Download Biochemical Properties and Mechanism of Action of Enterocin LD3

Probiotics & Antimicro. Prot. (2016) 8:161–169
DOI 10.1007/s12602-016-9217-y
Biochemical Properties and Mechanism of Action of Enterocin
LD3 Purified from Enterococcus hirae LD3
Aabha Gupta1,2 • Santosh Kumar Tiwari1,2,3 • Victoria Netrebov3
Michael L. Chikindas3,4
•
Published online: 5 May 2016
Ó Springer Science+Business Media New York 2016
Abstract Enterocin LD3 was purified using activity-guided multistep chromatography techniques such as cationexchange and gel-filtration chromatography. The preparation’s purity was tested using reverse-phase ultra-performance liquid chromatography. The specific activity was
tested to be 187.5 AU lg-1 with 13-fold purification.
Purified enterocin LD3 was heat stable up to 121 °C (at 15
psi pressure) and pH 2–6. The activity was lost in the
presence of papain, reduced by proteinase K, pepsin and
trypsin, but was unaffected by amylase and lipase, suggesting proteinaceous nature of the compound and no role
of carbohydrate and lipid moieties in the activity. MALDITOF/MS analysis of purified enterocin LD3 resolved m/z
4114.6, and N-terminal amino acid sequence was found to
be H2NQGGQANQ–COOH suggesting a new bacteriocin.
Dissipation of membrane potential, loss of internal ATP
and bactericidal effect were recorded when indicator strain
Micrococcus luteus was treated with enterocin LD3. It
inhibited Gram-positive and Gram-negative bacteria
including human pathogens such as Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa,
& Santosh Kumar Tiwari
[email protected]
1
Department of Genetics, Maharshi Dayanand University,
Rohtak, Haryana 124001, India
2
Department of Bioscience and Biotechnology, Banasthali
University, Tonk, Rajasthan 304022, India
3
Health Promoting Naturals Laboratory, School of
Environmental and Biological Sciences, Rutgers State
University, New Brunswick, NJ 08901, USA
4
Center for Digestive Health, New Jersey Institute for Food,
Nutrition and Health, New Brunswick, NJ 08901, USA
Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (urogenic, a clinical
isolate) and Vibrio sp. These properties of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics.
Keywords Enterocin LD3 Enterococcus hirae LD3 Bacteriocin Biopreservation Membrane potential
Introduction
Lactic acid bacteria (LAB) are known to produce ribosomally synthesized antimicrobial proteins as a part of their
defense mechanism. Many of these molecules inhibit closely related bacteria, and they are active mostly against
Gram-positive microorganisms; however, there are reports
that show several LAB bacteriocins inhibiting Gram-negative bacteria [1–4]. Presently, only nisin and pediocin PA1/AcH found their commercial use as food preservatives,
while many more were characterized for application in
food safety [5, 6].
Based on their properties such as charge, hydrophobicity
and molecular weight, several methods have been utilized
for purification of bacteriocins. One of the most common is
the use of salt precipitation and chromatography in combination with the bioassay [7]. Most of the reported LAB
bacteriocins are stable at a broad range of pH and temperatures, sensitive to some proteolytic enzymes and generally of a small molecular weight [2, 8–10].
Bacteriocins kill sensitive cells through different
mechanisms. Although structure–function relationships
have only been determined for certain bacteriocins, many
bacteriocins produced by LAB act by pore formation or
inhibition of cell wall biosynthesis [2, 11, 12]. Pore
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162
Probiotics & Antimicro. Prot. (2016) 8:161–169
formation can lead to cell death as a consequence of proton
motive force (PMF) depletion through dissipation of the pH
gradient and membrane potential. Furthermore, cell death
can result from efflux of phosphate, amino acids or other
substances [13].
In our previous study, we have demonstrated the bacteriocin production by a putative probiotic strain, E. hirae
LD3, isolated from a fermented food, Dosa [14]. Here, we
report the molecular mass, partial amino acid sequence,
stability under different conditions and mode of action of
enterocin LD3 purified from culture-free supernatant (CFS)
of E. hirae LD3.
Materials and Methods
Bacterial Strains, Culture Media and Growth
Conditions
E. hirae LD3 was grown in Lactobacillus MRS medium
[15] at 37 °C in an incubator shaker with agitation at
200 rpm (Scigenics Biotech, Chennai, India). Reference
LAB strains were obtained from the ARS Culture Collection (USDA) as mentioned in Table 1. E. coli (urogenic),
Pseudomonas fluorescence Psd and P. aeruginosa clinical
isolates were grown in Luria–Bertani Broth (LB) at 37 °C.
Micrococcus luteus MTCC 106, Staphylococcus aureus,
Salmonella typhi, Shigella flexneri, Listeria monocytogenes
and Vibrio sp. were grown in nutrient broth (NB) medium
at 37 °C. M. luteus MTCC106 was used as an indicator
strain for assay of antimicrobial activity. All the media
components were purchased from Hi-Media (Mumbai,
India), Sisco Research Laboratory (SRL, Mumbai, India)
and Sigma-Aldrich (St-Louis, USA).
Bacteriocin Purification and Assay
The CFS (2 L) of E. hirae LD3 was subjected to ammonium sulfate (SRL, Mumbai, India) precipitation at different saturation levels (0–30, 30–50 and 50–90 % w/v).
Salt was added slowly by continuous stirring on a magnetic
stirrer at 4 °C. The precipitate was collected by centrifugation at 7000g, 10 min, 4 °C (Sigma, Germany) and
suspended in minimum volume of 20 mmol l-1 sodium
acetate buffer, pH 4.6 (buffer A). The samples were
desalted by dialysis (2.0 kDa cutoff membrane, Sigma, St.
Louis, USA) against buffer A for 24 h. The dialyzed
samples were filter-sterilized through 0.2-lm membrane
(Axiva, New Delhi, India) and tested for antimicrobial
activity. Protein concentration was determined according to
the Bradford method [16]. The desalted active sample
(Fraction I) was further processed using cation-exchange
Table 1 List of microorganisms, their sources and reason to test against purified enterocin LD3
S.
No.
Microorganisms
Origin (source)
Reason for selection
References
1
Lactobacillus curvatus NRRL B4562
Milk
Target strain
ARS Culture Collection, USA
2
L. delbrueckii NRRL B4525
Target strain
ARS Culture Collection, USA
3
L. acidophilus NRRL B4495
Emmental
cheese
Human
Target strain
ARS Culture Collection, USA
4
L. plantarum NRRL B4496
Pickled cabbage
Target strain
ARS Culture Collection, USA
5
Lactococcus lactis subsp. lactis NRRL
B1821
Not available
Target strain
ARS Culture Collection, USA
6
L. lactis subsp. cremoris NRRL B634
Not available
Target strain
ARS Culture Collection, USA
7
Enterobacter cloaceae NRRL B14298
Soybean
Related to producer
ARS Culture Collection, USA
8
Enterococcus faecium NRRL B2354
Cheese
Related to producer
ARS Culture Collection, USA
9
Staphylococcus aureus
Human
Local hospital
10
E. coli (urogenic)
Human
Food-borne
pathogen
Pathogen
11
E. coli O157:H7
Human
Food-borne
pathogen
Dr. K. R. Matthews lab, Rutgers State
University
Local hospital
12
Pseudomonas fluorescens
Soil
Gram-negative
Dr. Sheela Srivastava lab, Delhi University
13
P. aeruginosa
Human
Pathogen
Local hospital
14
Salmonella typhi
Human
Pathogen
Local hospital
15
Shigella flexneri
Human
Pathogen
Dr. J. S. Virdi, Delhi University
16
Listeria monocytogenes
Human
Pathogen
Dr. J. S. Virdi, Delhi University
17
Vibrio sp.
Human
Pathogen
Local hospital
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Probiotics & Antimicro. Prot. (2016) 8:161–169
chromatography (CEC) with the benchtop column filled
with SP-Sepharose fast flow (Sigma, St. Louis, USA)
controlled by a peristaltic pump (MacFlow, New Delhi,
India). The column was equilibrated and washed with
buffer A, and Fraction I was applied to the column at a flow
rate of 0.5 ml min-1. The active compound was eluted
with buffer B (0.2, 0.5 and 1.0 mol l-1 NaCl in buffer A).
Active fractions were pooled, dialyzed against the doubledistilled (DD) water, lyophilized at -75 °C under vacuum
(10-3 torr) for 24 h (Allied Frost, New Delhi, India) and
resuspended in minimum amount of sterile DD water
(Fraction II).
Fraction II was then loaded into a gel-filtration column
(GFC) Sephadex G-25 (Amberson Biosciences, USA)
equilibrated and eluted with DD water. In these fractions,
protein concentrations were monitored at 280 nm and
antimicrobial activity was determined. The active fractions
were pooled, lyophilized and resuspended in minimum
amount of sterile DD water (Fraction III). The antimicrobial activity of bacteriocin at each step of purification was
determined using agar well diffusion assay (AWDA) [17]
and AU ml-1. One activity unit (AU) was defined as the
reciprocal of the highest dilution of bacteriocin causing
50 % growth inhibition [18].
Determination of Purity of Bacteriocin
To determine the level of purity in GFC-eluted sample
(Fraction III), it was applied on BEH C18 column (1.7 lm,
2.1 9 50 mm) fitted in Acquity RP-UPLC (reverse-phase
ultra-performance liquid chromatography) (Waters, Massachusetts, USA). The column was equilibrated with water
containing 0.1 % TFA (trifluoroacetic acid, Buffer A1)
(SRL, Mumbai, India), and sample was run with a step
gradient of Buffer B1 (acetonitrile (SRL, Mumbai, India)
containing 0.1 % TFA), initially 5 % B then 20 % B up to
3 min and 40 % B up to 6 min. The flow rate was maintained at 1.0 ml min-1 throughout the run, and absorbance
was monitored at 280 nm using a UV detector (Waters,
Massachusetts, USA).
Molecular Mass and Amino Acid Sequence
Purified enterocin LD3 was submitted for the determination of molecular mass and amino acid sequencing using
custom facilities available. Molecular mass (m/z) was
determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)
on LTQ Orbitrap Velos LC–MS/MS at the Center for
Advanced Proteomics Research, New Jersey Medical
School of the Rutgers Biomedical and Health Sciences
(Newark, New Jersey). The amino-terminal sequencing
was carried out on an Applied Biosystems Procise Protein
163
Sequencer equipped with an online HPLC system at New
Jersey Medical School Molecular Resource Facility
(NJMS-MRF). The sample was processed according to the
manufacturer’s recommendations; briefly, it was loaded
onto a polyvinylidene fluoride membrane, dried and placed
into the reaction cartridge. The instrument was operated
according to the standard protocol for Edman Sequencing
that initially runs a blank cycle and a standard cycle, followed by sequencing cycles. The data were collected using
SequenceProTM software.
Biochemical Characterization
Similar to other bacteriocins, purified enterocin LD3 was
characterized for stability at different temperatures, pH and
in the presence of organic solvents, detergents and surfactants. In addition, its activity was tested against the
selected range of related as well as pathogenic microorganisms as listed in Table 1. Purified enterocin LD3 was
treated with different hydrolyzing enzymes such as amylase, lipase, trypsin, pepsin, papain (Sigma-Aldrich, USA)
and proteinase K (SRL, Mumbai, India) at a final concentration of *1000 units ml-1. Following incubation at
37 °C for 2 h, enzymatic activity was terminated by heating the sample at 100 °C for 5 min. Untreated bacteriocin
sample was used as a control, and the residual antimicrobial activity was measured using AWDA method [17].
Effect of Enterocin LD3 on Viability of the M. luteus
MTCC 106 Indicator Strain
To understand the bactericidal or bacteriostatic nature of
enterocin LD3, M. luteus cells were incubated with the
bacteriocin as suggested by Kumar et al. [19]. About *106
actively growing cells of the indicator strain were resuspended in 0.8 % NaCl containing 100 AU ml-1 of bacteriocin and incubated at 37 °C. The bacteriocin’s effect on
actively growing cells was studied as well. For this, M.
luteus cells were inoculated in NB medium containing
100 AU ml-1 of bacteriocin at A600 0.02. Samples were
withdrawn at regular time intervals of 2 h up to 8 h for the
determination of viable cell counts (CFU ml-1) and growth
(A600). Control set was incubated without bacteriocin
treatment.
Detection of Intracellular ATP and Membrane
Potential
The effect of enterocin LD3 on the intracellular ATP
content and transmembrane electrical potential (Dw) of the
indicator strain M. luteus was determined using the method
as previously reported [20, 21]. ATP measurements were
taken using the commercially available ATP
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164
1.2
16
(a)
14
12
A280
0.8
10
0.6
8
6
0.4
4
0.2
0.0
2
0
5
10
15
20
25
30
35
Bacteriocin activity
(inhibition zone in mm)
1.0
0
Fraction numbers
(b)
10
0.6
A280
8
6
0.4
4
0.2
2
0.0
0
1
2
3
4
5
6
7
8
9
10 11 12 13
Bacteriocin activity
(inhibition zone in mm)
12
0.8
0
Fraction numbers
0.30
(c)
0.573
0.25
0.20
AU
Bioluminescent Assay Kit (Sigma, St. Louis, USA).
Briefly, M. luteus cells were washed with 50 mmol l-1
HEPES buffer, pH 6.5 and energized with 0.2 % glucose.
For time-dependent assay, extracellular ATP level of the
energized cells treated with enterocin LD3 (100 AU ml-1)
was measured at 15, 30 and 45 min after bacteriocin
addition. The control set was run without bacteriocin
treatment. To determine total (intracellular and extracellular) levels of ATP, 20 ll of the cell suspension was
mixed with 80 ll of dimethylsulfoxide (DMSO) (SigmaAldrich, St-Louis, USA) and kept at room temperature for
5 min to permeabilize the cells. Light emission was
detected using a luminometer (Luminoskan TL Plus,
Labsystems Oy, Helsinki, Finland). Intracellular ATP was
determined by calculating the difference in the total and
extracellular ATP concentrations.
For determination of Dw, fluorescence measurements
were initiated followed by the addition of 5 lM of the
fluorescent probe, 3,30 -dipropyltyiadicarbocyanine iodide
[Di-S-C3-(5)] (ThermoFisher, NY, USA) and 10 ll of the
M. luteus cell concentrate. After the stabilization of the
fluorescence signal, enterocin LD3 (100 AU ml-1) was
added. Further, valinomycin (10 lmol l-1) was added to
collapse any remaining residual Dw. A PerkinElmer fluorescence spectrometer LS50B (Fremont, CA, USA) with
excitation and emission wavelengths of 643 and 666 nm,
respectively, with a 10-nm slit width and an 1800-s assay
duration with reading every 0.1 s was used for all assays.
Probiotics & Antimicro. Prot. (2016) 8:161–169
0.15
0.10
0.05
Statistical Analysis
Experiments were performed in triplicate, mean values
were plotted along with standard deviation (mean ± SD),
and p \ 0.05 was considered as statistically significant.
The membrane potential assay was done in singlet but
repeated three times to monitor the reproducibility of the
result.
Results
Purification, Mass Spectrometry and N-Terminal
Sequencing
Two initial saturation levels of ammonium sulfate precipitates (0–30 and 31–50 %) did not show antimicrobial
activity and were discarded. However, saturation level
(51–90 %) demonstrated antimicrobial activity and was
further purified. The elution profile on CEC revealed two
separate peaks where only the first peak demonstrated
antimicrobial activity (Fig. 1a). The fractions corresponding to the active peak were pooled and applied on gelfiltration chromatography (GFC) where bacteriocin was
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0.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Minutes
Fig. 1 Elution profile of protein concentration (open circle) and
bacteriocin activity (closed circle) of enterocin LD3 on cationexchange (a) and gel-filtration chromatography (b). Single absorbance peak was found on RP-UPLC. Absorbance unit (AU) was
detected at 280 nm (c)
resolved into a broader peak with shoulder appearance. The
antimicrobial activity was present only in fractions (fraction no. 4 and 5) corresponding to major peak (Fig. 1b).
Further, the active fractions eluted from GFC were pooled
which demonstrated single sharp peak (Fig. 1c) when
applied on RP-UPLC. This indicated the homogeneity of
the purified bacteriocin sample. These active fractions were
further collected in repeated experiments and used for
characterization of the enterocin LD3.
Table 2 represents the complete purification scheme of
enterocin LD3. The total protein content in 2 L CFS was
2.8 9 104 lg and activity 4.0 9 105 AU. After ammonium
sulfate precipitation, volume was reduced to 50 ml and
specific activity was increased by 3.5-fold. Thereafter,
Probiotics & Antimicro. Prot. (2016) 8:161–169
165
Table 2 Summary of purification of enterocin LD3 from culture-free supernatant of Ent. hirae LD3
Purification steps
Volume (ml)
Total protein (lg)
Total activity
(AU)
Specific activitya
Purification foldb
Yieldc (%)
Culture supernatant
2000
2.8 9 104
4.0 9 105
14.2
1
100
2
4
Ammonium sulfate precipitation
50
4.0 9 10
2.0 9 10
50
3.5
5
Cation-exchange chromatography
30
90
6.0 9 103
66.6
4.6
1.5
Gel-filtration chromatography
1.5
8
1.5 9 103
187.5
13.2
0.37
a
Specific activity (SA) = Total activity/Total protein
b
Purification fold = SA of sample/SA of crude
c
Yield (%) = Total AU of 9 100/Total AU of crude
subsequent increase in the specific activity and purification
fold was recorded. In purified active fraction (GFC eluted),
specific activity was found to be 187.5 AU lg-1 with a
total yield of 0.37 % indicating about a 13-fold purification. This sample was considered to be pure and tested for
further molecular properties of enterocin LD3. The
MALDI-TOF/MS analysis of purified enterocin LD3
resolved to be single peak with m/z 4114.6 suggesting the
homogeneity and a small peptide (Fig. 2). Using Edman
degradation method, only seven amino acid residues were
identified at amino-terminal group: H2N-QGGQANQ–
COOH.
Biochemical Characterization
Purified enterocin LD3 was active in a range of pH 2.0–6.0;
however, its activity decreased at pH 7.0 and was completely lost at pH 8–10. Bacteriocin activity was retained
one hundred percent when the pH was adjusted back to pH
4.0. Buffers of related pH were used as a control and did
not show any growth inhibition. Enterocin LD3 remained
100 % active after 15 min at 100 °C and autoclaving at
121 °C under 15 psi pressure (Table 3). Thus, enterocin
LD3 was found to be highly thermostable and active at
acidic to near-neutral pH range. Similar stability was
observed when the sample was treated with different
organic solvents, surfactants and detergents. The bacteriocin activity was not affected by the reagents tested
(Table 3). When bacteriocin sample was treated with proteolytic enzymes such as pepsin, trypsin and proteinase K,
the inhibitory activity was reduced and completely lost in
the presence of papain; however, amylase and lipase did
not influence the activity of enterocin LD3 (Table 3).
Enterocin LD3 inhibited Gram-positive and Gram-negative bacteria such as M. luteus, Lactobacillus curvatus
NRRL B4562, Lact. delbrueckii NRRL B4525, Lact. acidophilus NRRL B4495, Lact. plantarum NRRL B4496,
Lactococcus lactis subsp. lactis NRRL B1821, L. lactis
subsp. cremoris NRRL B634, Enterococcus faecium
NRRL B2354 and Enterobacter cloaceae NRRL B14298.
In addition, enterocin LD3 inhibited some human pathogens including those of food origin, specifically Staphylococcus aureus, E. coli (urogenic), E. coli O157:H7
Pseudomonas fluorescens Psd, Ps. aeruginosa, Salmonella
typhi, Shigella flexneri, Listeria monocytogenes and Vibrio
sp. (Table 3).
Effect of Enterocin LD3 on Viability of the M. luteus
MTCC 106 Indicator Strain
Fig. 2 MALDI-TOF/MS spectrum of purified enterocin LD3 showing m/z 4114.6
M. luteus cells were treated with purified enterocin LD3
(100 AU ml-1), and turbidity at OD600 and loss of viable
cell count in terms of CFU ml-1 were monitored. There
was no change in CFU ml-1 of the control cells populations. However, the loss in the viable count of indicator
strain was recorded after 2 h of treatment, and almost fourlog decrease in the CFU ml-1 was observed at the end of
8 h (Fig. 3). The observed loss in cell viability suggested
the bactericidal mode of action of enterocin LD3. There
was no growth (A600) of the indicator cells observed when
treated with bacteriocin in growth medium, whereas control was growing normally (Fig. 3).
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Probiotics & Antimicro. Prot. (2016) 8:161–169
Table 3 Biochemical and antimicrobial properties of enterocin LD3 purified from culture-free supernatant of E. hirae LD3
S. No.
Treatments
Bacteriocin activity (mm)
1.
pH 2.0
14
2.
3.
4.
pH 4.0 (control, no pH adjustment)
14
pH 6.0
12
pH 7.0
8
pH 8.0
nil
pH 10.0
nil
Control (without heat treatment)
14
80–121 °C 15 min, 15 psi
14
Control (without solvent treatment)
14
Ethanol, methanol, isopropanol, acetone, ethyl acetate, SDS, Tween 80, urea, Triton X-100
14
Control (without enzyme treatment)
Amylase, lipase (1000 units ml-1)
14
14
Pepsin, trypsin (1000 units ml-1)
12
Proteinase K (1000 units ml-1)
9
-1
Papain (1000 units ml )
5.
nil
Micrococcus luteus MTCC106
14
Lactobacillus curvatus NRRL B4562
8
Lact. delbrueckii NRRL B4525
8
Lact. acidophilus NRRL B4495
8
Lact. plantarum NRRL B4496
nil
Lactococcus lactis subsp. lactis NRRL B1821
8
L. lactis subsp. cremoris NRRL B634
nil
Enterococcus faecium NRRL B2354
8
Enterobacter cloaceae NRRL B14298
8
Staphylococcus aureus
8
E. coli (urogenic)
8
E. coli O157:H7
Pseudomonas fluorescens
11
8
Ps. aeruginosa
10
Salmonella typhi
8
Shigella flexneri
8
Listeria monocytogenes
13
Vibrio sp.
8
Bacteriocin activity was measured in terms of the diameter size of the zone growth inhibition in mm
Measurement of Intracellular ATP and Dissipation
of the Membrane Potential
Loss of internal ATP and dissipation of the membrane
potential (Dw) were studied to further elucidate the mode
of action of enterocin LD3. The DMSO-treated cells were
used to determine total ATP concentration. Here, external
ATP concentration was measured after bacteriocin treatment. The internal ATP concentration was calculated by
subtracting the external with that of total ATP concentration. There was an increase in external ATP concentration,
and corresponding decrease in internal ATP was recorded.
123
The loss of internal ATP concentration in bacteriocintreated cells was noticed after 5 min of treatment, and this
loss continued to increase with the incubation time (Fig. 4).
The untreated cells showed almost similar internal ATP
concentration without significant change throughout the
incubation period.
For measurement of dissipation of Dw, probe-loaded
cells were treated with purified enterocin LD3. There was
an increase in the fluorescence, when probe was added
which was stabilized later. The addition of indicator cells
in probe solution caused a decrease in fluorescence. The
fluorescence was found stable after the energization of cells
Probiotics & Antimicro. Prot. (2016) 8:161–169
167
1.0
8
7
6
0.6
A600
log10 CFU mL
-1
0.8
5
0.4
4
0.2
3
2
0
2
4
6
8
0.0
Time (h)
Fig. 3 Loss of CFU mL-1 of treated (open square) and untreated
(closed square) M. luteus cells. The growth inhibition (A600) of
treated (closed circle) and untreated (open circle) cells of indicator
strain Micrococcus luteus by enterocin LD3
Internal ATP conc (µM)
35x10-12
30x10-12
25x10-12
20x10-12
15x10-12
10x10-12
0
15
30
45
Time (min)
Fig. 4 Loss of intracellular ATP of M. luteus cells treated with
enterocin LD3 (open circle). The untreated cells (closed circle) show
almost similar ATP content throughout the incubation period
with glucose and further ionophore (nigericin) treatment.
However, an increase in fluorescence was recorded just
after enterocin LD3 was added, indicating the release of
probe into medium. There was no further increase in the
fluorescence recorded after the addition of valinomycin,
suggesting complete dissipation of Dw in the targeted cells
treated with purified enterocin LD3 as shown in Fig. 5.
Discussion
Only two LAB bacteriocins, nisin and pediocin, are commercially utilized as food preservatives (Perez et al. [2].
Therefore, there is a need to characterize other bacteriocins
of LAB and elucidate their potential applications in food
safety as well as in therapeutics. The mechanism of action
of these bacteriocins is known to be pore formation and/or
membrane perturbation [22]. Here, we report on the
purification of enterocin LD3 from the previously characterized E. hirae LD3 strain with probiotic potential [14]. In
addition, for the bacteriocin’s mode of action, targeted
biochemical and molecular properties were elucidated.
The use of activity-guided multi-step chromatography
techniques had been quite common practice and successful
for purification of several bacteriocins [2, 18, 23, 24].
Therefore, these methods were used for purification of
enterocin LD3. It was purified to homogeneity and confirmed by RP-UPLC and mass spectrometry showing a
single peak. The presence of a single peak identified with
reverse-phase column and MALDI-TOF/MS analysis
shows the purity of the molecule which has also been
suggested by others [4, 18, 23]. During the course of
purification, increase in specific activity (up to
187.5 AU lg-1) and purification fold (13-fold) validated
the feasibility of the process.
Purified enterocin LD3 was characterized for targeted
biochemical properties. It was active after exposure to acidic
and near-neutral pH and completely lost its antimicrobial
activity in alkaline pH ranges and found to be stable at high
temperatures such as 100 and 121 °C at 15 psi. Similarly,
bacteriocins purified from several LAB strains were also
found to be stable at different pH and higher temperatures
[18, 25, 26]. Temperature and pH stability are one of the
most important properties for an effective food preservative.
Such stability of enterocin LD3 at different pH levels,
temperatures and in various organic solvents would also
facilitate the purification process of the bacteriocin.
MALDI-TOF/MS analysis of enterocin LD3 was found to
be m/z 4114.6 which is unique. To the best of our knowledge, no other bacteriocins are reported with the same m/z
ratio. Therefore, we speculate that enterocin LD3 may be a
new bacteriocin. Further, N-terminal amino acid sequencing
revealed first seven amino acid residues and demonstrated
lack of homology with known bacteriocins when compared
using pBLAST (NCBI). Therefore, mass analysis and partial
amino acid sequence of enterocin LD3 support our speculation on a new bacteriocin from Ent. hirae LD3.
Inhibition of Gram-negative bacteria is the unique feature of a few LAB bacteriocins [4, 17, 27, 28]. Enterocin
LD3 was able to inhibit some human pathogens such as S.
aureus, P. fluorescens Psd, P. aeruginosa, S. typhi, S.
flexneri, L. monocytogenes, E. coli O157:H7, E. coli
(urogenic) and Vibrio sp. This indicated its possible use in
food preservation and as a therapeutic agent. Inhibition of
food-borne pathogens would be an additional benefit for
the putative probiotic strain LD3 if utilized in food products. Bactericidal nature of enterocin LD3 was confirmed
by the loss of cell viability and further confirmed by the
loss of internal ATP and dissipation of membrane potential
of indicator cells. There was an increase in the external
123
Probiotics & Antimicro. Prot. (2016) 8:161–169
Fluorescence (au)
168
1000
950
900
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0.1
Micrococcus luteus cells
Valinomycin
Enterocin LD3
Probe
[DiSC3(5)]
Nigericin
Glucose
0.0
100
200
300
400
500
600
700
800
Time (s)
900
1000
1100 1200
1300
1400
1500
Fig. 5 Dissipation of membrane potential of indicator strain Micrococcus luteus by enterocin LD3
ATP, and subsequent decrease in internal ATP concentrations suggested the formation of pores and efflux of ATP.
Further, dissipation of membrane potential of M. luteus
cells also indicated the pore-forming nature of enterocin
LD3 which is a typical mechanism of action found in many
bacteriocins [21, 29–31]. The role of some bacteriocins in
therapeutics and other related areas has been recently
reviewed, and these bacteriocins were suggested for further
characterization and industrial scale production [9, 32].
Enterocin LD3 inhibited a range of Gram-positive and
Gram-negative bacteria including food-borne and human
pathogens suggesting its possible applications in food
biopreservation and therapeutics as well.
Conclusions
Enterocin LD3 purified to homogeneity has a unique mass,
and its N-terminal sequence suggests a novel compound.
Enterocin LD3 has bactericidal activity against tested
sensitive strains and causes dissipation of membrane
potential and the efflux of ATP, suggesting pore-forming
nature of its activity. The broad range of pathogen inhibition suggests its possible applications in food safety as well
as in therapeutics. This is the first study that demonstrated
the characterization of a new enterocin produced by E.
hirae LD3 strain isolated from a fermented food, Dosa. The
biochemical properties and host range of purified enterocin
LD3 suggest its applications as a food biopreservative and
as an alternative to clinical antibiotics.
123
Acknowledgments The present work and AG were financially supported by University Grant Commission, New Delhi, under major
research project (No. 36-38/2008 SR). SKT was supported by IndoUS Science and Technology Forum, New Delhi, and was truly
acknowledged.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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