its tertiary structure (the presence of sodium chloride considerably changes the fluorescent spectrum of the enzyme molecules). We have determined the optimum environmental factors for the functionality of the phage 8161 enzyme: pHopt, NaClopt, KH2PO4opt. It has been demonstrated that the different functional dynamic of the enzyme activity from composition parameters may be called by the aggregation of particles or the change of the tertiary structure. The study was supported by Russian Scientific Foundation (grant number 14-13-00731).