Accepted Manuscript Title: Bacteriophage phi11 lysin: physicochemical characterization and comparison with phage phi80␣ lysin Author: Lyubov Y. Filatova David M. Donovan Juli Foster-Frey Vladimir G. Pugachev Natalia F. Dmitrieva Tatiana A. Chubar Natalia L. Klyachko Alexander V. Kabanov PII: DOI: Reference:
S0141-0229(15)00056-3 http://dx.doi.org/doi:10.1016/j.enzmictec.2015.03.005 EMT 8740
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
10-12-2014 26-3-2015 30-3-2015
Please cite this article as: Filatova LY, Donovan DM, Foster-Frey J, Pugachev VG, Dmitrieva NF, Chubar TA, Klyachko NL, Kabanov AV, Bacteriophage phi11 lysin: physicochemical characterization and comparison with phage phi80rmalpha lysin, Enzyme and Microbial Technology (2015), http://dx.doi.org/10.1016/j.enzmictec.2015.03.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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A physicochemical investigation of lysins of phages phi11 and phi80α was carried out.
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Me++ ions and cysteine residues maintain the active conformation of phage phi11 lysin.
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Lysin of phage phi11 is much more stable than lysin of phage phi80α.
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Schemes of inactivation of lysins of phages phi11 and phi80α are suggested.
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Bacteriophage phi11 lysin: physicochemical characterization and comparison with
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phage phi80α lysin
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Lyubov Y. Filatovaa*, David M. Donovanb, Juli Foster-Freyb, Vladimir G. Pugachevc,
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Natalia F. Dmitrievad, Tatiana A. Chubara, Natalia L. Klyachkoa,e, Alexander V.
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Kabanova,f
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a
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University, Moscow, Russia.
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b
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NEA, ARS, USDA, Beltsville, MD, USA
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e
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f
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Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, USA.
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*Corresponding author: telephone number: +74959393476; fax number: +74959395417; e-
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mail address:
[email protected] (Lyubov Y. Filatova)
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Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center,
FBSE SRC of Virology & Bioengineering «Vector», Novosibirsk, Russia.
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I.M. Sechenov First Moscow State Medical University, Moscow, Russia.
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Pirogov Russian National Research Medical University (RNRMU), Moscow, Russia
Division of Molecular Pharmaceutics, Center for Nanotechnology in Drug Delivery, UNC
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Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State
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Abstract
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Phage lytic enzymes are promising antimicrobial agents. Lysins of phages phi11
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(LysPhi11) and phi80α (LysPhi80α) can lyse (destroy) cells of antibiotic-resistant strains of
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Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical
28
use possible. The objectives of the study were to investigate the stability of lysins of phages
29
phi11 and phi80α in storage and functioning conditions, to identify optimum storage
30
conditions and causes of inactivation. Stability of the recombinant LysPhi11 and LysPhi80α
31
was studied using turbidimetry. CD-spectroscopy, dynamic light scattering, and
32
electrophoresis were used to identify causes of inactivation. At 37°C, pH 7.5 and
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concentration of NaCl not higher than 150 mM, LysPhi11 molecules contain a high
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percentage of random coils (43%). However, in spite of this the enzyme has high activity
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(0.4-0.8 OD600nms-1mg-1). In storage conditions (4°C and 22°C, pH 6.0-9.0, 10-500 mM NaCl)
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LysPhi11 is inactivated by a monomolecular mechanism. The optimum storage conditions for
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LysPhi11 (4°C, pH 6.0-7.5, 10 mM NaCl) were selected under which the time of the enzyme
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half-inactivation is 120-160 days. LysPhi80α stability is insufficient: at 37°C the enzyme
39
loses half of its activity almost immediately; at 4°C and 22°C the time of half-inactivation of
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LysPhi80α varies in the range from several hours to 3 days. Despite the common properties in
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the manifestation of antistaphylococcal activity the kinetic behavior of the enzymes is
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different. LysPhi11 is a more promising candidate to be used as an antimicrobial agent.
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Highlights
A physicochemical investigation of lysins of phages phi11 and phi80α was carried out. Me++ ions and cysteine residues maintain the active conformation of phage phi11
45 46
lysin.
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Lysin of phage phi11 is much more stable than lysin of phage phi80α.
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Schemes of inactivation of lysins of phages phi11 and phi80α are suggested.
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Keywords: bacteriolytic enzymes, phages, staphylococcal infections, activity, stability,
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structure Abbreviations:1
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1. Introduction
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Gram-positive Staphylococcus aureus causes a large number of dangerous (often fatal)
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diseases of the human body, such as pneumonia, osteomyelitis, endocarditis, toxic shock, and
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sepsis [1]. Furthermore, S. aureus rapidly develops resistance to a broad spectrum of
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antibiotics used in clinical medicine, including reserve antibiotics. Mortality from infections
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caused by methicillin-resistant (MRSA) and vancomycin-resistant (VRSA) strains of
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staphylococci reached 20 thousand people in 2008 in the United States [2]. In addition to
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controlling of the use of over-used antibiotics there is also a need to develop alternative
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methods of antibacterial treatment. One reasonable alternative is the use of phage lytic
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enzymes (lysins, lysozymes) which are employed by the phages during the initial and final
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stages of their life cycle [3-7].
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Currently, a large number of lysins for Gram-positive bacteria are reported in
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recombinant form and a strategy to expose the purified protein to the target pathogen and
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obtain lysis (lysis from without) is well known. There are several advantages of phage lysins,
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such as their near-species selectivity, their fundamentally new mechanism of acting externally
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(avoiding intracellular resistance mechanisms); activity against antibiotic-resistant strains of
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microorganisms, and the low probability of the emergence of resistant pathogens due to
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coevolution of host and phage [3-5].
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1
LysPhi11 – phage phi11 endolysin; LysPhi80α – phage phi80α endolysin; MRSA – methicillin-resistant Staphylococcus aureus; VRSA – vancomycin-resistant Staphylococcus aureus; OD600nm – optical density at 600 nm; PPB – 20 mM potassium phosphate buffer with pH 7.5; PPB-NaCl –20 mM potassium phosphate buffer, pH 7.5, 150 mM NaCl; DLS – dynamic light scattering, CD – circular dichroism; kin – first-order inactivation constant; ksec-in – second-order inactivation constant; n – inactivation order; effective hydrodynamic radius, s – second.
τ
1/2
– half-inactivation time; Rh –
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Lysins of phages phi11 (LysPhi11, GenBank accession number YP_500516.1) and
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phi80α (LysPhi80α, ABF71642.1) are monosubunit proteins with a molecular weight of ~54
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kDa, which molecules contain two catalytic domains (CHAP, Amidase 2) and one C-terminal
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cell wall binding domain (SH3_5). LysPhi11 and LysPhi80α hydrolyze chemical bonds in the
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peptidoglycan through the activities of both types D-Ala-Gly-endopeptidase and N-
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acetylmuramyl-L-Ala amidase [6-8]. A common property of these enzymes is that LysPhi11
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and LysPhi80α reveal the same activity in the live cells lysis of more than 30 strains of
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Staphylococcus aureus. In addition, these enzymes exhibit a similar efficacy in the lysis of
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staphylococcal biofilms [8]. Apart from the microbiological experiments described above [6-
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8] an enzymological approach was applied to evaluate the applicability of lysins of phages
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phi11 and phi80α.
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As far as both theory and practice are concerned, it is important that the enzymes not
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only reveal the lytic activity, but can also maintain it for long periods of time (or manifest
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stability). Lysins of phages phi11 and phi80α, likewise most biocatalysts, require specific
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storage and transportation conditions and can be subject to various inactivating effects when
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functioning. Currently neither optimum storage conditions of these enzymes have been
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identified nor their stability has been estimated.
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The objective of this study was to carry out a physicochemical investigation of lysins
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of phages phi11 and phi80α. It contained the study of the lysins activity and stability in
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physiological conditions; the study of storage stability of the enzymes and identifying of the
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optimum storage conditions; the determination of the enzymes inactivation causes and the
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comparison of inactivation parameters.
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2. Materials and methods
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2.1. Materials
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Recombinant lysins (C-terminally 6xHis tagged proteins) were prepared according to
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the procedures reported previously [8]. LysPhi11 (19.0 mg/mL) and LysPhi80α (18.0 mg/mL)
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were stored at -20°C in an elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM
97
imidazole, 30% glycerol, рН 8.0).
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The culture of Staphylococcus aureus bacteria (strain 209 B-580) was grown in L-
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broth up to the exponential phase (4.0x108 cells/mL), was titrated to determine the cells
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concentration, and was autoclaved during 30 minutes at a pressure of 0.6-0.7 atm. The cooled
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suspension was precipitated by centrifugation at 9.0x103 rpm during 20 minutes. The
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precipitate was washed twice with physiological saline and dissolved in 1/20 of the original
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volume of the saline. The resulting suspension was examined for the presence of live bacteria
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by plating the samples in an agar nutrient medium.
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Due to the need of eliminating the use of infectious material in the chemical
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laboratory, a special culture of Staphylococcus aureus cells was to be prepared: the bacteria
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should lose the ability to grow and reproduce, but the cells (cell wall peptidoglycan) should
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not be disrupted (Fig. S1 A). Apart from that the methods of heat treatment of the cells used
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should not cause a change in the enzyme activity (Fig. S1 B). The method of autoclaving of
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Staphylococcus aureus cells was selected subject to the requirements of these two conditions.
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The use of the autoclaved Staphylococcus aureus cells ensures reproducibility of the test
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results of the enzymes preparations activity that allows to objectively evaluate both
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techniques to prepare them and the kinetic parameters of inactivation.
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To prepare the buffer solutions the following was used: Tris, potassium
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dihydrophosphate and potassium citrate, sodium chloride and sodium hydroxide, and
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hydrochloric acid produced by Sigma. All measurements using instrumental methods were
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performed in triplicate.
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2.2. Measurement of LysPhi11 activity
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Enzyme activity was estimated in a turbidity reduction assay from time-dependent
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turbidity changes in a suspension of Staphylococcus aureus cells. The cell suspension of S.
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aureus with OD600nm, which equals to 0.6 absorbance units, was prepared using PPB. 10-20
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µL of the enzyme solution (0.2-0.8 mg/mL) was added to 0.5 mL of the cell suspension
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(content of LysPhi11 solution was within limits of 2-4% of the final reaction volume).
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Measurement of the optical density change over time was determined on the
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spectrophotometer Jenway 6405 UV/Vis at 37°C. The initial reaction rates typical for the
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catalytic activity of LysPhi11 were determined based on the initial linear regions of the
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kinetic curves of the optical density (OD600nm) versus time (s) (Fig. S1 B).
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2.3. Investigation of the influence of salt concentration in solution media on the LysPhi11 activity at 37°C
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The cell suspension of S. aureus with OD600nm = 0.6 absorbance units was prepared
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using PPB with NaCl concentration of 10-1000 mM. 10-20 µL (0.4 mg/mL) of the enzyme
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was added to 0.5 mL of the cell suspension, followed by measurement of turbidity at 37°C.
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2.4. Study of LysPhi11 inactivation at 37°C
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The thermal stability of LysPhi11 was studied at 37°C in two different buffers: PPB-
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NaCl or PPB. The enzyme (0.1-0.4 mg/mL) was pre-incubated at 37°C during selected time
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intervals. After pre-incubation, aliquots of 10-20 µL were taken and activity measurement
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under standard conditions was performed (2.2).
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2.5. Investigation of the influence of pH, temperature and buffer on LysPhi11
stability under storage conditions
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For the selection of the optimum storage conditions the following parameters were
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varied: temperature (4°C or 22°C), pH (6.0-9.0), enzyme concentration (0.2-0.8 mg/mL), and
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low-molecular weight electrolyte (NaCl, 10-500 mM). To maintain the constant pH of the
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lysin solutions a universal buffer was used, containing 20 mM of each compound: Tris,
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potassium phosphate and citrate, respectively. Solutions of LysPhi11 were pre-incubated at
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4°C and 22°C during chosen time intervals and then aliquoted for measurement of the activity
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under standard conditions (2.2). 2.6. Electrophoresis
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Electrophoresis of LysPhi11 samples was performed according to the procedures
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described in [9]. SDS-PAGE electrophoregrams were analyzed using Biospectrum AC Chemi
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HR-410.
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2.7. Investigation of the aggregation kinetics of LysPhi11 by dynamic light
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scattering
The effective hydrodynamic radius of LysPhi11 was determined using Zetasizer Nano
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ZS (He-Ne laser, 5 mW, 633 nm) at temperatures of 22°C and 37°C and enzyme
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concentrations of 0.2-0.8 mg/mL.
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2.8. Circular dichroism spectroscopy of LysPhi11
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Circular dichroism spectra for LysPhi11 (0.4 mg/mL, PPB or PPB-NaCl) were
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recorded at 37°C. The optical path length of the quartz cuvette was 1 mm, the measurements
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were carried out in the far UV region (195-260 nm) on the device Jasco J815 with a scanning
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speed of 2 nm/sec. Before recording the CD spectra enzyme solutions were filter sterilized via
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0.22 μM Millex filter (Millipore). The predicted secondary structure elements were
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determined via analysis of the spectra using the program CDNN [10].
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2.9. Testing reactivation of LysPhi11 with Ca++ and Mg++cations The cell suspension of S. aureus with OD600nm = 0.6 absorbance units was prepared in
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deionized water followed by the addition of calcium and magnesium cations up to the
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concentration of 1-10 mM. 10 µL of the inactivated enzyme solution in deionized water with
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concentration of 0.4 mg/mL was added to 0.5 mL of the cell suspension, followed by
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measuring of LysPhi11 activity at 37°C.
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2.10. Study of the inactivation of LysPhi80α
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Study of the inactivation of LysPhi80α was held following the procedures similar to
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those for LysPhi11 (2.2-2.7). 3. Results
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3.1. Investigation of the influence of NaCl on LysPhi11 activity and stability at 37°C
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It is important to investigate the activity and stability of lytic enzymes under the
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functioning conditions from the viewpoint of practical use. NaCl is the major component of
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both blood plasma and tissue fluids of humans and animals. The dependence of LysPhi11
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activity on NaCl concentration was examined under the medium conditions close to
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physiological (37°C, pH 7.5). The activity of phage phi11 endolysin decreases with increasing
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of NaCl concentration up to nearly zero at a salt content of 750 mM (Fig. 1). At physiological
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concentration of NaCl (about 150 mM) LysPhi11 loses ~ 40% of its activity in relation to the
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“salt-free” level.
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The loss of the enzyme activity may be associated with structural changes caused by
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the increase in salt concentration. LysPhi11 structure was described by CD-spectroscopy (the
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spectra examples are shown in Fig. S2, their deconvolution results are given in Table 1). To
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determine the causes of the secondary structure enzyme changes CD spectra were obtained at
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two temperatures, 37°C and 22°C (one of the enzyme storage temperatures), and in two
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buffers (PPB and PPB-NaCl). The shape of the CD spectra obtained for phage phi11
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endolysin is typical for β-structural proteins with a minimum at 210-215 nm and a positive
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value of molar ellipticity at 195 nm [11].
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Content of random coils in LysPhi11 molecule at 22°C is by ~20% lower than at 37°C
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(Table 1). The effect of NaCl on the secondary structure of the enzyme at both temperatures
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was evaluated by comparing the results of the enzyme CD spectra deconvolution in a medium
Page 9 of 36
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without NaCl and in a medium with NaCl. The content of different elements of the secondary
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structure of the enzyme molecule in the medium with NaCl is identical to that in the solution
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without NaCl (Table 1). It is essential to emphasize that the enzyme LysPhi11 in unfolded conformation has a
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non-zero value of the activity at 37°C (Fig. 1). LysPhi11 enzyme activity is comparable in
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value with staphylolytic enzyme activity with an ordered structure in similar conditions
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(37°C, pH 7.5). The activity of LysK, which molecule contains only 23% of random coils at
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37°C, equals to 1.6 OD600nms-1mg-1 [12], which is comparable with the activity of LysPhi11
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(0.8 OD600nms-1mg-1, Fig. 1).
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As shown above, the structural changes in LysPhi11 molecules at 37°C lead to the
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unfolding of enzyme molecules. The unfolding results in additional interactions between the
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enzyme molecules, which causes aggregation. At 22°C (a storage temperature) LysPhi11
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particle size is 5±1 nm, the enzyme heating to 37°C causes an increase in the particle size up
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to 100-150 nm in PPB and 300-500 nm in a medium containing NaCl. Aggregation of the
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enzyme molecules is the reason for the decrease of LysPhi11 activity with increasing of salt
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concentration.
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Changing the salt composition of the environment (introduction of sodium and
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chloride ions) affects not only the activity, but also the mechanism of LysPhi11 inactivation at
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37°C. In PPB-NaCl, the dependence of LysPhi11 residual activity versus time is a function of
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the enzyme concentration (Fig. S3 A). Increase of LysPhi11 concentration from 0.1 to 0.4
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mg/mL leads to the enzyme stability decrease (Table 2), which is to say that intermolecular
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interactions is likely the main reason of the enzyme inactivation. The obtained dependences of
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the residual activity versus time were straightened in the coordinates of the second order
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equation (Fig. S3 B):
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1 = 1 + k sec in [E] 0 t A / A0
(Eq. 1)
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where A/A0 is the residual activity of the enzyme, [E] 0 is the initial concentration of
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the enzyme, ksec-in is the constant of inactivation of the second order, t is the time of the
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enzyme to achieve the residual activity A/A0. The second order kinetics of the enzyme inactivation includes the bimolecular
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interactions between the enzyme molecules (autolysis or aggregation). Using electrophoresis
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under denaturing conditions showed that the molecular weights of the active and inactivated
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enzymes are virtually identical (Fig. 2). It can be concluded that in PPB-NaCl inactivation of
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LysPhi11is caused by non-covalent aggregation.
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ln( A / A0 ) = k in t
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where A/A0 is the residual activity of the enzyme, kin is the constant of the first order
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inactivation, t is the time of the enzyme to achieve the residual activity A/A0. First-order
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kinetic corresponds to enzyme unfolding.
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As stated above, at the initial time moment (37°C) aggregates of LysPhi11 are formed
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with the hydrodynamic radius of 100-150 nm in PPB and 300-500 nm in PPB-NaCl. Further
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incubation at 37°C causes an increase of the particle size of LysPhi11, wherein in PPB-NaCl
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the process is more intense (Fig. 3). After one-hour incubation at 37°C the particle radius of
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LysPhi11 in PPB does not exceed 400 nm, in PPB-NaCl, the effective hydrodynamic radii of
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particles are over one micron. The temperature initially induces very fast denaturation
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processes and the formation of initial aggregates (particles of radius less than 100 nm), which
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further form aggregates of higher order [13]. Aggregation depends on the concentration of
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enzyme, at higher concentrations it is more intense (Fig. 4) presumably due to increasing of
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the frequency of collisions between molecules.
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In the simple form, the scheme of enzyme inactivation in PPB can be depicted as follows: N→D↔A
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In PPB-NaCl, the enzyme is inactivated by the following scheme:
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2N→D2↔A
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N – native, D – denatured, A – aggregated forms of LysPhi11.
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Irreversible process of inactivation was confirmed by measurement of activity of
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3.2. Stability of phage phi11 lysin under storage conditions
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Thermal stability of enzyme is one of the most important parameters that can limit its
253
practical application. There are many ways to represent stability data, but the kinetic of
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thermal inactivation is the most objective data that can accurately predict the stability of the
255
enzyme through any period of time. An important point in the study of thermal stability is to
256
consider a wide range of conditions e. g. pH, composition of the media, and the presence of
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substrates and reaction products. To optimize storage conditions the influence of the
258
following factors on LysPhi11 stability was investigated: the effect of pH (6.0-9.0),
259
temperature (4°C and 22°C), ionic strength (10-500 mM NaCl) and concentration of the
260
biocatalyst (0.2-0.8 mg/mL). The range of enzyme concentrations available to study depends
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on whether the enzymatic reaction rate can be measured. The concentration range of NaCl and
262
the pH was adjusted dependent on the possibility of non-zero enzyme activity (Fig. 1, [14]).
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Most pharmaceutical drugs based on proteins are stored at temperatures of 1-25°C, so the
264
study of enzyme stability was held using two temperatures close to the boundary values of
265
this interval.
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Under storage conditions the dependence of the residual activity of LysPhi11 versus
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time is irrespective of the enzyme concentration and can be described by a first order equation
Page 12 of 36
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(Eq. 2). Irreversible process of inactivation was confirmed by measurement of activity of
269
LysPhi11 cooled solutions, which corresponds to different degrees of inactivation. The dependences of effective values of the constants of first order inactivation of
271
LysPhi11 on pH and salt concentration at 4°C and 22°C are shown on Fig. 5 A, B. The values
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of the inactivation constants at 4°C are much lower than at 22°C, i.e. at lower temperatures
273
the rate of unfolding of the polypeptide chain is likely to decrease, reducing the inactivating
274
interactions.
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In the pH range of 6.0-8.0, a rise of pH does not affect the first order inactivation
276
constants (Fig. 5 A, B), but a dramatic increase was observed between pH 8.0-9.0. Unlike pH,
277
ionic strength of the solution is not a parameter of the medium that affects the inactivation
278
constant of LysPhi11 at 4°C and 22°C (Fig. 5 A, B). The concentration range of NaCl
279
required for inactivation (i.e. degradation of electrostatic interactions) is 500-1500 mM [15].
280
The salt concentrations selected in this study are insufficient to exhibit similar effects.
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From the perspective of novel applications, the enzyme stability could be most clearly
282
estimated using the time of half-inactivation by determining the dependence of the LysPhi11
283
residual activity versus time. As Fig. 6 A shows, at 22°C, the half-inactivation time of the
284
enzyme does not depend on the NaCl concentration and decreases with increasing pH from 6-
285
8 days (5x105-7x105 s) to 1-3 days (9x104-3x105 s).
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At 4°C, the half-inactivation time of the enzyme increases with decreasing pH and
287
NaCl concentration (Fig. 6 B). The maximum time of LysPhi11 half-inactivation at 4°C (120
288
– 160 days, 1.0x107-1.4x107 s) was observed at pH 6.0-7.5 and 10 mM NaCl. These
289
conditions can be considered as optimal for the storage of the enzyme (of those examined).
290
The effective hydrodynamic radius of the molecules with enzyme activity of 100%
291
equals to 4-5nm (Fig. S5 A). After 1 day at 22°C the particle size of LysPhi11 remains 4-5
292
nm (the enzyme activity decreases to 50-80%); after 12 days at 22°C LysPhi11 particles
Page 13 of 36
enlarge to the effective hydrodynamic radius of 300-400 nm (remaining enzyme activity is
294
10-30%) and after 24 days of incubation particle size becomes 400-500 nm (with 5-15%
295
remaining activity) (Fig. S5 B, C). After 30 days of incubation at 4°C the particle size of
296
LysPhi11 equals to 4-5 nm (50-90% of enzyme activity remains); after 120 days of incubation
297
both particles with effective hydrodynamic radius of 5 nm and the formation of particles of
298
radius of 100-400 nm (with 10-60% remaining enzyme activity) are observed; after 270 days
299
under these conditions the particle radius becomes more than 1 micron (2-20% enzyme
300
activity) (data not shown). When LysPhi11 is inactivated under the storage conditions after
301
reaching zero residual enzyme activity, precipitation is observed.
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As the particle size of LysPhi11 remains the same after storing during 1 day at 22°C
303
and during 30 days at 4°C, with a measurable loss of activity, it is likely that the limiting step
304
is the unfolding (denaturation) of enzyme molecules, with the next step being nucleation (the
305
formation of aggregates sized up to 100 nm), which are then combined into aggregates of
306
higher order. We thus propose that the irreversible inactivation of the enzyme in the storage
307
conditions could be described by the following classical scheme:
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By means of electrophoresis in denaturing conditions it was indicated that the
310
molecular weight of LysPhi11 does not change during inactivation (Fig. S6, to identify
311
formation of –S-S- bonds between LysPhi11 molecules denaturing sample buffer without 2-
312
mercaptoethanol was used). Therefore, the formation of aggregates is provided by non-
313
covalent interactions, such as electrostatic, hydrophobic, and hydrogen bonds. The inactivated
314
enzyme was partially reactivated by adding Ca++ and Mg++ cations to the reaction media to a
315
concentration >0.6 mM (Fig. 7).
316
3.3. A comparison of the kinetic properties of LysPhi11 and LysPhi80α
Page 14 of 36
The kinetic behavior of lysins of phages phi11 and phi80α at 37°C (PPB-NaCl) differ
318
both in the velocity and mechanism of inactivation. The process of LysPhi80α inactivation at
319
37°C with a sufficiently high accuracy can be described by a first order equation (Eq. 2),
320
which corresponds to a monomolecular mechanism with the inactivation constant of 0.01 s-1
321
(Fig. 8). Lysin of phage phi80α loses its half activity for less than 1 minute (60 s) which is
322
significantly lower than half-inactivation time for LysPhi11 (Table 2) inactivated under these
323
conditions according to a bimolecular mechanism.
cr
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317
LysPhi80α in both storage conditions (4°C and 22°C, 10-500 mM NaCl, enzyme
325
concentration 0.2-0.8 mg/mL) and functioning conditions (37°C, PPB-NaCl) is much less
326
stable than the LysPhi11. The constants of the first order irreversible inactivation for
327
LysPhi80α are much higher than the inactivation constants of LysPhi11 (Fig. 9). The half-
328
inactivation time for LysPhi80α does not exceed 3 days (3x105 s) at 4°C and 1/4 day (2x104 s)
329
at 22°C, which is 1-2 orders of magnitude lower than the same τ1/2 for LysPhi11 (Fig. 6).
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In addition to the enormous difference instability, another difference in the kinetic
331
behavior of the enzymes is that NaCl stabilizes LysPhi80α under storage conditions (Fig. 9).
332
The increased NaCl concentration disrupts the electrostatic interactions and enhances the
333
hydrophobic ones. Due to the increased hydrophobic interactions the compaction of the
334
protein globule might lend itself to the appearance of additional stability to unfolding,
335
resulting in a decrease of the constants of monomolecular process.
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336
te
330
In solutions of partially or completely inactivated LysPhi80α, as well as LysPhi11, the
337
formation of aggregates of denatured molecules with the effective hydrodynamic radius from
338
several hundred nanometers to 1-2 microns is observed (data not shown). The difference is
339
likely due to the aggregation rate, which is often caused by the difference in denaturation rate
340
of enzymes.
Page 15 of 36
Higher-ordered aggregates of molecules of inactivated LysPhi80α are likely to be
342
formed by non-covalent interactions; it is evident from the absence of bands with high
343
molecular weights on SDS-PAGE electrophoregram (Fig. 10 A, lanes 3-6). For inactivated
344
LysPhi80α the presence of dimers with a molecular weight of about 110 kDa was revealed
345
(Fig 10 A, lanes 4 and 6), that are likely to be formed through –S-S- bonds, and active
346
enzyme doesn’t demonstrate such dimers (Fig. 10 A, lane 2).
cr
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341
Based on these data, we propose a scheme of LysPhi80α inactivation, shown in Fig.
348
10 B. The irreversible denaturation, i.e. the transition N → D (storage conditions), described
349
by the first order kinetic equation (Figs. 5 and 9), is the limiting stage of inactivation of lysins
350
of phages phi11 and phi80α. Molecules of LysPhi11 and LysPhi80α in the unfolded state are
351
aggregated due to the appearance of additional areas of (hydrophobic) interaction between
352
them (Fig. S5).
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4. Discussion
354
The study of the activity and stability of LysPhi11 at 37°C clearly shows that changing
355
the salt composition of the solution can be a way to control the properties of the biocatalyst.
356
We found that the activity of LysPhi11 decreases with increasing of NaCl concentration.
357
Previously the cases of endolysins losing their activity with increasing of salt concentration
358
were identified. The deletion construct expressing the staphylolytic LysK CHAP catalytic
359
domain loses 30% of activity at 150 mM NaCl and is almost completely inactivated at 500
360
mM electrolyte [16]. The activity of peptidoglycan hydrolase HydH5 is reduced twice at 200-
361
500 mM NaCl [17]. Endolysin PlyGRCS exhibits maximum activity at 125-500 mM NaCl
362
[18]. Endolysin LysK exhibits maximum activity at 200-400 mM NaCl [19].
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353
363
Using the analysis of the enzyme CD spectra under various conditions it was
364
established that high levels of random coils (more than 40%) at 37°C is the result of thermal
365
denaturation of the enzyme molecules. The unfolding results in additional areas of interaction
Page 16 of 36
between LysPhi11 molecules, which causes aggregation. Presence of NaCl enhances
367
intermolecular hydrophobic interactions, thereby intensifying the process of aggregation and
368
enlarging of particles. Aggregation is the reason for the decrease of the enzyme activity with
369
increasing of salt concentration, since there are steric constraints to access the substrate.
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Modifying the enzyme activity as a function of NaCl concentration can be associated
371
with the state of the cell wall of S. aureus. Inside Staphylococcus aureus bacterial cells the
372
salt concentration is very high and can reach 6% w/v [21]. The salt content, equal to 6% w/v,
373
corresponds to ~1000 mM NaCl, i.e. under the measurement conditions of the enzyme
374
activity the salt concentration in the external medium is lower than inside the bacterial cells.
375
This results in a salt concentration gradient and cells swelling. The degree of swelling of the
376
cells, which increases with decreasing the salt concentration in the external medium, affects
377
the degree of stretching of the cell walls, which determines the peptidoglycan conformation
378
and accessibility of chemical bonds for the hydrolysis and hence the activity of the enzyme.
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370
Inactivation of LysPhi11 (37°C) according to first order mechanism occurs quickly
380
enough: the enzyme loses half of its activity after 10 minutes (6x102 s) due to denaturation.
381
Inactivation by aggregation of the enzyme in PPB-NaCl is slower than in PPB and can be
382
suppressed by diluting: LysPhi11 at a concentration of 0.4 mg/mL loses half of its activity in
383
10 minutes (6x102 s) and at a concentration of 0.1 mg/mL loses half of its activity in 150
384
minutes (9x103 s). It is worth noting the importance of having a high-ordered secondary
385
structure of lytic enzymes. Lytic enzyme LysK is inactivated at 37°C and pH 7.5 by the
386
aggregation mechanism and loses half of its activity after 360-900 minutes (2x104-5x104 s)
387
[12]. The secondary structure of LysK contains 24% of random coils, which is twofold less
388
than the more rapidly inactivating LysPhi11.
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389
Under storage conditions (4°C and 22°C, 10-500 mM NaCl, enzyme concentration
390
0.2-0.8 mg/mL, pH 6.0-9.0) dramatic increase of the constants of first order inactivation of
Page 17 of 36
LysPhi11 was observed between pH 8.0-9.0. This could mean that increasing pH promotes
392
the transition of the enzyme from one form to another. This transition may be caused by
393
deprotonation of any group of the enzyme, resulting in a drop of lysin stability. The following
394
groups have pK values above 8.0 in the protein molecule: the thiol group of a cysteine residue
395
(8.5-8.8), the amino group of a lysine residue (10.0-10.2), the guanidinium group of arginine
396
residue (>12.0), and the hydroxyl group of tyrosine residue (9.6-10.0) [21]. The thiol group of
397
a cysteine residue has the closest pK value to the observed interval of pH where the decrease
398
of the first order inactivation constants was observed. It was established previously that this
399
residue is essential for catalytic activity of lytic enzymes. Amino acid substitutions of cysteine
400
residues cause loss of activity of lysin PlyC (Cys333) [22], the catalytic CHAP domain of
401
staphylolytic enzymes LysK (Cys54) and LysGH15 (Cys54) [23, 24]. In this case, the
402
importance of a cysteine residue for the functioning of phage phi11 lysin was indirectly
403
confirmed by kinetic studies. Catalytic CHAP domain of LysPhi11 molecule contains only
404
one cysteine residue (Cys32), which is likely to be important for activity.
te
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391
X-ray analysis revealed that CHAP domains of staphylolytic enzymes LysK and
406
LysGH15 contain Ca-binding sites, in which Ca++ ions are coordinated by residues Asp (45,
407
47, 56), Tyr (49), and His (51) [23, 24]. Amino acid substitutions of Asp residues lead to
408
complete loss of CHAP domains activity, which helps confirm the importance of Ca++ ions
409
for catalysis. In the process of LysPhi11 inactivation a violation of the spatial configuration of
410
Me++ binding site occurs as a result of unfolding of the protein. The loss of activity of the
411
enzyme is likely to be caused by either dissociation of Me++ or limiting the possibility of
412
binding to the substrate. Because of the proximity of the Ca++ and Mg++ ionic radii the
413
recovery process of the enzyme activity is probably nonselective.
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Page 18 of 36
414
The maximum time of LysPhi11 half-inactivation (120 – 160 days, 1.0x107-1.4x107 s)
415
was observed at 4°C, pH 6.0-7.5 and 10 mM NaCl. It can be concluded that LysPhi11 enzyme
416
is very convenient for the long-term storage. Lysins of phages phi11 and phi80α are only homologous in the first 181 amino acids.
418
Molecules of LyPhi11 and LysPhi80α contain the same number of charged amino acid
419
residues, 97 and 95, respectively. The content of hydrophobic amino acid residues in the
420
molecules of the enzymes is different: there are 191 in LysPhi11, and 176 in LysPhi80α. The
421
higher content of hydrophobic amino acids provides a higher degree of compaction of the
422
protein globule in an aqueous medium and imparts resistance to unfolding. The main reason
423
for the difference in the kinetic behavior of the enzymes is the difference in their amino acid
424
composition. The similarity in the manifestation of staphylolytic activity may be due to the
425
presence of areas with a high degree of homology in the molecules of the enzymes (from the
426
1st to the 181st amino acid). It is in this area of the amino acid sequences of both lysins where
427
the CHAP domain is located containing groups responsible for catalysis [8]. We can conclude
428
that non-homologous domains such as Amidase and SH3_b play the key role in maintaining
429
the structure of LysPhi11 and LysPhi80α molecules which is the reason for differences in the
430
parameters of enzymes inactivation.
432
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431
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5. Conclusion
At 37°C lysins of phages phi11 and phi80α are inactivated following various
433
mechanisms. Under storage conditions LysPhi11 and LysPhi80α are inactivated by a
434
monomolecular mechanism, but the constants of inactivation of the enzymes differ
435
considerably. For phage phi11 lysin there are optimum storage conditions under which the
436
time of half-inactivation of LysPhi11 is 120-160 days. To ensure a long-term storage of
437
LysPhi80α it needs to be stabilized (e.g., by forming enzyme-polymer complexes and by
438
adding a low molecular weight additives, etc.). Differences in the parameters of inactivation
Page 19 of 36
of the enzymes are the result of the difference in their amino acid composition. The common
440
properties in the antimicrobial activity are due to the fact that in both LysPhi11 and
441
LysPhi80α molecules there is an area with a high degree of homology (from the 1st to 181st
442
amino acid), where the catalytic CHAP domain is located.
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439
6. Acknowledgements
444
The study was supported by Ministry of Science and Education of Russian Federation
445
(Project 11.G34.31.0004) and Russian Foundation for Basic Research (grant number 15-04-
446
07995).
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443
7. References
448
[1]. Lowy FD. Staphylococcus aureus infections. The New England Journal of
452 453 454 455 456
M
aureus infection. Clinical Infectious Diseases 2008;46:350 - 59.
d
451
[2]. Gordon RJ, Lowy FD. Pathogenesis of methicillin-resistant Staphylococcus
[3]. Schmelcher M, Donovan DM, Loessner MJ. Bacteriophage endolysins as novel
te
450
Medicine 1998;339:520 - 32.
antimicrobials. Future Microbiol 2012;10:1147 - 71. [4]. Nelson DC, Schmelcher M, Rodriguez-Rubio L, Klumpp J, Pritchard DG, Dong S
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an
447
et al. Endolysins as antimicrobials. Advances in Virus Research 2012;83:299 - 365. [5]. Szweda P, Schielmann M, Kotlowski R, Gorczyca G, Zalewska M, Milewski S.
457
Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus. Applied
458
Microbiology and Biotechnology 2012;96:1157 – 74.
459
[6]. Donovan DM, Lardeo M, Foster-Frey J. Lysis of staphylococcal mastitis
460
pathogens by bacteriophage phi11 endolysin. FEMS Microbiology Letters 2006;265:133 – 39.
461
[7]. Navarre WW, Ton-That H, Faull KF, Schneewind O. Multiple enzymatic
462
activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-
463
alanyl-glycine endopeptidase activity. Journal of Biological Chemistry 1999;274:15847 – 56.
Page 20 of 36
[8]. Schmelcher M, Shen Y, Nelson DC, Eugster MR, Eichenseher F, Hanke DC et al.
465
Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage
466
sites protect mice from MRSA infection. Journal of Antimicrobial Chemotherapy 2015;(in
467
press).
471 472 473
cr
470
bacteriophage T4. Nature 1970;227:680 - 85.
[10]. Bohm G, Muhr R, Jaenicke R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein engineering 1992;5:191 - 95.
us
469
[9]. Laemmli UK. Cleavage of structural proteins during the assembly of the head of
[11]. Greenfield NG. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 2006;1:2876 – 90.
an
468
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[12]. Filatova LY, Becker SC, Donovan DM, Gladilin AK, Klyachko NL. LysK, the
475
enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards
476
stabilization. Biochimie 2010;92:507 - 13.
479
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te
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[13]. Kurganov BI. Kinetic of thermal aggregation of enzymes. Biochemistry (Moscow) 1998;63:430 - 32.
[14]. Filatova LY, Donovan DM, Becker SC, Priyma AD, Kabanov AV, Klyachko
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474
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NL. An investigation of the structure and function of antistaphylococcal endolysins using
481
kinetic methods. Moscow University Chemistry Bulletin 2014;69:107 – 111.
482
[15]. Zhang J. Protein-Protein Interactions in Salt Solutions. In Cai W, Hong H,
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editors. Protein-Protein Interactions - Computational and Experimental Tools, Croatia:
484
InTech; 2012, p. 359 - 376.
485
[16]. Fenton M, Ross RP, McAuliffe O, O’Mahony J, Coffey A. Characterization of
486
the staphylococcal bacteriophage lysin CHAPK. Journal of Applied Microbiology
487
2011;111:1025 – 35.
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488
[17]. Rodríguez L, Martínez B, Zhou Y, Rodríguez A, Donovan D, García P. Lytic
489
activity of the virion-associated peptidoglycan hydrolase HydH5 of Staphylococcus aureus
490
bacteriophage vB_SauS-phiIPLA88. BMC Microbiology 2011;11:138. [18]. Linden SB, Zhang H, Heselpoth RD, Shen Y, Schmelcher M, Eichenseher F et
492
al. Biochemical and biophysical characterization of PlyGRCS, a bacteriophage endolysin
493
active against methicillin-resistant Staphylococcus aureus. Appl Microbiol Biotechnol
494
2015;99:741-52.
496
cr
[19]. Becker SC, Foster-Frey J, Donovan DM. The phage K lytic enzyme LysK and
us
495
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491
lysostaphin act synergistically to kill MRSA. FEMS Microbiol Lett 2008;287:185 – 91. [20]. Shlegel G. Microbiology. 6th ed. Moscow: Mir; 1987.
498
[21]. Kantor Ch, Shimmel P. Biophysical chemistry. Moscow: Mir; 1984.
499
[22]. Nelson D, Schuch R, Chahales P, Zhu S, Fischetti VA. PlyC: a multimeric
M
bacteriophage lysin. PNAS 2006;103:10765 – 10770.
d
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[23]. Sanz-Gaitero M, Keary R, Garcia-Doval C, Coffey A, Raaij MG. Crystal
502
structure of the lytic CHAPK domain of the endolysinLysK from Staphylococcus aureus
503
bacteriophage K. Virology Journal 2014;11:133.
Ac ce p
504
te
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[24]. Gu J, Feng Y, Feng X, Sun C, Lei L, Ding W et al. Structural and Biochemical
505
Characterization Reveals LysGH15 as an Unprecedented ‘‘EF-Hand-Like’’ Calcium-Binding
506
Phage Lysin. PLOS Pathogens 2014;10:e100410.
507 508 509 510 511 512
Legends to figures
Fig. 1. Influence of NaCl on LysPhi11 activity. Experimental conditions: 37°C, pH 7.5, bacteria OD600nm = 0.6 absorbance units, LysPhi11 8 μg/mL. Fig. 2. SDS-PAGE analysis of LysPhi11. Lane M, protein molecular weight markers; lane 2, LysPhi11 before inactivation; lane 3, LysPhi11 after inactivation at 37°C.
Page 22 of 36
514 515 516
Fig. 3. Aggregation of LysPhi11 in different media. Experimental conditions: 37°C, LysPhi11 0.4 mg/mL, PPB (white squares), PPB-NaCl (black squares). Fig. 4. Influence of incubation time on the particle size of LysPhi11. The enzyme was incubated at 37°C for 60 minutes; PPB-NaCl (grey columns); PPB (white columns).
ip t
513
Fig. 5. Inactivation of LysPhi11 under storage conditions (kin values).
518
Experimental conditions: LysPhi11 0.2-0.8 mg/mL, universal buffer (Tris-phosphate-citrate),
519
22°C (A), 4°C (B).
cr
517
Fig. 6. Inactivation of LysPhi11 under storage conditions (τ1/2 values).
521
Experimental conditions: LysPhi11 0.2-0.8 mg/mL, universal buffer (Tris-phosphate-citrate),
522
22°C (A), 4°C (B).
an
us
520
Fig. 7. Reactivation of LysPhi11 by Ca++ and Mg++. Experimental conditions: 37°C,
524
deionized water, bacteria OD600nm = 0.6 absorbance units, LysPhi11 8 μg/mL. Ca++ (white
525
columns), Mg++ (grey columns).
528
d
te
527
Fig. 8. Dependence of LysPhi80α residual activity on time. Experimental conditions: 37°C, PPB-NaCl, LysPhi80α 0.4 mg/mL. Fig. 9. Inactivation of LysPhi80α under storage conditions. Experimental
Ac ce p
526
M
523
529
conditions: LysPhi80α 0.2-0.8 mg/mL, universal buffer (Tris-phosphate-citrate), pH 7.5. 22°C
530
(black circles), 4°C (white circles).
531
Fig. 10. Analysis of LysPhi80α inactivation by SDS-PAGE. A. SDS-PAGE
532
electrophoregram of LysPhi80α before and after inactivation. Lanes 1, 2, LysPhi80α before
533
inactivation; lanes 3, 4, LysPhi80α after inactivation at 22°C; lanes 5, 6 LysPhi80α after
534
inactivation at 4°C; lanes M, molecular weight protein markers. Lanes 1, 3, 5 correspond to
535
the enzyme samples mixed with denaturing sample buffer without 2-mercaptoethanol, lanes 2,
536
4, 6 correspond to the enzyme samples mixed with denaturing sample buffer with 2-
Page 23 of 36
537
mercaptoethanol. B. Schematic representation of LysPhi80α inactivation process. N – native
538
enzyme, D – denatured enzyme, A – aggregated enzyme, D-S-S-D – dimer.
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539
Page 24 of 36
539
Table 1.
540
Content of secondary structure elements (%) in LysPhi11 molecule as deconvoluted from CD spectra. 22°C
Secondary
37°С
No NaCl
150 mM NaCl
No NaCl
150 mM NaCl
Helix
14.4±1.3
13.4±1.0
12.1±0.5
12.3±1.1
Antiparallel
19.9±4.9
18.8±1.0
13.7±1.8
13.0±4.6
Parallel
17.3±1.7
16.9±1.5
12.9±1.0
β-turns
21.5±0.6
23.5±1.6
18.3±0.5
18.6±0.5
Random coils
26.8±4.2
27.4±1.2
43.0±1.3
43.1±4.0
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13.0±1.6
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541
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structure element
Page 25 of 36
542
Table 2.
543
Kinetic parameters of LysPhi11 inactivation at 37°C.
Buffer
τ
n
Inactivation constant
PPB-NaCl
2
ksec-in = (6.2±0.5)x10-3 s-1M
10-150
PPB
1
kin = (7.8±0.2)x10-4 s-1
10
cr
min
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544
1/2,
ip t
Parameters of inactivation
Ac ce p
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