ISOLATION AND PURIFICATION BIOACTIVE PROTEINS BROWN
ALGAE Sargassum duplicatum FROM LAE - LAE ISLAND, SOUTH
SULAWESI AS ANTIBACTERIA
Zulviana Sudirman, Rauf Patong, Ahyar Ahmad
Department of Chemistry, Hasanuddin University,
UNHAS Tamalanrea, Makassar 90245
ABSTRACT
A research has been conducted to study the bioactivity of protein fraction isolated
from brown algae Sargassum duplicatum, as antibacterial taken from Lae-Lae
Island located in South Sulawesi. The fraction was isolated by using buffer 0.1 M
Tris (hydroxymethyl) amino methane. The fractionation of protein from the crude
extract used a salting-out method by adding ammonium sulfate at saturation
levels of 0–20%, 20–40 %, 40–60 % and 60–80 %. Protein was purified by a
dialysis method using a cellophane membrane. The protein content was
determined by the Lowry method with the concentration of each fraction
respectively were were 0-20%, 20-40%, 40-60% and 60-80% is 0.82 mg/mL,
0.59 mg/mL, 0.72 mg/mL, 0.67 mg/mL. Test antibacterial activity is using agar
diffusion method with iron pecandang. The results showed that all protein
fractions from the brown algae Sargassum duplicatum isolated protein showed
bioactive compounds have inhibitory effects on the growth of pathogenic bacteria
such as Staphylococcus aureus and Echerchia coli. At 60-80% fraction of the
brown algae Sargassum duplicatum have inhibitory effects on the growth of the
largest 20.9 mm and 13.2 mm Staphylococcus aureus bacteria on bacterial growth
Echerchia coli after dialysis.
Keywords: Algae; antibacterial; protein fractions; Lowry method.
Introduction
Indonesia has been known as
an archipelago that most of its territory
is sea and has the longest coastline in
the world around 80791.42 km . In the
sea, there are many varieties of living
things like aquatic plants and aquatic
animals . The magnitude of the
potential of marine make scientists and
producers antibiotic compounds begin
to look to the sea world as a source of
potential antibiotics because most of
the natural resources in the sea has not
been exploited to the fullest and also
the needs of the world to new types of
antibiotics is increasingly urgent ,
because the standard antibiotics more
less effective because many pathogenic
bacteria resistant to that antibiotics.
High rates of both infections are
endemic and epidemic, and use of
drugs continuously suspected as the
cause of resistance ( Dali et al , 2011) .
Some marine life such as
sponges and algae have been
researched, explored and developed to
be used as a source of raw materials of
drugs in the pharmaceutical industry.
Exploration and research about marine
life for pharmaceutical has grown
rapidly within 30-40 years. This is
evidenced by the increased of
awareness from industry and consumer
of medicine (pharmaceutical) at our
country and abroad to prioritize the use
of drugs from natural products known
as " back to nature " (Dali et al, 2011) .
The natural drugs are the result
of secondary metabolites from living
organisms that have an unique
chemical compounds. Compounds
result of secondary metabolites found
in living organism is the elements that
is used as a deterrent to disease and
survival of the organism. Secondary
metabolites is then collected ,
processed , and used as a new drug
formula . Some secondary metabolites
byoorganism has become well known
drugs, such as aspirin, morphine,
digitalis,
penicillin,
and
taxol
(Anonymous, 2003).
There are several species of
macroalgae, such as Turbinaria
decurrens
Bory,
Sargassum
echinocarpum
JG
Agardh
and
Laurencia cartilaginea which has
many chemical compounds that can be
used for humans. The chemical
compound macroalgae Turbinaria
decurrens is sodium alginate and
iodine compounds. Sodium alginate
compound can be used in the
manufacture of antibacterial drugs,
antitumor, lowering hypertension and
overcoming glandular disorders. Iodine
content contained by macroalgae in
Indonesia reached 2000 times higher
than that found in sea water. The
content of iodine is more commonly
found on Turbinaria and Sargassum.
The content of iodine is useful to
address iodine deficiency in the
society, which decrease the level of
intelligence (Anonymous, 2002).
Macroalgae
Sargassum
duplicatum easily obtained in the
waters of Indonesia , the main
chemical compound as a source of
alginate and contain protein, vitamine
C, tannins, iodium, fenol as medicine
to goiter, antibacterial and tumors
(Trono & Ganzon, 1988). Sargassum
duplicatum contains Mg, Na, Fe,
tannin, iodine and phenol potential as
antimicrobial agents to several types of
bacterial pathogens that can cause
dysentery ( Sastry and Rao, 1994).
The ability of crude extracts
from several species of marine algae as
an antibacterial against Staphylococcus
aureus has been investigated by Val, et
al (2001). This research specifically
found that a species of macroalgae
Caulerpa taylori, Halimeda discoidea ,
Ulva rugida, Dictyota sp and
Osmundea hybrid effectively inhibit
the growth of bacteria Staphylococcus
aureus
with
a
diameter
of
approximately 14 mm. So far, it is not
much research data to explore group
protein compounds of macro algae as a
raw material for medicine in human
and animal diseases. The use of
proteins as raw material for the drug
has several advantages, including:
protein compounds can be received
well by the body and cause fewer side
effects (Huang, 1999) as well as genes
of protein compounds can be cloned
that can be produced on a large scale
on an industrial scale through genetic
engineering techniques .
This research done to explore
and characterize several bioactive
protein fractions from one type of
macro algae in South Sulawesi are
brown algae, brown algae contain the
pigment chlorophyll a and c, alpha beta
carotene, and alginate. Alginate
compound is can be used in
manufacture
antibacterial
drug,
antitumor, hypertension and cope with
glandular disorders (Parven and viqar,
2004). This algae has been consumed,
especially Sargassum duplicatum, this
type of brown algae contain 2.97%
protein, 0.26 % fat , antitumor agents,
algin, minerals (Ca, K, Na, Cu, Zn,
Mg, I, S and P, phenol. Algae also
contains antibacterial and anti viral
(Mulyo, 2013). Derived bioactive
proteins as antibacterial activity was
tested by agar diffusion method. From
the results of this reasearch are
expected emergence of knowledge and
a better understanding of the bioactive
protein components of macroalgae,
where the protein may function as an
antibacterial that can be used as a base
of new antibacterial drugs.
Materials and Methods
The materials used are brown
algae (Sargassum duplicatum), pure
cultures of bacteria Echerechia coli,
consisting of buffer A (0.1 M Tris (
hydroksimetil ) amino methane pH 8.3
, 2 M NaCl , 0.01 M CaCl2 ; β mercaptoetanol 1 % Triton X - 100
0.5%), consisting of buffer B (0.1 M
Tris (hydroksimetil) amino methane
pH 8.3 , 0.2 M NaCl , 0.01 M CaCl2) ,
buffer C consisting of (0.01 M Tris
(hydroksimetil) amino methane pH 8.3
, 0.2 M NaCl , 0.01 M CaCl2),
aquadest, NA medium (Nutrient Agar),
MHA medium (Muller Hilton Agar),
klorampenikol , BSA (Bovine Serum
Albumin), ammonium sulfate, Lowry
A consists of (phosphotungstat phosphomolybdat acid solution with
distilled water 1:1) , Lowry B consists
of (2 % Na2CO3 ; 0.1 N NaOH ;
CuSO4.5H2O 1 %, sodium potassium
tartrate 2 %), alcohol 70%, cotton,
aluminum foil, aquadest, rubbing
alcohol and a tissue roll.
Sampling and Sample Preparation of
Brown Algae
Sampling was carried out in the
waters of Lae - Lae island, Makassar,
South Sulawesi, with a depth of 1-2
meters . Samples of Algae were taken
using a knife and then stored in ice
box.
Sample Preparation
Isolation of bioactive proteins
algae using procedures modified from
previous methods (Moore , 2011) as
follows: The species of algae that has
been cut into small pieces collected
and weighed as much as 500 g fresh
weight, pulverized in a blender with
500 mL of solvent using buffer A
filtered using gauze . Then the filtrate
obtained by freeze / melt as much as 23 times .
Fractionation
Fractions containing proteins
and are thought to have antibacterial
and antifungal affinity fractionated
using ammonium sulfate saturation at
each level : 0-20 % ( F1 ) , 20-40 %
(F2) , 40-60 % ( F3 ) and 60-80 % (F4)
Dialysis
The precipitate obtained after
fractionation of the highest levels of
saturation
ammonium
sulphate
dissolved in a buffer B and
subsequently dialyzed in a buffer C.
The protein fractions were inserted into
a cellophane bag that does not leak or
damaged . Cellophane that has been
filled
with
protein
fractions
incorporated into a beaker containing a
solution of buffer C and then stirred
with a magnetic stirrer stirrer . Dialysis
is continued until a colorless buffer
solution again
Determination of Protein Content
For the determination of protein
content of each fraction using the
Lowry method with bovine serum
albumin ( BSA ) as a standard solution
Rejuvenation Bacteria Test
The bacteria Escherichia coli
and Staphylococcus aureus derived
from pure cultures, each taken as a
loop and then inoculated with inscribed
on NA medium slant . Culture of
bacteria on each agar slant incubated at
37 ° C for 18-24 hours .
Preparation of Bacterial Suspension
Test
The bacteria Escherichia coli
and Staphylococcus aureus has been
rejuvenated for 18-24 hours , each
taken one loop and suspended in
physiological saline solution of 0.9 %
sterile dilution is then performed to test
the bacteria suspension was obtained
against the blank transmittance 25 %
NaCl solution 0,9 % sterile .
Antibacterial Activity Tests
First of all mediums Saboroud
dextrose agar ( SDA ) for the two test
bacteria was poured into petri dishes
and allowed to solidify ( Base Layer ) ,
then 5 mL of medium NA 0.1 mL of
bacterial suspension was mixed
Staphylococcus aureus and then poured
on top of the base layer and allowed to
half solidified ( seed layer ) . The same
thing is done on Echerchia coli
bacterial culture test using sterile SDA
medium . 6 pieces pencadang placed
on the seed layer . Pencadang used has
an inner diameter of 6 mm , an outer
diameter of 8 mm and height 10 mm
and set each pencadang . Each
pencadang in a petri dish filled each
protein fraction with BSA as a negative
control and sklorampenikol as a
positive control . Both Petri dishes
were incubated in the incubator room
temperature . Observed and measured
the diameter of inhibition zone is
formed .
Results and Discussion
Extraction,
Isolation
Determination
of
Concentration of Algae
and
Protein
The results of measurements of
the protein content of the red algae me
crude extract and ammonium sulfate
fraction carried by the Lowry method
of use of a bovine serum albumin
(BSA) as the standard liquid . Protein
distribution pattern of brown algae in
the crude extract and fractions of
proteins of varying degrees of
saturation of ammonium sulfate in full
can be seen in Table 1 .
The
results
of
protein
concentration measurements that have
been done show that the highest
protein concentration of the protein
fractions 0-20 % of the brown algae
Sargassum duplicatum 0.18 mg / mL
to 35.90 mg total protein is the volume
fraction of as much as 44 mL
The concentration of different
proteins in each fraction, indicating
that the protein precipitated from each
fraction was different proteins . The
protein is precipitated by differences in
water solubility, higher solubility in
water, the more ammonium sulfate was
added. The highest concentration of
protein in the brown algae sargassum
duplicatum found in 0-20 % fraction (
F1 ) is equal to 0.82 mg / mL . From
these data indicate that the protein
fraction which has the highest
concentration of protein is a type of
protein that is low solubility in water .
Table 1. Distribution patterns fractionation of protein at various levels of
saturation of ammonium sulfate Sargassum duplicatum
Spesies
Algae
Sargassum
duplicatum
Level of
saturation
(NH4)2SO4
Volume
every
Fraction
(mL)
Concentration
of Protein
(mg/mL)
Total of
protein
(mg)
F1
44
mL
0,82 mg/mL
35,9 mg
F2
41,5 mL
0,59 mg/mL
24,7 mg
F3
43
mL
0,72 mg/mL
30,8 mg
F4
42,5 mL
0,67 mg/mL
28,3 mg
Protein fractions obtained from
the dialysis tested the ability of
bioactive compounds against several
species of pathogenic bacteria . The
test bacteria used in this research are
gram-negative bacteria ( E. coli ) while
gram-positive bacteria ( S. aureus ),
both of which are human pathogens .
Testing the inhibition of the
growth of bacteria carried by the agar
diffusion method . Agar diffusion
method is done by putting pencadang
on medium Mueller Hinton Agar (
MHA ) that has been soaked in a
solution of various concentrations of
protein fractions from brown algae .
Bioactivity test Antibacterial Protein
Fraction from Algae Sargassum
duplicatum
The results of measurements of
the diameter of the protein fractions
inhibitions brown algae sargassum
duplicatum against the two test
bacteria after an incubation period 1x
24 hours and 2 x 24 hours obtained the
results as listed in Table 2 .
Table 2 shows each the fraction
of protein at various levels of
saturation of ammonium sulfate
showed
bioactivity
antibacterial
against
Ecerchia
coli
and
Staphylococcus aureus, characterized
by the presence of a clear zone on each
test medium which proved that the
studied samples containing algael
protein compounds that could inhibit
the growth of pathogenic bacteria.
The strongest antibacterial
activity of brown algae Sargassum
duplicatum the incubation period of 1 x
24 hours against S. aureus bacteria
present in fraction 60-80 % ( F4 ) with
inhibition zone diameter of 17.8 mm
and the lowest activity found in
fraction 0-20 % ( F1 ) with inhibition
zone diameter of 11.8 mm. Positive
control (chloramphenicol) showed a
diameter of inhibition 21.5 mm.
Table 2. Diameter of inhibition average from protein fraction before dialysis browun
algae Sargassum duplicatum against test bacteria for 1 x 24 jam and 2 x 24 jam.
Diameter Of Inhibition (mm)
No
Level of
Saturation
(NH4)2SO4
E.coli
S.aureus
1 x 24 jam
2 x 24 jam
1 x 24 jam
2 x 24 jam
1
F1
10,9 mm
11,8 mm
11,8 mm
12,2 mm
2
F2
11,7 mm
12
mm
13,9 mm
14,2 mm
3
F3
12,0 mm
12,2 mm
15,2 mm
15,6 mm
4
F4
12,3 mm
12,9 mm
17,8 mm
18,2 mm
5
Kontrol (+)
16,7 mm
16,9 mm
21,5 mm
`21,8 mm
6
Kontrol (-)
7,00 mm
7,00 mm
7,00 mm
7,00 mm
After an incubation period of 2 x
24 hours, an increase in the diameter of
zone of inhibition against S. aureus in all
protein fractions, namely fractions of 020 % ( F1 ), the fraction of 20-40 % ( F2
), the fraction of 40-60 % ( F3 ) and
fraction of 60-80 % ( F4 ). This suggests
that the bioactive protein compounds
contained in all the protein fractions that
are bactericidal efficacious at usual
doses can kill germs (Tan and Kirana,
2002). Bactericidal generally inhibit
bacterial growth by irritating the cell
wall , bacterial proteins coagulate
because of differences in acidity, as well
as hydrolysis and liquid diffusion cell
that causes the osmotic pressure (
Siswandoyo , 1995) .
The
strongest
antibacterial
activity against Ecerchia coli bacteria
during incubation contained 1 x 24 hours
at fraction 60-80% (F4) with inhibition
zone diameter of 12.3 mm and the
lowest activity found in fractions 0-20%
(F1) with a diameter of inhibition zone
of 10.9 mm. Positive control inhibitions
forming a diameter of 16.7 mm.
After an incubation period of 2 x
24 hours , it turns out there was an
increase in diameter of inhibition zone
against Ecerchia coli bacteria in all
protein fractions as well as the positive
control. An increase in the diameter of
inhibition zone indicates that the
bioactive protein is bacteriosidal namely
the usual dose can kill bacteria .
Biological capabilities of each
bacterial has different response to
antibacterial ingredients. One of the
most influential factors is the existence
of structural differences between the
bacterial cell wall of gram-negative and
gram-positive
bacteria.
Specific
components owned by gram-positive
bacteria is composed of teikhioat acid,
teikhuronat acid, and polysaccharides
components, where as the specific
component of gram-negative bacteria
consists of lipoproteins, outer membrane
and lipopolysaccharide. Outer membrane
of gram-negative bacterial cell wall
membrane phospholipid bilayer is
largely replaced by lipopolysaccharide
molecules. Outer membrane has a
permeability to low molecule solutes that
the existing active substances can not
enter the cells of bacteria , the bacteria
more difficult consequently destroyed or
inhibited growth (Gupta , 1990) .
Antibacterial compounds as one
of antimicrobial agents have 3 different
forms of work, which is bacteriostatic,
bactericidal
and
bacteriolitic.
Mechanism of action is bacteriostatic
inhibits protein synthesis by binding to
ribosomes, whereas bactericidal prevent
growth and cause death , but does not
cause cell lysis of bacteria become. In
contrast to the bactericidal, bacteriolitic
works by making the bacterial cell lysis .
The process of bacterial cell lysis seen
from the decrease in the number of cells
or turbidity after such material is added
(Brock and Madigan, 1994). Third of the
antibacterial mechanism of action in
accordance with the results of the
research conducted is an antibacterial
agent is bacteriocidal that can prevent
the growth of microorganisms and cause
death .
Bioactivity test Antibacterial Highest
Protein Fraction Results Dialysis
Precipitated proteins after testing
antibacterial activity before dialysis have
the highest inhibitory activity was
dialyzed with the highest fraction
entering into a cellophane bag.
Cellophane that has been filled with a
suspension of protein solution was
dialyzed in dialysis buffer C. Fractions
were tested antibacterial results as was
done before dialysis to prove that the
antibacterial activity of the compound is
a protein, because the antibacterial
activity testing before dialysis was no
other substances that can inhibit the
growth of pathogenic bacteria in addition
to protein . From the results of
measurements of the diameter of the
inhibitions
against
the
bacteria
Echerchia coli and Staphylococcus
aureus obtained the data in Table 3.
Table 3. Inhibiton Zone of antifungal after dialysis from protein fraction after
dialisys from brown algae Sargassum duplicatum to several test bacteria for 1 x 24
jam and 2 x 24 jam
Diameter of Inhibition (mm)
Level of
E.coli
S.aureus
No
Saturation
(NH4)2SO4
1 x 24 jam
2 x 24 jam
1 x 24 jam 2 x 24 jam
1
F4
12,8 mm
14,1 mm
19,72 mm
20,96 mm
2
Kontrol (+)
18,6 mm
18,95 mm
22,03 mm
22,07 mm
3
Kontrol (-)
9,00 mm
9,00 mm
9,00 mm
9,00 mm
In general , from the observation
that the protein fraction obtained 60-80
% (F4) of algae Sargassum duplicatum
showed antibacterial activity against
Staphylococcus aureus largest with
inhibition of 20.96 mm and the
Echerchia coli bacteria with inhibition of
14.1 mm the incubation period of 2 x 24
hours after the dialysis process. Zone of
inhibition at the highest protein fraction
after dialysis rose higher than before
dialysis . It is proved that a protein
compound after dialysis highest protein
fractions have antibacterial activity . It
can be seen from the increase in
antibacterial resistance zone that has
been dialyzed protein fraction . This is
caused by ions bullies and small
molecules through the membrane after
dialysis out a cellophane bag , resulting
in an increase in bioactivity of protein ,
so that pathogenic bacteria inhibition
zone increased after dialysis process .
The ability of the inhibition may be
effective despite obstacles showed
smaller diameter than the positive
control , because according to
Cappuccino , JG and Sherman , N. , (
1992) that an antibiotic can be
considered effective to inhibit bacterial
growth when the diameter of the
obstacle is indicated ≥ 14 mm.
and 12.4 mm against the test bacteria
Echerchia coli , after dialysis zone of
inhibition of protein fraction 60-80 % (
F4 ) of algae Sargassum duplicatum ie
increased 20.9 mm against the test
bacteria Staphylococcus aureus and 13.2
mm against the test bacteria Echerchia
coli.
Preference
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A. M., 2005, Mikrobiologi
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dan
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Conclusion
All protein fractions from the
brown algae Sargassum duplicatum
isolated compounds showed a bioactive
protein that has the inhibition of the
growth of pathogenic bacteria such as
Sthpylococus aureus and Echercia coli.
Protein fraction 60-80 % ( F4 ) of algae
Sargassum duplicatum before dialysis
have inhibition zone of 18.2 mm against
the test bacteria Staphylococcus aureus
Gupta, M. D. S., 1990, Mikrobiologi
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Substance From Marine Algae.
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Using
Benzene,
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