ИСТИНА

At present, Staphylococcus aureus is one of the most dangerous pathogens of various diseases. About 90% of Staphylococcus aureus strains are resistant to one or more antibiotics. One of the most promising methods of staphylococcal infections treatment is the use of bacteriophage lytic enzymes. These enzymes are produced by the phages to destroy the peptidoglycan layer of bacteria. The enzyme LysK lyses cells of Staphylococcus aureus, including strains resistant to methicillin and vancomycin. LysK is not used in medicine due to its low stability at storage temperatures (4, 20-25°C) and temperature of action (37°C). Previously, it was found that LysK is inactivated by aggregation mechanism and usage of polycations is effective technique for LysK stabilization. So, the aim of this study was to investigate the interaction of the enzyme LysK with the positively charged block copolymers of polyethylene glycol and polylysine (EG114Lys30 and EG114Lys10), polylysines 2.5, 9.6 and 55.2 kDa, polyarginines 5-15, 15-75 and > 70 kDa, an to determine optimal conditions for the stabilization of the LysK in the discussed temperature range. Activities and stabilities of series of samples with different charge ratios polyelectrolyte/enzyme (from 1 to 100) and low molecular weight electrolyte concentrations (11, 300, 500 mM NaCl) at pH 9.0 and temperatures of 4, 22 and 37°C were investigated. Activities of samples were determined by turbidimetry. In all cases, the activity of LysK in mixtures with polymers is higher than (10 times) that of the enzyme without them. It was shown that the addition of polymers to the enzyme leads to an increase in its stability under all temperatures in the presence of polylysines and their block copolymers with polyethylene glycol. Half inactivation time was chosen as quantitative parameter for characterization of the LysK stability. LysK half inactivation time was increased from 2 days to 2 months at 22°C by addition of polylysine with mass of 2.5 kDa and from 0.5 h to 100 h at 37°C in the presence of block copolymer EG114Lys10 in 300 mM NaCl. By dynamic light scattering the formation of complexes between the enzyme and polylysines and PL-PEG block copolymers was detected, due to which, apparently, and the stabilizing effect is achieved. Hydrodynamic radius of polymer molecules corresponds to ~ 4 nm, the addition of the enzyme lead to its increase to 20-200 nm. These particles were named “bacteriolytic nanozymes”. By means of IR spectroscopy it was shown that that interactions of the LysK with polylysines and their block copolymers with polyethylene glycol lead to the structuring of the enzyme molecules, the proportion of alpha helices and beta structures increases and the content of disordered structures decreases. The presence of polyarginines causes destabilization and destructuring of the enzyme. So, polylysines and their block copolymers with polyethylene glycol are effective additives for the LysK stabilization for using in medicine. Investigations were supported by Russian Ministry of Science and Education (grant № 11.G34.31.0004).