A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal () conditions: pH 6.0-10.0, t 20-30 °C, NaCl 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.
Location of phosphodiester bonds essential for ammoacylation of bovine tRNATn' was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21. 22, 36-38. 57-59, 62 and 64 were critical for aminoacylation capacity of tRNA'IP -transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and m the regions presumably outside the contact area as well. These results indicate that for mamtenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non-modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed m this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.
The aim of this research was to study the influence of magnetic field on the growth of microorganisms. In our experiments this influence was tested using Escherichia coli strain BT21. The experiments were carried out in a shaker flask at different intensities of magnetic field. The application of magnetic drives and joints in the bioreactors imposes strong requirements on the hermetic sealing of the testing vessels, which is utterly important from the sterility point of view. Our experiments have convincingly shown that the magnetic influence results in higher growth rate of biomass. In addition, some indications have been obtained that as the magnetic strength exceeds a definite level its positive influence on the bacterial growth decreases.
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