Isolation and Characterization of Bioactive Protein from Several
Species of Sponges as Antimicrobial Agent
Andi Ilham and Ahyar Ahmad
Faculty of Natural Sciences, Hasanuddin Unioversity, Makassar, 90245 INDONESIA
ABSTRACT
A research on anti-bacterial bioactivity of protein fraction isolated from several species of
sponges of Barang Lompo island in South Sulawesi by using polar solvent buffer of 0.1 M TrisHCl with pH 8.3 containing 2 M NaCl 0.01 M CaCl2, 1 % -mercaptoethanol, 0.5 % Triton X-100
has been conducted. The concentration of protein was determined by the Lowry method and the
bioactivity test was investigated by a gel diffusion method. Results showed that the concentration
of protein in the crude extracts isolated from 500 g of fresh samples of four species of sponges
(BLS 02, BLS 06, BLS 07, and BRL 01) was 7.080 mg/mL; 8.400 mg/mL; 16,624 mg/mL,
respectively. Initial purification of protein uses conducted by using the fractionation method with
ammonium sulfate, followed by a dialysis process. All protein fractions showed bioactivity to
Salmonella typhy, where the highest inhibition zone was found in the protein fraction with the
saturation level of ammonium sulfate of 40-60 % from sponge BRL 01 with the inhibition zone of
26.48 mm and the protein fraction with the saturation level of ammonium sulfate of 30-40 % from
sponge BLS 06 with the inhibition zone of 26,18 mm. Test of the inhibition level in the various
concentration of bioactive protein with the highest inhibition zone showed that the maximum
activity was in 4000 g/mL of protein from BRL 01 40-60 % and BLS 04 30-40 % with inhibition
zones of 23.54 and 20.56 mm, respectively. The results indicated that bioactive protein from
sponges is very potential as the basic material for the new anti-bacterial drug, especially to
Salmonella typhy.
Keywords : Bioactivity, sponges, bioactive protein, anti-bacterial, inhibition zone.
necessarily to be carried out. Untill recently,
INTRODUCTION
marine natural resources have not been
Indonesia, known as a maritime country
with ocean area of 75% covering the country,
has abundant source of marine biota, among
others are a lot variety of sponge
species.
Some species have been reported containing
bioactive compounds that have been widely
applied in pharmatical industries (Ahmad et al.,
optimally utilized. Therefore, any effort to
identify potential bioactive compounds from
marine natural resources will be of a great
interest (Caraan, 1994 dan Nybakken, 1993).
It is widely accepted that marine natural
rersources can be clasified into 2 classes i.e.,
marine biota or plants such as algae, seagrass
and marine animal such as fish, mollusca, soft
1995).
In paralel with the trend of disease
pattern changes such as the resistance of
disease germs towards a certain medicine, the
efforts to find new medicines is therefore
coral, sponge, echinodermata, aschidin, and
tunichata. Some marine animal species have a
good resource of vitamin, protein, and mineral
compounds. In addition to that, several certain
species have a capability to synthesize and
Intensive researches have found that using
accumulate toxic compounds in a certain part of
chloroform as a non polar solvent, some
the body (Rachmaniar, 1996). Such compounds
sponges
are
metabolite
compounds of antimicroba, but others did not
compounds that are usually used as a self
show any bioactivity. Such sponges have been
defence system from other organism in its
reported having bioactive compounds such as
environment
activities.
cesterpene extracted from Hyatella intestinalis
Therefore, such compounds have a promising
(Karuso et al., 1989), methyl steroid extracted
prospect to be extracted and isolated and
from Agelas flabellaformis (Gunasekara et al.,
utilized as new potential medicines (Sardjoko,
1989),
1996).
officinalis, Ircinia variabilis, spongia gracilis,
considered
as
and
secondary
pharmacologic
each
In general, the natural medicines are
Erylus
having a specific chemical characteristic. The
resulted
from
ketosteroid
lendenfeldi
and
terpenoid,
extracted from
Dyctionella
insica
1998; Fusetani et al., 1999) that can be utilized
in
existence of the living organisms. These
pharmatical
industries
and
deseases
medicines for human and animal. Until recently,
secondary metabolite compounds are then
however, research on the exploration of certain
collected, processed, and used as a new drug
group of protein compounds derived from
metabolite
sponges as a raw material for medicines
compounds derived from bio-organisms have
designated to human and animal has not been
been used as popular medicines such as
published.
asphirine, morphine, digitalys, peniciline, and
taxol (Anonim, 2003).
Protein as anti bacteria medicines has been
promising since it can be well accepted by
Nybaken (1993) reported that sponges have
human body and has a small side effect.
ability to screen the bacteria in its environment
Therefore, research on the use of protein as a
up to 77 % through the utility of digested food
source of medicines has been tremendously
enzymatically. Bioactive compounds in sponges
growing (Huang, 1999). Moreover, the gens in a
have advantage in digesting process such that
the bioactive compounds
cesterpene,
Spongia
Microscleroderma sp. (Schmidt and Faulkner,
desease attacks as well as to defend the
secondary
and
comunis,
cyclo peptide extracted from Theonella sp. and
organisms are used as a defence mean against
Some
containing
metabolite
(Cimminiello et al., 1989), short peptide and
secondary
metabolite reactions compounds in the living
formula.
secondary
Hipospongia
variabiline
derived from secondary metabolite compounds
coumpounds
contained
group of protein can be clonned such that it can
yielded from such
be produced in an industrial scale through
process will be varied according to the eating
genetic engineering.
habit of each sponge.
The goal of this research was to explore and
characterize some bioactive protein fractions
2
extracted from several types of sponges in
BSA
(Bovine
Serum
Albumin)
4
mg/mL,
South Selatan. The extracted bioactive protein
amphiciline 30 ppm, cotton, and aluminium foil.
was tested its activity as an anti bacterial agent.
The research result shows that bioactive protein
Extraction and isolation of sponge bioactive
protein
Extraction and isolation of sponge bioactive
compounds obtained at saturated ammonium
sulphate solution of 40-60% extracted from
sponges
sponge species of BRL 01 and saturated
ammonium sulphate solution of
previous
have been collected were cut into small peices,
and each species was weighed into 500 g of
zones of 26.48 and 26.18 mm respectively
fresh sample, homogenized with blender using
toward Salmonella typhy.
buffer solution A (Tris-HCl 0,1 M pH 8,3, NaCl 2
M, CaCl2 0,01 M,  - merkaptoetanol 1 %,
From the results of resistance testing at
protein
Triton X- 100 0,5 %), filtered with buchner, and
concentrations, the highest bioactivity toward
the filtrate obtained was then freezed and
Salmonella
protein
liquified between 2 or 3 times, and then
concentration of 4,000 g /mL. This research
centrifugized at 12.000 rpm and 4oC for about
was also expected to be able to contribute to a
30
better understanding of knowledge regarding
obtained was stored in a refrigerator before
bioactive protein compounds derived from
being tested for anti bacterial agent and further
sponges since such protein compounds have
purification stage.
typhy
of
using
modified) as folows; four sponge species that
indicated the strongest activity with resistence
variations
conducted
methods (Schroder et al., 2003; Ely et al., 2004
30-40%
extracted from sponge species of BLS 06
several
were
was
bioactive
found
at
minutes,
and
finally
the
supernathan
optimal and effective resistance forces against
Protein Concentration Determination
bacterial growth so that they can be used as a
The
raw material of a new anti microba medicine.
calculation
of
bioactice
protein
concentration in the buffer A (Tris-HCl 0,1 M pH
MATERIALS AND METHODS
8,3 NaCl 2 M, CaCl2 0,01 M,  -merrmined
Materials used in this research were four
using kaptoetanol 1 %, Triton X-100 0,5 %) was
species of sponges, pure bacterial fertile of
Echerichia
coli,
Salmonella
typhi,
Aquades, MHA
Staphylococcus
and
Vibrio
determined based on Lowry method (Colowick
aureus,
and
cholerae,
Kaplan,
1957)
using
bovine
serum
albumine (BSA) as a standard.
(Muller Hinton Agar) media,
buffer A (Tris-HCl 0,1 M pH 8,3, NaCl 2 M,
Anti Bacterial Activity Testing
CaCl2 0,01 M,  - merkaptoetanol 1 %, Triton
The testing of resistance power of bioactive
X-100 0,5 %), buffer B (Tris-HCl 0,1 M pH 8,3,
protein
NaCl 0,2 M, CaCl2 0,01 M), buffer C (Tris-HCl
(Echerichia
0,01 M pH 8,3, NaCl 0,2 M, CaCl2 0,01 M),
Salmonella typhi, and Vibrio cholerae) was
3
against
coli,
the
growth
of
bacteria
Staphylococcus
aureus,
conducted with diffusion method (Ely et al.,
(BLR 01). Sponges were taken from their
2004) using sterile filter-paper disk. filter-paper
habitates at the sea depth of 5-15 meters from
disk was placed above the seed layer in the
sea surface level at temperature of 29 0C.
media of MHA and then the sample (4 mg/mL
The identification of sponge species was
each) was poured into the filter-paper disk with
conducted in perfect manner and the result was
the amount of 250  L. The sample was then
presented in Table 1. Such identification is very
incubated for 24 hours at temperature of 37oC
importance to be carried out to easily trace
and then observed and measured. The testing
back the source of bioactive compounds
was conducted in duplo modes and repeated
yielded from the marine biota.
three
times
to
produce
representative
Table 1. The identification results of four
sponge species (George and George, 1999;.
Marine Filtration Laboratory, Faculty of Marine
Science and Fishery, Hasanuddin University).
experimental data.
Fractionation and Protein Dialysis
The supernathan (raw extracts) containing
No
protein and having anti bacterial activities was
then fractionated using amonium sulphate at
Species
Code
Name of species
1
BLS 02
Ianthella flabelliformis
2
BLS 06
Gelliodes sp.
3
BLS 07
Cribrochalina sp.
4
BRL 01
Phylospongia foliancens
saturated levels of 0 – 30 %, 30 – 40 %, 40 –
60 % and 60 – 80 %, respectively.
The precipitates obtained after fractionation at
each saturation level of amonium sulphate was
then suspensized in a cetain amount of buffer B
(Tris-HCl 0,1 M pH 8,3, NaCl 0,2 M, CaCl2 0,01
M), and then dialysed in buffer solution C (Tris-
The main reason of selecting four species was
HCl 0,01 M pH 8,3, NaCl 0,2 M, CaCl2 0,01 M)
based on research result conducted by Razak
using selofan pocket (sigma) until obtaining
and Ridhay (2004) using 29 sponge species
colorless buffer. After dialysis testing, each
including four species (BLS 02, BLS 06, BLS
protein fraction was then undergoing anti-
07, BRL 01) extracted using chloroform as non
bacterial testing similar to the previous testing
polar solvent showing that
on the preparate of raw extract protein.
during inhibition
testing those species did not indicate any anti
microbial activity. Based on that finding, it can
RESULTS AND DISCUSSIONS
be
Sampling and Preparation of Sponges
predicted
that
there
might
be
other
compounds in the four sponge species having
Sponges were sampled at two locations i.e., 3
anti microbial activity.
species sampled at South Barang Lompo Areas
Extraction, Isolation and Determination of
Protein Concentration from Sponges
(BLS 02, BLS 06 dan BLS 07), and 1 spesies
sampled at North East Barang Lompo Areas
4
Extraction and isolation of sponge bioactive
protein
were
carried
out
using
modified
previous method (Schroder et al., 2003; Ely et
2
BLS 06
430
8.400
3612.00
3
BLS 07
420
16.624
6982.08
4
BRL 01
450
5.640
2538.00
al., 2004). The four sponge species were
Testing of Anti Bacterial of
Preparate in the Whole Extracts
collected, cut into small pieces, and then
weighed
into
500
g
of
fresh
Protein
sample,
homogenized with blender using buffer solvent
The testing of resistance power of the
A (Tris-HCl 0,1 M pH 8,3, NaCl 2 M, CaCl2 0,01
bioactive protein against the bacterial growth
M,  -merkaptoetanol 1 %, Triton X-100 0,5 %)
(Salmonella
Staphylococcus aureus
to break the cells such that protein in the cells
activity as reflected by large inhibition zone
freezed and liquified 2 or 3 times and then
toward the tested bacterial growth as seen in
centrifugized at the rate of 12,000 rpm and
Table 3.
temperature of 4oC for 30 minutes, and finally
Table 3 shows that very strong inhibition
determined the protein content and antibacterial
toward Salmonella typhy occurred on sponge
testing to prove that the raw extract contained
anti
BLR 01 where the inhibition zone reached to
bacterial
39,45 mm, larger than other species and even
activity.
amphiciline.
The protein content in the sponge
that
bovine serum albumine (BSA) as standard.
Table 2. Protein Concentration in Whole
Extract from Four Sponge Species
BLS 02
7.080
3079.80
positive
control
or
Table 4. The bioactivity testing of anti
bacterial agent in raw extract
6982.08 mg.
1
as
inhibition zone.
located at BLS 07 containing total protein of
Total
Protein
(mg)
used
the
sponge species of BRL 01 has a strong
concentration (16,624 mg/mL) found in species
Protein
Conc.
(mg/mL
be
The protein preparate of raw extract from
derived
from sponge species was varied with highest
No
might
concentration need to be increased.
Experimental result in Table 2 shows that the
Whole
Extract
Volume
(mL)
435
phenomenon
has undergone resistence toward amphiciline
method (Colowick and Kaplan, 1957) using
protein concentration in raw extract
Such
caused by the bacterial pathogene in the media
samples was calculated based on Lowry
Sponge
Species
dan Echerichia coli)
species showed very strong anti bacterial
residues and filtrates. The filtrates was then
having
cholerae,
raw extracted protein of the four sponge
was then filtered using buchner to separate cell
compounds
Vibrio
was conducted with agar diffusion method. The
will easily dissolve in buffer solvent. The sample
protein
thyphi,
5
The raw extract having anti bacterial activity
was fractionated using ammonium sulphate at
Such strong inhibition might be caused by
the saturation level of 0 – 30 %, 30 – 40 %, 40
severe environmental zone i.e., swallow aquatic
– 60 % and 60 – 80 %.
zone at which its living environment located at
ammonium sulphate salts from low to high
tidal areas in the depth of 0 - 5 meters from
concentration resulted in different protein types
surface sea level. Open biological environment
precipitated. Higher concentration ammonium
condition and big tidal has caused the physical
sulphate caused a lot of hydrophobic functional
growth of bacteria becoming short and small.
groups neutralized by ammonium salts so that
Such extreem condition resulted in forming a
water could no longer bounded turning to the
good endurance toward its environment that
decrement of protein solubility in water causing
may lead to the stimulation of the marine biota
the protein precipitated. The protein distribution
in
metabolite
patterns of the four sponge species at each
compounds. Consequently, such protein will
fractionation is presented in table 5. Table 5
have ability to protect or inhibit the growth of
indicates that highest protein concentration
pathogene
found in sponge species BLS 06, fractions from
producing
large
bacteria
secondary
particularly
against
Salmonella typhy.
The addition of
0 to 30 % i.e., 22,32 mg/mL, while the lowest
High concentration of raw extract protein does
protein concentration found in the fraction of 30
not always show a strong anti bacteria activity.
– 40 % from sponge species BLS 02 i.e., 4,08
Table 3 shows that although the sponge
mg/mL.
species BLS 07 that having highest protein
Table 5. The protein distribution patterns at
various saturation level of ammonium
sulphate
concentration (16.624 mg/mL), the activity does
not exhibit strongest activity. Such phenomenon
may occur since not all protein present in the
raw extract of species BLS 07 functions as anti
microbial protein. The sponge species BLR 01,
on the other hand, a lot of anti microbial
proteins are accumulated in that species such
that the anti bacterial activity exhibits stronger
inhibition power than others. In addition to that,
the species BRL 01 possibly contains not only
protein fraction but also non protein polar
compounds that contributes in inhibiting the
bacterial growth.
Protein Fractions Containing Anti Bacterial
Activity
6
Anti Bacterial Bioactivity of
Fractions Yielded From Dialysis
Protein
precipitates
The inhibition zone of antibacterial protein of
sponge BLS 02 can be seen in figure 1 showing
after
that the inhibition zone at each protein fraction
fractionation with ammonium sulphate were
derived from species BLS 02, is also higher
dissolved with buffer B (Tris-HCl 0,1 M pH 8,3,
than BSA, and the inhibition levels are almost
NaCl 0,2 M, CaCl2 0,01 M), until perfectly
the same at each fraction at which the strongest
emulsified. Each protein suspension was then
inhibition zone found at fraction of 0 – 30 % i.e.,
poures
13,8 mm towards Echerichia coli.
into
selofan
obtained
Protein
pocket.
The
selofan
containing protein suspension was dialysed in
The inhibition zone of each protein fraction
buffer solution C (Tris-HCl 0,01 M pH 8,3, NaCl
derived from species BLS 06 is higher than
0,2 M, CaCl2 0,01 M). The fractions resulted
BSA (see figure 2), except the activities of
from dialysis was tested its anti bacterial
fraction 60 – 80 % towards Staphylococus
properties using method described previously
aureus, dimana
for raw extract protein.
ditemukan pada fraksi 30 – 40 % sekitar 26,18
The objective of this
procedure was to prove that compound having
zona hambatan terkuat
mm terhadap Salmonella typhy.
anti bacterial activity derived from protein
compounds. If the anti bacterial testing applied
to saturated ammonium fraction does not show
any activity, therefore it can be concluded that
the compound contributing to inhibition effect on
the pathogenic bacterial growth is caused by
polar compounds and not due to protein.
However, all ammonium sulphate fractions give
positive result as indicated by inhibition zone at
Figure 2. The Diagram of Antibacterial
Inhibition Zone Derived from Sponge
Species BLS 06
each treatment.
The inhibition zone of each protein fraction
derived from species BLS 07 is higher than its
controlling agent (-)
BSA (see figure 3), at
which strongest inhibition zone found at the
fraction of 30 – 40 % i.e., 17.90 mm towards
Echerichia coli.
Figure 1. Zone Diagram of Antibacterial
Inhibition of Sponge Species BLS 02
7
Such phenomenon might be caused by
non protein polar contained in the raw extract
protein but having antibacterial activity towards
Salmonella typhy, in particular. Therefore,
futurev research is needed to trace the
existence of such compounds to strengthen the
above
hypothesis.In
the
experiment
of
antibacterial testing, it was obvious that each
protein fraction at various level of saturated
Figure 3. The Diagram of Inhibition Zone of
Antibacterial Derived from Sponge Species
BLS 07
ammonium sulphate exhibited inhibition activity
for all sponge samples as indicated by clear
zone for every tested media. This proved that
The inhibition zone at each protein fraction was
all
comparable of related positive control of
sponge
samples
contained
protein
compounds having ability to inhibit pathogenic
amphiciline 30 ppm, even at protein fraction of
bacterial growth.
40-60%, it has inhibition zone of about 200%
In the antimicrobial testing, the greatest
stronger than the amphiciline in bacterial testing
inhibition level against the bacterial growth of
against Salmonella typhy. In the raw extract
Salmonella typhy found in sponge species BLR
protein, on the other hand, the inhibition zone is
01 (see figure 4) i.e., 26.48 mm. This indicates
higher than protein fraction at various level
that
saturated ammonium sulphate using similar
such
species
contains
very
strong
antimicrobial protein compounds in inhibiting
bacterial testing agent.
the bacterial growth of Salmonella typhy that
precipitated in the fraction of amonium sulfat
with saturation level of 30 – 40 %. Therefore, it
can
be
concluded
that
protein
having
antimicrobial activity tend to precipitate at
saturated ammonium sulphate of 30 – 40 %.
The bioactivity of protein fractions at
various level of saturated ammonium sulphate
derived
from
sponge
species
BRL
01
underwent activity decrement compared to then
activity derived from raw extract protein, as can
be seen in figure 4, inhibition zone found very
Figure 4. The Histogram of Inhibition Zone
of Antibacterial Derived from Sponge
Species BRL 01
strongly
at
raw
extract
protein.
Such
phenomenon might be caused by several
reasons; first, raw extract protein contained non
8
protein polar compounds that also functioning
The testing results show that the
as antibacterial growth agent by synerging with
inhibition level at various bioactive protein
protein polar compounds. After being separated
concentration
through protein purification with fractionation at
exhibited
various level of saturated ammonium sulphate
concentration of 4000 µg/mL with inhibition
and dialysis, the inhibition level bocames
zone of 23.54 mm and 20,56 for each proteinn
decreased or weaker.
fraction of 40-60% derived from sponge BRL 01
towards
maximum
Salmonella
activity
at
typhy
protein
and protein fraction of 30-40% extracted from
sponge
Inhibition Testing of Various Bioactive
Protein Concentration at Optimum Fraction
BLS
06.
The
activity
decrement
comparted to the initial testing (see figure 2 and
Figures 2 and 4 show that both active
4), might be caused by storage factor resulting
protein (fraction 40-60%) isolated from sponge
in the instability of bioactive protein. At lower
BRL 01 and active protein (fraction 30 – 40 %)
protein
isolated from sponge BLS 06 exhibit strongrest
relatively exhibited a similar activity.
concentration
i.e.,
40-400
µg/mL,
Based on the results and discussions
activity with inhibition zones of 26.48 and 26.18
mm respectively toward Salmonella typhy. To
described
previously,
the
antibacterial
identify the effect of protein concentration at
mechanism derived from sponges can be
various fractions toward antibacterial activity,
predicted as follow; first, the protein consists of
inhibition testing was carried out at various
group of enzyme protein at which the inhibition
proteinn concentrations i.e., 4000, 400, 200,
mechanism is by degrading bacterial cell walls
100 and 40 µg/mL. The reslut is presented in
composing of peptidoglikan and lipoprotein
figure 5.
catalysed by hydrolase or lipase enzym.
Second possibility is that antimicrobial protein
consists of generic protein where the inhibition
mechanism is by bonding with essential metals
such as iron that is needed to bacterial growth.
Such assumptions are open to be proved in
further research.
Unfortunately,
reports
or
scientific
articles on antibacterial activity of protein
fraction derived from sponge is limited such that
insufficient literatures can be refered to support
Figure 7. The Diagram of Inhibition Zone
Towards Salmonella typhy at Various
Protein Concentration Derived from Sponge
Species BRL 01 and BLS 06
the discussion of this research results.
Conclusion
9
Based on the result and discussion described
inhibition zone of 26.48 and 26.18 mm
previously, it can be concluded as follow:
respectively toward Salmonella typhy.
1. All
isolated
sponge
species
contain
3. The result of inhibition testing towards
bioactive protein compounds having ability
Salmonella
to inhibit pathogenic bacterial growth such
bioactive protein concentration shows that
as
the highest activity found at the maximum
Salmonella typhy, Vibrio cholerae,
typhy
at
various
level
of
protein concentration of 4,000 g /mL
Staphylococus aureus and Echerichia coli.
2. Bioactive protein at saturation level of
ammonium sulphate of 40-60% derived
Acknowledgement
from sponge species BRL 01 and saturated
We thank to the head of Pharmaceutical
ammonium sulphate of 30-40% extracted
Microbiology
from
University for antimicrobial testings.
BLS 06 show stongest activity with
Laboratory
of
Hasanuddin
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