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Applied Biochemistry and Biotechnology

, Volume 180, Issue 3, pp 544–557 | Cite as

A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers

  • Lyubov Y. Filatova
  • David M. Donovan
  • Nadiya T. Ishnazarova
  • Juli A. Foster-Frey
  • Stephen C. Becker
  • Vladimir G. Pugachev
  • Nadezda G. Balabushevich
  • Natalia F. Dmitrieva
  • Natalia L. Klyachko
Article

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: pHopt 6.0–10.0, topt 20–30 °C, NaClopt 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.

Keywords

Fusion enzyme Staphylolytic activity Enzyme kinetics Applied biocatalysis 

Notes

Acknowledgments

The study was carried out by SkolTech N 182-MRA and was supported by BARD grant no. IS-4573-12R/58-1265-2-132.

Supplementary material

12010_2016_2115_MOESM1_ESM.docx (23 kb)
Fig. S1 (DOCX 22 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Lyubov Y. Filatova
    • 1
  • David M. Donovan
    • 2
  • Nadiya T. Ishnazarova
    • 1
  • Juli A. Foster-Frey
    • 2
  • Stephen C. Becker
    • 2
  • Vladimir G. Pugachev
    • 3
  • Nadezda G. Balabushevich
    • 1
  • Natalia F. Dmitrieva
    • 4
  • Natalia L. Klyachko
    • 1
    • 5
  1. 1.Department of Chemical Enzymology, Faculty of ChemistryM.V. Lomonosov Moscow State UniversityMoscowRussia
  2. 2.Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, NEA, ARS, USDABeltsvilleUSA
  3. 3.Federal Budget Institution of Science, State Research Center of Virology and Bioengineering “Vector”NovosibirskRussia
  4. 4.I.M. Sechenov First Moscow State Medical UniversityMoscowRussia
  5. 5.Division of Molecular Pharmaceutics, Center for Nanotechnology in Drug Delivery, UNC Eshelman School of PharmacyUniversity of North Carolina at Chapel HillChapel HillUSA

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