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Journal of Basic Microbiology 2012, 52, 1 – 10 1
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
Research Paper
Antibacterial and Anticancer activity of ε-poly-L-lysine (ε-PL)
produced by a marine Bacillus subtilis sp.
Nermeen A. El-Sersy1, Abeer E. Abdelwahab2, Samia S. Abouelkhiir1, Dunja-Manal Abou-Zeid3
and Soraya A. Sabry3
1 National Institute of Oceanography and Fisheries, Microbiology Laboratory Environmental Division,
Alanfushy Qayt bay, Alexandria, Egypt
2 Medical Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI),
City for Scientific Research and Technology Application, Borg El-Arab, Alexandria, Egypt
3 Faculty of Science, Botany and Microbiology Department, El-Shatby, Alexandria, Egypt
A marine Bacillus subtilis SDNS was isolated from sea water in Alexandria and identified using
16S rDNA sequence analysis. The bacterium produced a compound active against a number of
gram negativeve bacteria. Moreover, the anticancer activity of this bacterium was tested
against three different human cell lines (Hela S3, HepG2 and CaCo). The highest inhibition
activity was recorded against Hela S3 cell line (77.2%), while almost no activity was recorded
towards CaCo cell line. HPLC and TLC analyses supported evidence that Bacillus subtilis SDNS
product is
ε
-poly-L-lysine. To achieve maximum production, Plackett-Burman experimental
design was applied. A 1.5 fold increase was observed when Bacillus subtilis SDNS was grown in
optimized medium composed of g/l: (NH4)2SO4, 15; K2HPO4, 0.3; KH2PO4, 2; MgSO4 ⋅ 7 H2O, 1;
ZnSO4 ⋅ 7 H2O, 0; FeSO4 ⋅ 7 H2O, 0.03; glucose, 25; yeast extract, 1, pH 6.8. Under optimized
culture condition, a product value of 76.3 mg/l could be obtained. According to available litera-
ture, this is the first announcement for the production of
ε
-poly-L-lysine (
ε
-PL) by a member of
genus Bacillus.
Keywords: ε-Poly-L-lysine / Bacillus subtilis / Plackett-Burman / Antibacterial / Anticancer
Received: June 16, 2011; accepted: August 18, 2011
DOI 10.1002/jobm.201100290
Introduction*
Multi-drug resistance is a world-wide problem, attrib-
uted to the extensive use of antibiotics, selection pres-
sure on bacterial strains and lack of new drugs, vac-
cines and diagnostic aids. These shortcomings lead to
an urgent global call for new antimicrobial drugs, par-
ticularly from natural resources [1].
Marine microorganisms represent a wide source of
yet undiscovered compounds that, besides unprece-
dented chemical structures, often possess interesting
biological activities [2]. They constitute a promising
source of unique metabolites with considerable phar-
maceutical and therapeutical potential. Common bio-
Correspondence: Prof. Dr. Nermeen Ahmed El-Sersy, National Insti-
tute of Oceanography and Fisheries, Microbiology Lab. Marine Environ-
mental Division, Qayetbay, Anfoushy, Alexandria, Egypt
E-mail: Nermeen_ok@yahoo.co.uk
Phone: 01006620217
logical assays usually focus on antimicrobial and cyto-
toxic activities as demonstrated throughout the litera-
ture [3].
Antimicrobial agents produced by Gram-positive bac-
teria have attracted much attention because of their
potential use as food preservatives. Some representa-
tives of Bacillus spp, such as B. subtilis and B. licheniformis,
are generally recognized as safe (GRAS) bacteria [4, 5].
ε
-Poly-L-lysine (
ε
-PL) is a basic homopolymer which
has strong antimicrobial activity against most gram-
positive and gram-negative bacteria, fungi and also
some kinds of viruses. Moreover, it is harmless to hu-
mans, biodegradable and has antitumor effect [6]. Thus,
it has been widely used in food manufacturing as a safe
preservative.
The nutritional and environmental conditions have a
great influence on production of antimicrobial agents.
To achieve maximum production, knowledge regarding
2 N. A. El-Sersy et al. Journal of Basic Microbiology 2012, 52, 1 – 10
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
the environmental factors affecting this process needs
to be well identified. Experimental designs are excellent
techniques for optimization of culture conditions to
achieve optimal production [7].
Because of
ε
-poly-L-lysine has been only detected by
members of family Streptomycetaceae, it was thus aimed
in this study to explore marine environment for strain
with capability to produce the polymer. Optimization
study will be applied to maximize polymer production.
Moreover, the study will be extended to prove its anti-
bacterial and anticancer activity.
Materials and methods
Bacterial strains
Bacillus sp. SDNS used in this study was isolated from
marine water samples of Alexandria, Egypt. Test bacte-
ria included Gram positive (Staphylococcus aureus, Strepto-
coccus faecalis) and Gram negative (E. coli, Pseudomonas
aeruginosa, Vibrio fluvialis, Vibrio vulnificus, Vibrio sp. N2).
Strains were kindly provided by Microbiological lab,
National Institute of Oceanography and Fisheries
(NIOF).
Chemicals and reagents
Bacteriological media components, chemicals, solvents,
reagents and dyes, of analytical grade were obtained
from commercial suppliers.
HeLa S3 cell line PCCL-2.2
(HeLaS3 cell line slides (human cervix adenocarcinoma),
Human HeLa S3 cells were cultured in Ham’s F12K
medium, HepG2 cell line ATTC#HB.8065(HepG2 Hu-
man hepatocellular liver carcinoma cell line) Whole
Cell Lysate, CaCo cell line HTB-37(Caco2 (Human colo-
nic carcinoma cell line) Whole Cell Lysate, were ob-
tained from (Abcam, USA).
Growth condition
M3G medium [8] was used for seed culture preparation
and production of the homopolymer in shake flasks. It
contained (g/l): glucose, 50; yeast extract, 5; (NH4)2SO4,
10; KH2PO4, 1.36; K2HPO4, 0.8; MgSO4 ⋅ 7 H2O, 0.5;
ZnSO4 ⋅ 7 H2O, 0.04; and FeSO4 ⋅ 7 H2O, 0.03. The pH was
adjusted to 6.8 with NH4OH solution (24–28%, w/v).
Glucose and yeast extract were autoclaved separately. A
standard inoculum (1%) was taken from previously
prepared seed cultures (OD600 ~ 0.8–1) and used to in-
oculate 50 ml portions of M3G medium dispensed in
100 ml flasks. Flasks were then incubated shaken at
160 rpm at 30 °C. Samples were taken regularly at time
intervals to measure growth and
ε
-PL concentration by
Itzhaki method [9].
Bacterial identification
DNA was isolated, purified and the region of 16S rDNA
was amplified using either universal primers or species-
specific primers. Genotypic characterization was per-
formed using 16S sequence analysis. Multiple align-
ments with sequences of most closely members and
calculations of levels of sequence similarity were car-
ried out using Bioedit [10]. Sequences of rRNA genes,
for comparison, were obtained from the NCBI database.
A phylogenetic tree was reconstructed by Bioedit [10].
Phenotypic characteristics were determined for the
selected isolate according to the standard methods [11].
Measurement of ε-poly-L-lysine
Samples were taken regularly at time intervals to
measure growth and
ε
-PL concentration by Itzhaki
method [9]. To measure the concentration of
ε
-PL, 1 ml
of 0.1 mM phosphate buffer and 2.0 ml 0.1 mM methyl
orange (MeO) solution were added to 1 ml of bacterial
supernatant. The mixture was vigorously shaken on a
reciprocal shaker at 30 °C and then centrifuged. The
optical density of resulting supernatant was measured
at 465 nm against an appropriate blank. Equivalent
ε
-PL
concentration was determined using the methyl orange
standard curve.
Optimization of nutritional factors
Cultivation medium optimization was performed using
the Plackett-Burman experimental design [12]. In this
experiment, seven independent variables were screened
in nine combinations organized according to the Plack-
ett-Burman design matrix. For each variable, a high (+)
and low (–) level was tested. All trials were performed
in triplicates and the averages of degradation observa-
tion results were treated as the response. The main
effect of each variable was determined with the follow-
ing equation:
Exi = (Σpi+ – Σpi–)/N
where Exi is the variable main effect, Σpi+ and Σpi– are
ε
-PL
production responses in trials where the independent
variable (Xi) was present in high and low concentra-
tions, respectively, and N is the number of trials divided
by 2. All trials were performed in triplicates and the
averages of percentages of
ε
-PL production results were
treated as the responses. Verification of validity of the
optimum medium compared to the basal medium and
the Plackett-Burman reverse medium was applied.
Antibacterial activity
The antibacterial effect of the bacteria isolated was
studied using the agar diffusion method [13]. Nutrient
Journal of Basic Microbiology 2012, 52, 1 – 10
ε
-Poly-L-lysine (
ε
-PL) produced by a B. subtilis sp. 3
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
agar plates were seeded with the test organisms includ-
ing Gram positive and Gram negative bacteria and
holes of 10 mm diameter were made in the plates using
a sterile cork borer. 50 μl of each supernatant were
transferred into each hole under aseptic conditions.
Plates were then incubated for 1 d at 37 °C. The anti-
microbial activity of the isolates was detected as clear
inhibition zone around holes and was measured (mm).
Anticancer activity
In order to evaluate the anticancer activity of the bacte-
rial filtrate, several steps were undertaken: lyophiliza-
tion, cytotoxicity test and effect of IC50 on three differ-
ent tumor cell lines. Lyophilization can be summarized
in three general steps: Freezing, primary drying and
secondary drying. 10 ml of filtrate were lyophilized and
converted into powder using a Telson lyophilizer
(Spain). Different concentrations (5, 25, 50, 75, 100 μg/
ml) of the powder were prepared and used in the sec-
ond step (cytotoxicity test). To measure cytotoxicity of
the examined compound, 5 × 104 lymphocyte cells were
seeded per well in 96 well plates and incubated in
RPMI1640 (Sigma-Aldrich Chemical Co) media contain-
ing different concentrations (5, 25, 50, 75, 100 μg/ml) of
the tested powder and incubated for 24 h in 5% CO2
incubator. Next, the media were removed and wells
were washed with Hank’s Balance Salt Solution (HBSS).
The fraction of viable lymphocytes was measured by
the MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetra-
zolium bromide).
In the MTT assay, yellow MTT is reduced to purple
formazan in the mitochondria of viable cells. 100 μl of
the MTT working solution (0.5 mg/ml) were added to
each well and incubated at 37 °C for 4 h. Next, media
were removed, wells were washed with PBS, and 100 μl
of dimethyl sulfoxide (DMSO) were added to solubilize
the formazan crystalline product. The absorbance was
measured with a plate reader at 570 nm and normal-
ized to the absorbance at 630 nm. Fraction of viable cell
was determined by dividing the normalized absorbance
of the test well to that of the control well [14]. Cytotox-
icity assay was performed on different human cell line
using the previously described method.
The anticancer activity of the substance was tested
using three different human cell lines (Hela S3, HepG2
and CaCo). Cells (2 × 104) were seeded per well in 96
well plates and incubated in Ham’s F-l2, RPMI and
DMEM, respectively for 24 h in 5% CO2 incubator for
cell attachment. Next, the media were changed with
media containing 10% of IC50 of the tested substances
and cells were incubated in that media for 24, 48 and
72 h. Media without any chemical was used as the nega-
tive control. At each time point, the media were re-
moved, wells were washed with PO4 Buffer Saline (PBS)
and fractions of viable cells were measured by the MTT
assay.
HPLC analysis
Culture supernatant of isolated bacteria, was subjected
to acid hydrolysis with 6 N HCl. The concentration of
L-lysine was determined using HPLC. Samples were pas-
sed through a Hypersil ODS, 5 μm column (4.6 × 250),
eluted with (A: 0.5 M CHCOOH at pH = 2.8; B: MeOH) at
a flow rate of 1 ml/min [15].
Partial purification and hydrolysis of Methyl Orange
(MeO) precipitating substance
The supernatant of isolated bacteria was adjusted to pH
2.5 with an HCl solution and saturated with a metha-
nol/acetone (3:1) mixture 30 to 70% after standing
overnight at 4 °C. The resulting precipitate was then
hydrolyzed as stated above. The hydrolyzed product
obtained was subjected to thin-layer chromatography
using ready prepared TLC plate’s silica gel 60 [MERCK].
Solvent system used as developer consisted of n-
butanol-acetic acid-pyridine-water (4:1 : 1 : 2); ninhydrin
reagent was used for visualization [15].
Results
Identification of bacterial isolate
DNA sequencing of 16S rDNA (the first1438 bp) of the
selected isolate, showed a highest similarity of 99.7% to
B. subtilis BAC-UFMT FJ025757.1 (Fig. 1) with GenBank
accession number of GU191141.
Bacillus subtilis SDNS formed mucoid creamy colonies.
Cells were Gram positive, endospore forming rods, Oxi-
dase and catalase positive. The bacterium hydrolysed
blood and was sensitive to all antibiotics tested (Flu-
cloxacillin FL5, Norfloxacin NOR10, Ampicillin/Sulbac-
tam SAM20, Gentamicin CN10, Imipenem IPM10, Eryr-
romycin E15, Ceftazidime CAZ30, Ampicillin AM10,
Ciprocin ciprofloxacin CIP5, Cephalerxin CL30 and
Cefadroxil CFR30).
Production of ε-PL in batch culture
As depicted in Fig. 2 in batch culture, cells exhibited
a lag phase of 3 h, followed by exponential growth
which extended till 14 h where maximum growth
(OD600 = 5.86) was achieved.
ε
-PL production started
after one hour of growth and showed highest titre
(71.7 mg/l) after 14 h of growth. On the other hand,
initial pH value (6.8) gradually decreased by time reach-
ing a value of 5.3 after 16 h.
4 N. A. El-Sersy et al. Journal of Basic Microbiology 2012, 52, 1 – 10
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
Figure 1. Phylogenetic tree of isolate EL5. 16S rDNA-based den-
dogram showing the phylogenetic position of isolate EL5 among
representatives of related bacterial species. The tree was construct-
ed by Bioedit method [10]. Percentages (right) indicate phylogenetic
similarities.
Application of statistical design
(Plackett-Burman design)
The application of Plackett-Burman statistical design
was carried out in a ‘two phases’ of optimization ap-
proach. The first step was to screen for important fac-
tors and their levels that affect production process in
shaked flasks (Table 1). The second was the verification
experiments to validate the results under specific opti-
Figure 2. Growth and poly-lysine production of Bacillus sp. SDNS
grown on M3G medium for 24 h at 30 °C under shaken conditions
(160 rpm).
mized experimental conditions. All the experiments
were carried out in duplicates according to a design
matrix (Table 2), which was based on the number of
variables to be investigated. In the analysis of these
designs, usually only main effects are estimated. The
ε
-PL concentration (or the residual MeO) was measured
after 4 days of incubation (Table 2).
The production of
ε
-PL by Bacillus subtilis SDNS was
positively affected by KH2PO4, MgSO4 ⋅ 7 H2O and
(NH4)SO4, and negatively affected by yeast extract,
ZnSO4 ⋅ 7 H2O, K2HPO4 and glucose (Fig. 3). The polymer
production was mainly dependent on yeast extract and
KH2PO4. i.e. the high concentration of yeast extract had
the most significant negative effect on
ε
-PL production
and the high concentration of KH2PO4 had the most
significant positive effect on
ε
-PL production by Bacillus
subtilis SDNS. Therefore, decreasing the yeast extract
concentration and simultaneously increasing the
KH2PO4 concentration in the culture medium can in-
creased the
ε
-PL production. The significant variables
were identified by statistical analysis of the Plackett-
Burman experiment using the t-test supported by Exel
Table 1. Main effect and t-value results with factors examined as independent variables affecting
ε
-PL production and their levels
in the Plackett-Burman experimental design. Medium components are given in g l–1.
Levels Factors Symbols
Low level (–1) Basal medium (0) High level (+1)
Main effect
t
-value
(NH4)SO4 NH 5 10 15 3.475 0.25
K2HPO4 K2 0.3 0.8 1.3 –12.375 –0.96
KH2PO4 KH 0.86 1.36 2 14.375 1.15
MgSO4 ⋅ 7 H2O Mg 0.1 0.5 1 6.775 0.50
ZnSO4 ⋅ 7 H2O Zn 0.0 0.04 0.08 –18.675 –1.62
Glucose G 25 50 75 –2.825 –0.20
Yeast extract YE 1 5 10 –19.275 –1.69
Journal of Basic Microbiology 2012, 52, 1 – 10
ε
-Poly-L-lysine (
ε
-PL) produced by a B. subtilis sp. 5
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
Table 2. The Plackett-Burman experimental design matrix for 7 factors.
Independent variable Eq. PL conc. (mg/l) Trials
NH K2 KH Mg Zn G YE
1 –1 –1 –1 +1 +1 +1 –1 59.7
2 +1 –1 –1 –1 –1 +1 +1 55.8
3 –1 +1 –1 –1 +1 –1 +1 24.1
4 +1 +1 –1 +1 –1 –1 –1 72.3
5 –1 –1 +1 +1 –1 –1 +1 76.3
6 +1 –1 +1 –1 +1 –1 –1 73.6
7 –1 +1 +1 –1 –1 +1 –1 73.6
8 +1 +1 +1 +1 +1 +1 +1 45.9
9 0 0 0 0 0 0 0 71.7
Figure 3. Elucidation of cultivation factors affecting ε-PL production
by Bacillus sp. SDNS (b) using Plackett-Burman experimental
design.
Microsoft Office to determine the statistical signifi-
cance of the measured response and calculate main
effects for Bacillus subtilis SDNS (Table 1). The anti- op-
timized medium was the opposite concentrations of the
optimized one and its growth medium formula for
Bacillus subtilis SDNS contained in (g/l): (NH4)SO4, 5;
K2HPO4, 1.3; KH2PO4, 0.86; MgSO4 ⋅ 7 H2O, 0.1; ZnSO4 ⋅
7 H2O, 0.08; glucose, 75; yeast extract, 10. FeSO4 ⋅ 7 H2O
was added with constant value 0.03 g/l and pH 6.8.
Verification experiment
In order to validate the obtained data and to evaluate
the accuracy of the applied Plackett-Burman statistical
design, a verification experiment was carried out in
triplicates to predict the near optimum levels of inde-
pendent variables. The data were examined and com-
pared to the basal and anti-optimized medium. Data
revealed that the poly-L-lysine production raised by 1.5
fold for Bacillus subtilis SDNS when growing in opti-
mized medium (Fig. 4). We thus predict that in order to
produce the highest amount of poly-L-lysine, the me-
dium formula should be formulated as follows (g/l):
Figure 4. Verification experiment of the applied Plackett-Burman
statistical design by comparing the concentration of ε-PL produced
by Bacillus sp. SDNS growing on the resulting optimized medium
(OP.M), the basal medium (BM) and the anti-optimized medium
(A.OP.M).
(NH4)2SO4, 15; K2HPO4, 0.3; KH2PO4, 2; MgSO4 ⋅ 7 H2O,
1; ZnSO4 ⋅ 7 H2O, 0; FeSO4 ⋅ 7 H2O, 0.03; glucose, 25;
yeast extract, 1 and the expected yield could be up to
76.3 mg/l. pH 6.8.
Antibacterial activity
Data in Fig. 5 reveal that
ε
-PL of Bacillus subtilis SDNS
exhibited antagonistic effect against all Gram negative
tested bacteria, with variable degrees depending on
bacterial species. No effect was observed with the Gram
positive Staphylococcus aureus. These data were recorded
after incubation of plates for 24 h, extending incuba-
tion time did not show any change in zone measure-
ment (data not shown).
Anticancer activity
Anticancer activity was performed by first testing the
toxicity of culture filtrates and evaluates its activity
against three different cell lines: human cervix adeno-
carcinoma (HeLaS3), Human hepatocellular liver carci-
noma cell line (HepG2) and Human colonic carcinoma
cell line (Caco2).
6 N. A. El-Sersy et al. Journal of Basic Microbiology 2012, 52, 1 – 10
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
Figure 5. Clear zone diameter expressed in cm produced by Bacil-
lus sp. SDNS against several microbes after 24 h incubation and at
37 °C.
The effect of culture supernatant of the bacterial
strain Bacillus subtilis SDNS on HepG2 tumor cell line
using MTT assay was employed giving rise a percentage
of inhibition of 56.2% after 72 h (Table 3).
Upon using the HelaS3 tumor cell line, the super-
natant of Bacillus subtilis SDNS showed almost 77.2%
inhibition after 72 h (Table 3).
The supernatant of the bacterial strain showed al-
most no activity towards Caco cell line (Fig. 6) Figs. 6A–
F represent photographs showing the effect of bacterial
extract on the three cell lines examined based on cell
count by microscopic observation.
HPLC and TLC analyses
HPLC was performed for the hydrolyzed products of
Bacillus subtilis SDNS strain. Fig. 7 shows the chroma-
tograms obtained in this experiment. Data reveal the
presence of L-lysine (the product of
ε
-PL hydrolysis) in
concentration of 9.15448 mg/ml. Hydrolysed product
was analysed by TLC which confirm the presence of
L-lysin (data not shown).
Discussion
It is widely accepted that new drugs, especially antibiot-
ics, are urgently required, and that the most propitious
source remains natural products. To explore new
sources of bioactive natural products the marine envi-
Table 3. The effect of culture supernatant of the bacterial strain
Bacillus subtilis SDNS on tumor cell line using MTT assay.
Time (h) % of Inhibition on tumor cell line
HepG2 Hela S3 CaCo
24 9.7 20.6 0.5
48 19.5 41.7 4
72 56.2 77.2 9.8
ronment wants particular attention, in view of the
remarkable diversity of microorganisms and metabolic
products [16].
ε
-PL possesses antimicrobial activity against a wide
spectrum of microbes and has been widely used in food
manufacturing as a safe preservative [17]. Its produc-
tion is limited to one family Streptomycetaceae [18]. In
this work, a screening study targeting bacterial strains
as producers of
ε
-PL was performed. Over one hundred
marine bacterial isolates were screened. This is the first
time to announce the isolation of marine Bacillus subtilis
SDNS capable of producing
ε
-PL, data not previously
reported according to literature available. Sea water
was required for growth of the bacterium and its me-
tabolism. Quantification of
ε
-PL was done using Itzhaki
method [9], a rapid and sensitive technique employed
by other investigator [19, 20]
Production of
ε
-PL by Bacillus subtilis SDNS began with
the beginning of bacterial growth and reached its
maximum at late exponential phase. This was accom-
panied by shifting of pH to acidic condition. The data
are in agreement with that previously reported by Yo-
shida & Nagasawa [21] with Streptomyces celluloflavus who
observed accumulation of the polymer under acidic
condition.
In order to maximize
ε
-PL production by Bacillus sub-
tilis SDNS an experimental design was employed. Plack-
ett-Burman design is one of the so-called “screening
designs”. Such designs are traditionally used for identi-
fying important factors from among many potential
factors [12]. In the analysis of these designs, usually
only main effects are estimated. M3G medium is the
conventional medium used for production of
ε
-PL as
reported in literature [20]. Under our experimental
conditions, glucose and ammonium sulfate appeared to
be the most important factors affecting
ε
-PL production
by Bacillus subtilis SDNS, on the contrary, yeast extract
had a negative effect. Our results agreed with those of
Shih and Shen [22] with Streptomyces albulus IFO 14147
except for Yeast extract which enhanced polymer pro-
duction. This could be due to the nature of the bacterial
species. We thus suggested an optimized formula con-
sisting of (g/l): (NH4)SO4, 15; K2HPO4, 0.3; KH2PO4, 2;
MgSO4 ⋅ 7 H2O, 1; ZnSO4 ⋅ 7 H2O, 0; FeSO4.7H2O, 0.03;
glucose, 25; yeast extract, 1 pH 6.8.
The amount produced by Bacillus subtilis SDNS could
reach up to 76.3 mg/l. The quantity produced is low
compared to that recorded in literature. S. albulus used
for commercial production produces 4–5 g/l [23],
whereas Kitatospora sp. produces 1.75 g/l without pH
control and 7.7 g/l after pH control as reported by Ouy-
ang et al. [24]. Also, Hirohara et al. [15] stated that
Journal of Basic Microbiology 2012, 52, 1 – 10 7
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
(A) (B)
(C) (D)
(E) (F)
Figure 6. Photographs illustrating the difference between the bacterial extract of Bacillus subtilis SDNS (B) on the growth inhibition of
HepG2 tumor cell line compared to control (A); the difference between the bacterial extract of Bacillus subtilis SDNS (D) on the growth
inhibition of HelaS3 tumor cell line compared to control (C); difference between the bacterial extract of Bacillus subtilis SDNS (F) on the
growth inhibition of CaCo tumor cell line compared to control (H).
8 N. A. El-Sersy et al. Journal of Basic Microbiology 2012, 52, 1 – 10
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
(a)
(b)
Figure 7. HPLC chromatograms of standard L-lysine (A), L-lysine produced by Bacillus sp. SDNS (B).
S. aureofaciens USE-82 produced the highest amount
among the isolated eight strains which produced
4.0 g/l. We thus speculate that further studies are re-
quired to improve the amount produced.
ε
-PL molecules are cationic, surface active agents due
to their positively charges amino groups in water, and
they have been shown to have a wide antimicrobial
spectrum by growth inhibition studies with yeast,
fungi, and Gram-positive or Gram-negative bacterial
species [25, 26]. Our data reveals that Bacillus subtilis
SDNS exhibited antagonistic effect against all Gram-
negative tested bacteria, with variable degrees depend-
ing on bacterial species. Whereas no effect was ob-
served with the Gram-positive Staphylococcus aureus,
growth of Streptococcus faecalis was inhibited.
Data obtained in the present study showed that Bacil-
lus subtilis SDNS metabolites potentially inhibited the
proliferation of HelaS3 cells with lethal concentration
LC71 and HepG2 cells with lethal concentration LC53.
These results may contribute in finding an alternative
for treatment of cancer and be used as a chemothera-
peutic drugs. With similarity to the data obtained in
this part of study Shlyakhovenko et al. [27] found after
extensive investigation that nucleoprotein fraction of
Bacillus subtilis 7025 (NPF) culture medium filtrate can
be effective as antitumor immunotherapeutic agents.
Another data obtained by Abe
et al
. [28], are in accord-
ance of our data as Bacillus sp. produced compounds
acting as antitumor agents. Parasporin-2 is a newly
classified Bacillus thuringiensis crystal toxin with strong
cytocidal activities toward human liver and colon can-
cer cells. Unfortunately, no data are available with
respect to the effect of metabolites produced by Bacillus
subtilis against both tumor cell lines studied through-
out this study HepG2 and HelaS3.
Journal of Basic Microbiology 2012, 52, 1 – 10
ε
-Poly-L-lysine (
ε
-PL) produced by a B. subtilis sp. 9
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
Data of HPLC revealed the presence of L-lysine
(the product of
ε
-PL hydrolysis) in concentration of
9.15448 mg/ml. The culture filtrate was partially puri-
fied as previously reported [29, 30]. The product was
hydrolyzed and subjected to thin layer chromatography
(TLC) technique which is very popular in separating
components of hydrolysis.
The hydrolysate produced showed the presence of
L-lysine. These data are similar to those published by
Nishikawa and Ogawa [17]. More future study is re-
quired for the identification of the product.
Conclusion
Marine environment is a good source for valued species
that need to be explored. The main aim of the present
study was to explore our natural environments search-
ing for species producing the polymer
ε
-PL, due to the
limited number of organisms known till now to pro-
duce this polymer which limits its large scale produc-
tion. This product is of great economic importance.
Producing such polymer industrially in Egypt will save
millions of dollars (it is now sold for 3500 LE/g).
References
[1] Sundaram, S., Priyanka D., Shalini, P., 2011. In vitro evalu-
ation of antibacterial activities of crude extracts of Witha-
nia somnifera (Ashwagandha) to bacterial pathogens. Asian
J. Biotechnol., 3(2), 194–199.
[2] Sepcic, K., Polona, Z., Nina, Gunde-C., 2011. Low water
activity induces the production of bioactive metabolites
in halophilic and halotolerant fungi. Mar. Drugs, 9, 43 –
58.
[3] Debbab, A., Amal, H.A., Wen, H.L., Peter, P., 2010. Mini
review. Bioactive compounds from marine bacteria and
fungi. Microbial Biotechnol., 3(5), 544– 563.
[4] El-Sersy, N.A., Ebrahim, H.A.H., Abou-Elela G.M., 2010.
Response surface methodology as a tool for optimizing
the production of antimicrobial agents from Bacillus liche-
niformis SN2. Curr. Res. Bacteriol., 3, 1– 14.
[5] Pakpitcharoena, A., Potivejklub, K., Kanjanavasa, P., Aree-
kita, S., Chansir, K., 2008. Biodiversity of thermotolerant
Bacillus sp. producing biosurfactants, biocatalyst and an-
timicrobial agents. Sci. Asia, 34, 424 – 431.
[6] Szende, B., Gy Szökán, E., Tyihá, K.P.R., Gáborjányi, M.A.,
Khlafulla, A.R., 2002. Antitumor effect of lysine-isopep-
tides. Cancer Cell International 2: 4. This article is avai-
lable from: http://www.cancerci.com/content/2/1/4, Cell x.
[7] El-Sersy, N.A., Abou-Elela, G.M., Abd-Elnaby, H., Ebrahim,
H.A.H., El-Toukhy, N.M.K., 2010. Optimization, economi-
zation and characterization of cellulase produced by ma-
rine Streptomyces ruber. Afr. J. Biotechnol., 9(38), 6355–
6364.
[8] Kahar, P., Iwata, T., Hiraki, J., Park, Y.E., Okabe, M., 2001.
Enhancement of
ε
-polylysine production by Streptomyces
albulus strain 410 using pH control. J. Biosci. Bioeng., 9(2),
190– 194.
[9] Itzhaki, R.F., 1972. Colorimetric method for estimating
polylysine and poly-arginine. Anal. Biochem., 50, 569 –
574.
[10] Hall, T.A., 1999. BioEdit: a user-friendly biological se-
quence alignment editor and analysis program for Win-
dows 95/98/NT. Nucl. Acids. Symp. Ser., 41, 95–98.
[11] Ventosa, A., Quesada, F., Rodríguez-Varela, F., Ruiz-Berra-
quero,F., Ramos-Cormenzana, A., 1982. Numerical taxo-
nomy of moderately Gram negative rods. J. Gen. Micro-
biol., 128, 1959– 1968.
[12] Plackett, R.L., Burman, J.P., 1946. The design of optimum
multifactorial experiments. Biometrika, 33(4), 305–325.
[13] Abou-Elela, G.M., El-Sersy, N. A., El-Sheuawy, M.A., Abd-
Elnabi, H., IbraInm, H.A.H., 2009. Bio-control of Vibrio flu-
vialis in aquaculture by mangrove (Avicennia marina) seeds
extracts. Res. J. Microbiol., 4(1), 38 – 48.
[14] Xuezhong, H., Junyu, M., Angel, E.M., Weijie, X., Esmaiel,
J., 2007. Cytotoxicity of paclitaxel in biodegradable self-
assembled core-shell poly (lactide-co-glycolide ethylene
oxide fumarate) nanoparticles. Pharmac. Res., 25(7),
1552– 1562.
[15] Hirohara, H., Saimura, M., Takehara, M., Miyamoto, M.,
Ikezaki, A., 2007. Substantially monodispersed poly(
ε
-L-
lysine) frequently occurred in newly isolated strains of
Streptomyces sp. Appl. Microbiol. Biotechnol., 76, 1009–
1016.
[16] Bull, A.T., Stach, J.E.M., 2007. Marine actinobacteria: new
opportunities for natural product search and discovery.
Trends Microbiol., 15, 491– 499.
[17] Nishikawa, M., Ogawa, K., 2002. Distribution of microbes
producing antimicrobial
ε
-poly-L-lysine polymers in soil
microflora determined by a novel method. Appl. Environ.
Microbiol., 68(7), 3575– 3581.
[18] Saimura, M., Takahara, M., Mizukami, S., Kataoka, K.,
Hirohara, H., 2008. Biosynthesis of nearly monodispersed
poly(
ε
-L-lysine) in Streptomyces species. Biotechnol. Lett.,
30, 377– 385.
[19] Takehara, M., Saimura, M., Inaba, H., Hirohara, H., 2008.
Poly (
γ
-L-diaminobutanoic acid), a novel poly (amino acid),
coproduced with poly(
ε
-L-lysine) by two strains of Strepto-
myces celluloflavus. FEMS Microbiol Lett., 286, 110– 117.
[20] Shih, I.L., Shen, M.H., Van, Y.T., 2006. Microbial synthesis
of poly (
ε
-lysine) and its various applications. Bioresource
Technol., 97(9), 1148– 1159.
[21] Yoshida, T., Nagasawa, T., 2003.
ε
-Poly-L-lysine: microbial
production, biodegradation and application potential.
Appl. Microbiol. Biotechnol., 62, 21– 26.
[22] Shih, I.L., Shen, M.H., 2006b. Optimization of cell growth
and poly(
ε
-lysine) production in batch and fed-batch cul-
tures by Streptomyces albulus IFO 14147. Proc. Biochem., 41,
1644– 1649.
[23] Shima, S., Oshima, S., Sakai, H., 1983. Biosynthesis of
ε-poly-L-lysine by washed mycelium of Streptomyces albulus
No-346. Nippon Nogeikagaku Kaishi, 57, 221–226.
10 N. A. El-Sersy et al. Journal of Basic Microbiology 2012, 52, 1 – 10
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com
[24] Ouyang, J., Xu, H., Li, S., Zhu, H. et al. 2006. Production of
ε
-poly-lysine by newly isolated Kitatospora sp. PL6-3. Bio-
technol., 1, 1459– 1463.
[25] Hiraki, J., Ichikawa, T., Ninomiya, S.I., Seki, H. et al., 2003.
Use of ADME studies to confirm the safety of
ε
-polylysine
as a preservative in food. Reg. Toxicol. Pharm., 37, 328–
340.
[26] Yu, H., Huang, Y., Huang, Q., 2009. Synthesis and charac-
terization of novel antimicrobial emulsifiers from
ε
-poly-
lysine. J. Agric. Food Chem., 58(2), 1290– 1295.
[27] Shlyakhovenko, V.A., Olishevsky, S.V., Kozak, V.V., Ya-
nish, Y.V., Rybalko, S.L., 2003. Anticancer and immu-
nostimulatory effects of nucleoprotein fraction of Bacillus
subtilis 7025 culture medium filtrate. Exp. Oncol., 25,
119– 123.
[28] Abe, Y., Shimada, H., Kitada, S., 2008. Raft-targeting and
oligomerization of parasporin-2, a Bacillus thuringiensis
crystal protein with anti-tumour activity. J. Biochem.,
143(2), 269-275.
[29] Hirohara, H., Takehara, M., Saimura, M., Masayuki, A.,
Miyamoto, M., 2006. Biosynthesis of poly(
ε
-L-lysine)s in
two newly isolated strains of Streptomyces sp. Appl. Micro-
biol. Biotechnol., 73(2), 321– 331.
[30] Shih, I.L., Shen, M.H., 2006. Application of response sur-
face methodology to optimize production of poly-
ε
-lysine
by Streptomyces albulus IFO 14147. Enz. Microbi. Technol.,
39(1), 15– 21.
