Microbiological
Communication
Biosci. Biotech. Res. Comm. 9(3): 539-545 (2016)
Biochemical characterization and identi cation of
enterocins produced by
E. hirae
And
E. faecalis
Zia H. Khan* and J.H.Anandani
Department of Biochemistry, Shri Shivaji College, Akola
ABSTRACT
The present study was an endeavour to partially purify and characterize enterocins produced by E.hirae and E.faecalis
from UTI patients.Maximum enterococcal isolates were obtained from females of 10-20years age group and compared
with males of same age group. Enterocin was partially puri ed by ammonium sulphate precipitation technique fol-
lowed by dialysis and analysis by SDS-PAGE revealed molecular mass of enterocin to be 4.5-5.5 kDa approximately
.Partially puri ed enterocin was heat and pH stable and sensitive to proteolytic enzymes, thus proving it to be of
proteinaceous nature and was therefore characterized as enterocin. Such bacteriocin have broad eld of application
including both food industry and medical sector.
KEY WORDS:
ENTEROCOCCUS HIRAE
,DIALYSIS,
E.FAECALIS
, ENTEROCIN, SDS-PAGE.
539
ARTICLE INFORMATION:
*Corresponding Author: ziakhan7862@rediffmail.com
Received 25
th
July, 2016
Accepted after revision 5
th
Sep, 2016
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007
Thomson Reuters ISI ESC and Crossref Indexed Journal
NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006
© A Society of Science and Nature Publication, 2016. All rights
reserved.
Online Contents Available at: http//www.bbrc.in/
INTRODUCTION
For years, Enterococci have been considered as harm-
less inhabitants of the gastrointestinal tracts of humans
and animals. The genus Enterococcus is a heterogenous
group of bacteria, which includes 20 different species.
The interest on Enterococci is raised in the last decades
mainly for 2 important characteristics; they are con-
sidered as infection agents especially in immune com-
promised hosts and they are used as useful probiotics
and starter cultures in various fermented food (Maki
and Agger 1998; Leroy et al., 2003; Manolopoulou
et al., 2003).
Enterococci produce bacteriocins, Known as enterocins
which are proteinaceous compounds produced by bacte-
ria that exhibit a bactericidal or bacteriostatic mode of
action against sensitive gram positive and gram nega-
tive bacteria. Enterococci are part of the normal intesti-
nal micro ora and may become opportunistic pathogens
in individuals with in serious diseases whose immune
systems are compromised. E.faecium and E.faecalis are
known to produce enterocins which possess heat sta-
bility and antilisterial activity, (Wilaipun et al., 2004,
Enan, 2006, Sawa et al., 2009, Jamaly et al., 2010).
Urinary Tract Infection (UTI) is a common infection
prevalent among various age groups. Although UTI is
540 BIOCHEMICAL CHARACTERIZATION AND IDENTIFICATION OF ENTEROCINS PRODUCED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Zia Khan and Anandani
common in women it can affect both gender and all age
groups. Untreated UTI in a long run can lead to many
complications such as pyelonephritis and renal damage.
Although Enterococci is commonly isolated from UTI it
is less often speciated. Speciation and antibiogram of
enterococci is gaining relevance because of emerging
antimicrobial resistance.The selection and identi cation
of a enterocin produced be Enterococcal strain isolated
from urine is of interest because it can be used as probi-
otic bacterium inhibit other bacterial pathogens (Gamal
et al., 2006 and Civamani et al., 2014).
Many enterococci produce enterocins which are short
peptide as a defense mechanism. Bacteriocins( enterocins)
and are used bioactive peptides and most are cationic at
physiological pH. This peptide is highly active against
pathogenic bacteria and it plays a dual mode of action
at high concentration, it produces localized holes in cell
wall and cellular membrane which leads to leakage of
macromolecule such as protein into external medium
and cause death of pathogenic organism; at lower con-
centration; it modi es the ion permeability of the cells,
discipating both components of proton motive force
(Minahk et al., 2004).Enterocins have developed a great
deal of interest as an approach to control food borne
diseases to be used as starter culture and biopreservative
in various food products. In some cases,enterocin are
used as probiotics as a result of their protective effects
in GIT (Khan et al. 2016)
Thus, considering all this, the study was undertaken to
characterize enterocins from E.hirae & E. faecalis and to
evaluate its inhibitory activity against indicator strain.
The antimicrobial activity was measured in Arbitrary
units(AU/ml). Molecular size was also determined along
with its activity against heat, pH, proteolytic enzymes
and storage conditions.
MATERIAL AND METHODS
STUDY DESIGN
A descriptive study was performed at Biochemistry
Dept., Shri Shivaji College of Arts, Commerce and Sci-
ence, Akola. The urine specimens were collected from
government hospital, private hospital and pathology
laboratory. Patients with complaints of fever, burning
micturition and pain lower abdomen were included in
the study. The urine samples were collected and pro-
cessed in the laboratory by standard methods. The name,
age, sex and date of onset of symptoms was noted. On
isolation of enterococci, the organism was speciated and
its activity was checked using different parameters. All
the data obtained was recorded using MS Excel Soft-
ware.The statistical analysis included statistics like fre-
quency percentage, mean and standard deviation.
BACTERIAL IDENTIFICATION
Total one hundred and eight urine samples were col-
lected for Enterococcal isolation. Samples were collected
in sterile broth medium and transferred immediately to
laboratory for further processing. Samples were inocu-
lated onto De Man, Rogosa and Sharpe broth for enrich-
ment purpose and incubated at 30
o
c for 24-48 hrs. The
enriched cultures were then analyzed for isolation of
relevant organism. The isolation was performed by the
routine microbiological procedure and inoculation was
performed on selective and differential media viz Ente-
rococcus Con rmatory Agar, De Man Rogosa, Sharpe
Agar and Bile Esculin Agar. All plates were then incu-
bated at 30
o
c for 24-48 hrs.
SCREENING OF ENTEROCIN PRODUCING
ISOLATES
All enterococcal isolates were screened for enterocin
production by Agar-well diffusion method against indi-
cator strain S. aureus. Enterococcal isolates were grown
in Brain Heart Infusion broth and incubated at 37
o
C for
16-18h. For extraction of enterocins, bacterial cells were
removed by centrifugation at 10,000x g, for 30min at
4
o
C. After centrifugation, the supernatant was adjusted
to pH7.0 with 0.1N NaOH. This is cell-free neutralized
supernatant, also designated as crude preparation. Brain
Heart infusion agar plates were overlaid with 3.0mL soft
agar containing 0.1mL (approximately1X-106CFU/mL)
of the indicator organism. Wells (5mm diameter) were
cut and 100μL of cell-free neutralized supernatants of
the test organism were poured into each well. Plates
were incubated at 37
o
C for overnight. A clear zone sur-
rounding the bacteriocin producer colonies after growth
of the indicator strain was consider as bacteriocin posi-
tive. Inhibition zone around the wells were measured
and recorded.
PARTIAL PURIFICATION OF ENTEROCIN
Ammonium Sulfate Precipitation and Dialysis
Partial puri cation of enterocins was carried out by
using ammonium sulphate precipitation method (Harris
et al., 1989 ) The enterocin producer isolates were grown
in Brain Heart Infusion broth at 37
o
C for 16-18hrs.
The bacterial cells were removed by centrifugation at
10,000x g, for 30min at 4
o
C and supernatant was col-
lected. The ammonium sulfate was added slowly to the
cell free neutralized supernatant with constant stirring
(using magnetic stirrer) till the level of 80% saturation
was achieved. The pellet obtained was then suspended in
20 mM sodium phosphate buffer in dialysis bag(Dialysis
tubing D0405 Sigma Aldrich) and was dialysed for over-
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOCHEMICAL CHARACTERIZATION AND IDENTIFICATION OF ENTEROCINS PRODUCED 541
Zia Khan and Anandani
night at 4
o
C with constant stirring The system was held
for overnight at 4
o
C and the precipitates were recovered
by centrifugation (10,000x g, for 30 min at 4
o
C). The
resulting pellet was solubilized in 20mM sodium phos-
phate buffer of pH6.8. The sample thus obtained was des-
ignated as crude preparation. The antimicrobial activity
of this sample was assayed by using agar-well diffusion
method and described in terms of AU/mL. One arbitrary
unit (AU) of enterocin was de ned as the reciprocal of
the serial dilution that showing a clear inhibition zone,
multiplied by a factor of 100 (to obtained AU/mL).
Quantitative Determination of Enterocin Activity
The agar well-diffusion method was performed, to deter-
mine antimicrobial activity of enterocin. Brain Heart
Infusion Agar plates were pre-inoculated with 3.0ml
soft agar containing (approximately 1x 106 CFU/ mL)
of indicator organism and wells of 6 mm diameter were
bored in it. Two-fold serial dilutions of cell-free neutral-
ized supernatant in sterile phosphate buffer (pH 0.7) were
made and 100μL of each two-fold dilution was pipet-
ted into each well. The plates were incubated at 37
0
C +
2
o
C for 16-18hrs and diameter of zone of inhibition was
measured in mm. The inhibitory strength was expressed
as arbitrary unites or activity units/ mL. One arbitrary
unit (AU) of enterocin was de ned as the reciprocal of
the serial dilution that showing a clear inhibition zone,
multiplied by a factor of 100 (to obtained AU/mL).
Effect of Physicochemical Treatments on
Enterocin Activity
Thermo Stability Test:-To evaluate the thermal stability,
1ml of enterocin preparations was exposed to different
temperatures viz.,60
o
C for (60 min), 80
o
C for (40 min),
100
o
C for (30min), and 121
o
C for (15 min). Activity was
checked by agar well diffusion assay (Iqbal et al., 1999).
Stability at Different pH Values: Enterocin activity
was also checked by placing it on wide range of pH.The
supernatant pH levels were adjusted between 2.0 and
12.0 using 1 N HCl and 1 N NaOH. The pH stability was
assayed at room temperature (25°C) after 1 and 24 hrs
of incubation of partially puri ed enterocin solutions.
After incubation, the tested supernatant was re-adjusted
to neutral pH and assayed for activity. Untreated sam-
ples were used as the control.
Effect of Proteolytic Enzymes: Sensitivity of
the enterocin to proteolytic enzymes trypsin,
-chymotrypsin,lipase, lysozyme and catalase was
tested against partially puri ed enterocin samples. Each
enzyme was dissolved in 10 mM sodium phosphate
buffer (pH 7.0) and the solutions were added to the
enterocin solution for a nal concentration of 1 mg/
ml following incubation at 37 °C for 2 hrs. Untreated
samples were used as the control. The residual enterocin
activity was determined by agar-well diffusion method
(Ahmad et al., 2003). Sensitivity to Chloroform: To test
for chloroform sensitivity the culture supernatant was
mixed with an equal volume of chloroform and kept at
room temperature for 4 hrs before assessing the anti-
microbial activity.Protein Quantitation: Protein estima-
tion from crude enterocin preparation was carried out by
using Lowry method (Lowry et al., 1951).
Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) Pro ling of Partially
Puri ed Enterocin
Molecular weight of different enterocins were determined
from fractions from ammonium sulfate precipitated frac-
tion by performing 16% Tris-Tricin Sodium dodecyl sul-
fate poly-acrylamide gel electrophoresis (SDS-PAGE).
Standard molecular weight marker procured from GeNei
TM(GeNei
TM
1500 - 10,000) was used as reference molec-
ular weight marker. Partially puri ed enterocin solutions
obtained from different isolates, were loaded on the gel.
After electrophorsis, the gel was xed with a solution
containing 15% ethanol and 1% acetic acid. The gel was
then washed with distilled water for 4 hrs. The gel was
stained with solution containing 0.15% Coomasie brilliant
blue R-250 in 40% ethanol and 7% acetic acid to iden-
tify the enterocin. The gel was then sequentially washed
with phosphate buffered saline for 1hr and subsequently
deionized water for 3 hrs. (Parbal 1988)
RESULTS AND DISCUSSION
Bacterial Isolation and Identi cation: In the present
study, total 108 urine samples were collected from UTI
patients. Mean values in Table 1 states that maximum
isolates obtained were from females as females are more
prone to urinary tract infections. The gures related to
standard deviation shows that heterogenity is more in
males of all age groups as compared to females with
respect to their dietary habits, physical workout and mode
of living. Morphological, physiological and biochemical
identi cation of enterococcal isolates were carried out
according to Standard microbiological technique. Out of
108 samples 40 enterococcal isolates were obtained and
identi ed as E. hirae 14 (35%) and E.faecalis 26 (65%)
Results were expressed in mean (standard deviation)
Max isolates(45.37%) were seen in 10-20yrs age
group followed by 21-40 yrs age group(34.25%). 62.03%
represents that max isolated from females as compared
to 41(37.96%) from males.
Preliminary screening of enterocin producing
enterococci
Screening of enterocin producing enterococci was done
by agar well diffusion method is summarized in table no.2
542 BIOCHEMICAL CHARACTERIZATION AND IDENTIFICATION OF ENTEROCINS PRODUCED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Zia Khan and Anandani
Table 1: Age and Sex distribution of Isolates
Age groups(yrs) Male Female Total Standard Deviation
10-20 yrs 20 (18.51%) 29 (26.85%) 49 (45.37%) 2.84
20-40 yrs 13(12.03%) 24(22.22%) 37 (34.25%) 5.13
40-60 yrs 8 (7.40%) 14(12.96%) 22 (20.37%) 3.56
Total 41(37.96%) 67(62.03%) 108 (100%)
Standard Deviation (13.16) (12.50) (12.71)
Table 2: Frequency of enterocin production screened by Agar Well
Diffusion Method
Identi ed strains No.of producer strains/
No.of tested strains
Frequency Percentage
(%)
E.hirae 8/14 57.14
E. faecalis 18/26 69.23
Total 26/40 65
Table 3: Partial Puri cation of Enterocin from cultural supernatent of E.hirae and E. faecalis
Sr. No. Sample / Step Volume
(ml)
Activity
units
(AU/ml)
Total
activity
(AU)
Protein Conc.
(mg/ml)
Total
Protein
(mg)
Speci c
activity
Activity
recovered
Fold
puri cation
1) E.hirae EHE 8
Culture Supernatent 1000 160 160000 3.80 3800 42.10 100 1
Ammonium sulphate
precipitation 80%
100 640 640000 2.8 280 2285.71 400 54.29
2 E. faecalis EFE 9
Culture Supernatent 1000 160 160000 4.5 4550 35.16 100 1
Ammonium sulphate
precipitation 80%
100 640 640000 4.6 460 1391.30 400 39.57
3 E.hirae EHE 10
Culture Supernatent 1000 160 160000 3.6 3600 44.44 100 1
Ammonium sulphate
precipitation 80%
100 640 640000 4.2 420 1523.80 400 34.28
4 E. faecalis EFE 15
Culture Supernatent 1000 160 160000 4.7 4700 34.04 100 1
Ammonium sulphate
precipitation 80%
100 640 640000 4.3 430 1488.37 400 43.72
5 E.hirae EHE 18
Culture Supernatent 1000 160 160000 3.5 3500 45.07 100 1
Ammonium sulphate
precipitation 80%
100 640 640000 4.1 410 1560.97 400 34.63
6 E.faecalis EFE 21
Culture Supernatent 1000 160 160000 4.3 4300 37.20 100 1
Ammonium sulphate
precipitation 80%
100 640 640000 5.0 500 1280.00 400 34.40
The obtained isolates were screened for their entero-
cinogenic potential against speci c indicator strain of
S.aureus. It was observed that out of 108 samples 40
enterococcal isolates were obtained showing strong
inhibitory activity. Amongst these 40 isolates, 8 (57.14%)
out of 14 were found to be E.hirae and 18 (69.23 %)
out of 26 of E.faecalis were found to be ef cient pro-
ducer of enterocin. For the assessment of antimicrobial
activity shown by ef cient enterocin producers six iso-
lates were selected for further study. These were desig-
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOCHEMICAL CHARACTERIZATION AND IDENTIFICATION OF ENTEROCINS PRODUCED 543
Zia Khan and Anandani
Table 4: Effect of different temperature on Enterocin activity
Sr. No. Enterocin Temperature treatment
60°C for
60 min
80°C for
40 min
100°C for
30 min
121°C for
15 min
1 EHE 8 + + + -
2 EFE 9 + + + -
3 EHE 10 + + + -
4 EFE 15 + + + -
5 EHE 18 + + + -
6 EFE 21 + + + -
(+) - Activity retained (-) – Activity lost
Table 5: Effect of different pH on Enterocin activity
Sr. No. Enterocin pH treatment
23456789101112
1 EHE 8 + + + + + + + - - - -
2 EFE 9 + + + + + + + - - - -
3 EHE 10 + + + + + + + - - - -
4 EFE 15 + + + + + + + - - - -
5 EHE 18 + + + + + + + - - - -
6 EFE 21 + + + + + + + - - - -
(+) - Activity retained (-) – Activity lost
Table 6: Effect of different Enzymes on Enterocin activity
Sr. No. Enterocin Catalase Trypsin -chymotrypsin Lipase
(pepsin A)
Lysozyme
1 EHE 8 + - - + +
2 EFE 9 + - - + +
3 EHE 10 + - - + +
4 EFE 15 + - - + +
5 EHE 18 + - - + +
6 EFE 21 + - - + +
(+) - Activity retained (-) – Activity lost
Abreviations: Enterocin from respective isolates,
1.EHE 8- E.hirae EHE8 , 2.EFE 9- E.faecalis EFE 9, 3.EHE 10- E.hirae EHE10
4.EFE 15-E.faecalis EFE 15, 5.EHE 18- E.hirae EHE18, 6 EFE 21-E.faecalis EFE 21
nated as E.hirae EHE8, E.faecalis EFE9 E.hirae EHE10
E.faecalis EFE15 E.hirae EHE18 E.faecalis EFE21. Their
enterocins were named by adding enterocin to speci c
isolate number. One of the most important characteris-
tic of enterocin is evaluation of susceptibility for differ-
ent antibiotics.The obtained isolates were susceptible to
commonly used antibiotics like ampicillin, amoxycilin
while resistant to nor oxacin only.Thus stating its use
as ef cient and safe for clinico-medical sector(Khan
et.al.2016)
Partial Puri cation of Enterocin: The partial puri ca-
tion of enterocin from cell free supernatant of E.hirae
and E.faecalis was done as mentioned in table no.3.The
antibacterial activity was determined in terms of activity
units AU/ml. The antibacterial activity for cell free neu-
tralized supernatant and partially puri ed enterocin was
found to be 160000 AU/ml and 640,000AU/ml respec-
tively. It was seen that the speci c activity of enterocin
EHE8 in cell free supernatant was 42.10(AU/mg) which
was increased upto 2285.71 (AU/mg) after ammonium
sulphate precipitation and dialysis. Speci c activity of all
remaining enterocins were found as EFE9 1391.30(AU/
mg) ,EHE10 1523.80(AU/mg), EFE15 1488.37(AU/mg),
EHE18 1560.97(AU/mg) EFE21 1280(AU/mg).Thus, spe-
Zia Khan and Anandani
544 BIOCHEMICAL CHARACTERIZATION AND IDENTIFICATION OF ENTEROCINS PRODUCED BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
PLATE 7. Bands 1 and 2 represents 4.8kDa and
5.3kDa of EHE8 and EFE9 respectively.
PLATE 8. Bands 1 and 2 represents 5.5kDa and
4.7kDa of EFE21 and EHE18 respectively
ci c activity after ammonium sulphate precipitation and
dialysis was found to be increase in all crude enterocins
as compared to cell free supernatant.
Effect of physicochemical treatment on Enterocin
activity: The effect of thermal treatment is as summa-
rized in table no 4. All crude enterocins were stable
following boiling for 30mins.The antimicrobial activ-
ity remained unaffected when heated at 100
o
C for
30min but activity gets reduced or lost beyond 121
o
C
for 15min. All the tested enterocins were stable to wide
range of pH from 2-8 as shown in table 5. Table 6 shows
the effect of various enzymes on inhibitory activity of
enterocins .It demonstrated sensitivity to proteolytic
enzymes trypsin and chymotrypsin while treatment
with catalase, lipase and lysozyme did not affect the
activity of enterocin.Thus,the obtained data suggests
that the loss of enterocin activity to proteolytic enzyme
is due to the fact that enterocin contained an essential
proteinaceous component while stability in presence of
both lipase and lysozyme indicates that it lacked lipid
or carbohydrate moieties. Similar enterocin activities to
different physicochemical parameters were also seen in
Cocolin et al.,(2007) Our results are also in accordance
with the ndings of Ahmad et al., (2003) and Dezwaan
et al.(2007).
SDS-PAGE Pro ling of Partially Puri ed Enterocin:
The crude enterocin obtained were further characterized
by SDS PAGE analysis. The results of partial puri cation
of obtained enterocin fraction from E.hirae and E.faecalis
are depicted in plate 7 and plate 8.SDS-PAGE pro ling
revealed protein bands having molecular mass in range
of approximately 4.5 to5.5kDa.Bands were observed in
lane 1 and 2 showed partially puri ed enterocin from
cultures of E.hirae EHE8 and E.faecalis EFE 9 represent-
ing molecular mass approximately 4.8kDa and 5.3kDa
in plate 7. Bands 1 and 2 in plate 8 represents molecu-
lar mass of bands from partially puri ed enterocins
from cultures E.faecalis EFE21 and E.hirae EHE 18 as
5.5kDa and 4.7kDa respectively. In the present study the
molecular weight observed is similar to those obtained
inearlier studies. Line et al., also characterized enterocin
by SDS-PAGE analysis which reveals 5.5kDa enterocin
fraction. Similarly, Park et al., identi ed partialy puri ed
bacteriocin produced by E.faecium JCM 5804
T
,enterocin
A and enterocin B of molecular mass 4.5kDa approxi-
mately.
CONCLUSION
The current study thus describes the biochemical charac-
terization of enterocin from E.hirae and E.faecalis from
urine samples.It was found that enterocin was heat sta-
ble and stable over wide range pH2-8 sensitive to prote-
olytic agents trypsin and chymotrypsin.Molecular size
ranges 4.5-5.5kDa which states that obtained enterocin
may belong to class IIa bacteriocin. Several enterocins
have been characterized to date,many of which are pro-
duced by Enterococci which have potential to cover a
very broad eld of applications including both the food
industry and clinico-medical sector.
ACKNOWLEDGEMENT
The help rendered for performing statistical analysis by
Dr. S.T.Khadakkar, Head Department of Statistics, Shri
Shivaji College, Akola is acknowledged.
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