title: A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers.
abstract: 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 (opt) conditions: pHopt6.0-10.0, topt20-30 °C, NaClopt400-800 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.
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia. luboff.filatova@gmail.com.
authors.forename: Lyubov Y
authors.initials: LY
authors.lastname: Filatova
authors.affiliation: Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, NEA, ARS, USDA, Beltsville, MD, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Donovan
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Nadiya T
authors.initials: NT
authors.lastname: Ishnazarova
authors.affiliation: Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, NEA, ARS, USDA, Beltsville, MD, USA.
authors.forename: Juli A
authors.initials: JA
authors.lastname: Foster-Frey
authors.affiliation: Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, NEA, ARS, USDA, Beltsville, MD, USA.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Becker
authors.affiliation: Federal Budget Institution of Science, State Research Center of Virology and Bioengineering "Vector", Novosibirsk, Russia.
authors.forename: Vladimir G
authors.initials: VG
authors.lastname: Pugachev
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Nadezda G
authors.initials: NG
authors.lastname: Balabushevich
authors.affiliation: I.M. Sechenov First Moscow State Medical University, Moscow, Russia.
authors.forename: Natalia F
authors.initials: NF
authors.lastname: Dmitrieva
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia.|Division of Molecular Pharmaceutics, Center for Nanotechnology in Drug Delivery, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Natalia L
authors.initials: NL
authors.lastname: Klyachko
mesh_terms: D000838:Anions / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000494:pharmacology*; D001433:Bacteriolysis / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008248:Lysostaphin / Q000494:pharmacology*; D010316:Particle Size; D011108:Polymers / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000166:cytology* / Q000187:drug effects; D013696:Temperature
chemical_list: D000838:Anions; D001426:Bacterial Proteins; D011108:Polymers; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D008248:Lysostaphin
keywords: Applied biocatalysis; Enzyme kinetics; Fusion enzyme; Staphylolytic activity