ELABORATION OF A METHOD OF LYSOZYME PURIFICATION FROM HUMAN SALIVA

Introduction. Growing antibiotic resistance dictates the need for search for alternative antimicrobial drugs. Antimicrobial polypeptides, including lyso-zyme, are among prospective prototypes. Existing methods of human lysozyme purification are less elaborated in comparison with methods of lyso-zyme purification from other species sources. The aim of the study. Development of a simple and rapid method of lysozyme purification from human saliva, which can be performed in laborato-ries equipped for research of peptides and low-molecular proteins. Material and methods. Human saliva was treated with NaCl, centrifuged, filtered through filter with pores diameter 0.45 μm, used for two-steps sol-id-phase extraction on C18 cartridges (elution with 20% and 60% acetonitrile), reversed-phase high-performance liquid chromatography on C18 column in acetonitrile gradient and re-chromatography. Homogeneity and molecular weight were confirmed by SDS-PAGE electrophoresis. Enzyme activity was confirmed in turbidimetric analysis in the presence of Micrococcus lysodeikticus. The presence of antimicrobial activity was confirmed by electrophore-sis under acidic conditions with subsequent gel overlay against Listeria monocytogenes. Results. Saliva treatment with 62.5—1000 mM NaCl did not affect total protein content in the supernatants after centrifugation. Treated with 0.5 M NaCl and filtered saliva was used for solid-phase extraction. After the separation of components eluted with 20% acetonitrile, components eluted with 60% acetonitrile were used for RP-HPLC. As the result, a major peak was obtained and the fractions used for further purification and analysis. The sample contained a protein with the molecular weight of lysozyme. The protein possessed muramidase activity towards Micrococcus lysodeikticus cell walls and antimicrobial activity towards L. monocytogenes. Conclusions. In the present study, a simple method of lysozyme purification from human saliva was elaborated (about 5 μg of lysozyme from 1 mL of saliva). The protocol preserves essential biological properties of lysozyme.