FUNDAMENTAL OF BIOPROSPECTION ASSIGNMENT

INVESTIGATION OF BIOACTIVE COMPOUNDS OF SEA

ANEMONE-ASSOCIATED BACTERIA AND THEIR
MOLECULAR IDENTIFICATION BASED ON 16S RDNA

BY :

SALSABILA LUQYANA

1710422023

BIOLOGY KBI

FACULTY OF MATHEMATIC AND NATURAL SCIENCE

DEPARTMENT OF BIOLOGY

ANDALAS UNIVERSITY

PADANG, 2018
 PREFACE

Praise be to Allah SWT who has helped his servant finish this paper with great ease.
Without help she may not be able to complete the author well.

This paper is prepared so that the reader gets information about the bioprospection of
bacteria found in sea anemones as bioactive compounds and bio pigments. This
paper is compiled by a compiler with various obstacles. Whether it comes from the
constituents themselves or who come from outside. But with patience and especially
God's help finally this paper can be completed.

This paper includes the “Investigation of Bioactive Compounds of Sea Anemone-

Associated Bacteria and Their Molecular Identification Based on 16s Rdna and is
deliberately chosen because the authors draw attention to the bioprospection of
molecular-organisms.

Hopefully this paper can provide a broader insight to the reader. Although this paper
has advantages and disadvantages. Authors beg for advice and criticism. Thank you.

Author
 TABLE OF CONTENTS

PREFACE
TABLE OF CONTENTS

CHAPTER I. INTRODUCTION
1.1 Background

1.2 Purpose

CHAPTER II. MATERIALS AND METHODS

2.1 Sea Anemone Sampling

2.2 Isolation of Bacteria

2.3 Pathogen Anti-Bacterial Activity Test

2.4 Bacterial Selection by Extraction

2.5 Characterization of Biopigmen

2.6 DNA extraction

2.7 PCR - PKS and NRPS

2.8 16S rDNA Polymerase Chain Reaction (PCR)

2.9 Visualization, Purification of 16s rDNA PCR

CHAPTER III. RESULTS AND DISCUSSION

CHAPTER IV. FINAL

3.1 Conclusion
3.2 Reccomendation

REFERENCES
 CHAPTER I. INTRODUCTION

1.1 Background

Sea anemones are one of the potential biota in coral ecosystems. The content
of secondary metabolites from these marine microorganisms known to be a
rich source of bioactive compound products and has potential applications
health and biotechnology (Thiel & Imhoff, 2003). Sea anemone tentacles are
used as catchers food (Friese, 1993). In the tentacles of sea anemones contain
stinging cells called nematocyst is a poison that contains various substances
such as peptide, protein, phospholipid, phospholipase, glycoproteins, sterols,
bioactive amines, and carbohydrates. Each nematocyst consists of bubbles
small which contains actinoporins, an inner filament and a small outer sensor
(Shiomi et al., 2003). The existence of associations of microorganisms with
marine organisms which also synthesize secondary metabolites such as host
organisms is a great potential for new alternative sources of exploration
(Proksch et al., 2002; Burgess et al., 1999).

Isolation and identification of bacteria associated with sea anemones

that are capable of producing compounds. Bioactive does not only provide
information about the diversity of bacteria in the microbial community
structure, but can also provide a stage from the solution to the problem of
providing bioactive compounds (Proksch et al., 2002). In this study, isolation
and molecular identification of bioactive compound-producing bacteria
including anti-bacterial compounds and biopigments associated with sea
anemones have been carried out.

1.2 Purpose

The purpose of this paper are :

1. Provide information about the diversity of bacteria in the microbial
community structure
2. Provide a stage from the solution to the problem of providing
bioactive compounds
 CHAPTER II. MATERIAL AND METHODS

2.1. Sea Anemone Sampling

Bacteria were isolated from green anemones taken from the waters of
Bandengan, Jepara, Central Java and anemones E. Medusivora that taken
from Lake Hajibuang, Maratua Island, Berau Regency, East Kalimantan.

2.2 Isolation of bacteria

Bacterial isolation was carried out by cutting the anemone's body

parts, then diluted and planted on Zobell 2216E agar medium and soybean
mannitol medium in a petri dish. As much as 1 L of ZoBell 2216E media is
made by mixing 15 g of bacto-agar (Merck); 2.5 g bacto-peptone (Merck);
0.5 g of yeast extract (Merck). The mannitol soybean medium was prepared
by mixing 20 g soy bean, 20 g mannitol (Manitol) and 15 g agar (Merck).
Incubation is carried out at room temperature for 48 hours (Radjasa et al.,
2007). Based on morphology the color of the growing bacterial colonies is
purified by the streak methodPetri dishes (Madigan et al., 2000).

2.3 Pathogen Anti-Bacterial Activity Test

The method used is the overlay method (Terkina et al., 2006) by

preparing petri dishes sterile containing 20 ml of media. Bacteria were grown
in mannitol soybean media and incubated for 4 days. On the third day, the
test bacteria were planted in liquid zobel media and shaken out with shakers
for 1 day. On the day fourth, the test bacteria which had been treated were
transferred into soft media with a ratio of 1% test bacteria volume to soft agar
media volume. The test bacteria were poured over the bacteria of the sample
that had been planted, and incubated at room temperature for 24 hours.
Observations are made by looking at whether there is a growth inhibition
zone bacteria around the sample.

2.4 Bacterial Selection by Extraction

For extraction and identification of pigments used acetone, diethyl

ether, methanol, acetonitrile, n-hexane, KLT silica gel 60 F plate 254
(Merck), Si-60 silica gel, N2 gas, distilled water, and aquabides Colored
green anemone bacteria are extracted using pigment cold solvent acetone (7:
2 v / v) (Kuki et al., 1994) and is authenticated (Britton, 1995). Bacterial
selection performed by observing the spectrum of crude extract dissolved in
acetone using ultraviolet spectrophotometer - visible Varian Cary 50 double
beam at a wavelength of 300–800 nm.

2.5 Characterization of Biopigmen

For the biopigment characterization process, crude extracts were

analyzed by high performance liquid chromatography (HPLC) using the ODS
RP-C18 stationary phase and the mobile phase of methanol: acetonitrile (7: 3
v / v), with flow rate of 1 ml. minute -1 (Maeda, 2005). The analysis was
carried out at a wavelength of 300-800 nm.

2.6 DNA extraction

DNA extraction was carried out using the freeze and thaw method
(Radjasa et al., 2007), by entering 1 (one) ose of bacterial culture in 100 μL
of sterile aquabides. Then the suspension is inserted in coolant at -70℃ and
heated in 95℃ for 10 minutes. The step is repeated 5 times.

2.7 PCR - PKS and NRPS

In this PKS PCR KSDPQQF primary is used (5 ’MGN GAR GCN

NWN ATG GAY CCN CAR CAN MG 3 ') and KSHGTGR (5' CCN ARN
SWN GTN CCN GTN CCN GTN CCR TG 3 ') (Piel, 2002). On PCR These
NRPS are used A2gamF primers (5 ’AAG GCN GGC GSB GCS TAY STG
CC 3’) and A3gamR (5 ’TTG GGB IKB GTS GIN CCS GAG GTG 3 CCG
'(mwgBiotech, Ebersberg, 34 Germany in Radjasa et al., 2007). DNA
amplification is done with the initial denaturation cycle at 94 ° C for 2
minutes, then denaturation successively (94℃ for 1 minute), annealing (55 °
C for 1 minute), and extension (72 ° C for 2 minutes). A series of
denaturations, annealing and extensions is repeated 45 cycles (Radjasa,
2005).

2.8 16S rDNA Polymerase Chain Reaction (PCR)

16s rDNA PCR amplification was carried out with temperature

treatment: denaturation at 94℃ for 3 minutes, then 30 cycles (annealing at
55℃ for 60 seconds, extension at 72℃ for 90 seconds and denaturation at
94℃ for 40 seconds), and 42℃ for 1 minute, 72℃ for 5 minutes and finally -
4℃. Primers used for 16s rDNA PCRs is a 27°F universal primer (5'-
AGAGTTTGATCMTG GC TCAG-3 ') and eubacteria 1492 R specific
primer (5'-TACG GYTACCTTGTTACGACTT-3 ') (Isnansetyo & Kamei,
2003).

2.9 Visualization, Purification of 16s rDNA PCR

Products and DNA Sequencing Visualization of 16S rDNA PCR

products was carried out by electrophoresis by inserting 5 µL of PCR
products into 1% agarose gel wells. On agarose gel 2%. Purification was
carried out to obtain pure DNA from 16S PCR amplification rDNA. The
materials used are High Pure PCR Product Purification Kit (Roche, Germany)
which consists of three solutions, namely solution 1 (binding buffer), solution
2 (washing buffer) and solution 3 (elution buffer). DNA from PCR is traced
through extension reactions using the primary 1492R and Big Dye terminator
V.3.1. The results of the reaction are read with (ABI 3130XL, Applied
Biosystem).
 CHAPTER III. RESULTS AND DISCUSSIONS

From E. Medusivora and Green Anemone obtained 10 bacterial isolates. Nine

isolates succeeded isolated from E. medusivora and 1 isolate from Anemone
Green. The appearance of bacteria that have been isolated can be seen in
Figure 3.

3.1 Antibacterial Sensitivity Test

Nine E. Medusivorous symbiont bacterial isolates were has been

tested against several test bacteria, obtained 1 bacterium that has antibacterial
activity (Figure 4). Specifications of these antibacterial activities shown in
Table 1.
3.2 Polymerase Chain Reaction - Polyketide Synthase -Non Ribosomal
Peptide Synthetase

Electrophoresis results indicate the presence of DNA files PKS

sequence in E. medusivore bacteria compared with Bacillus subtilis 168 (K)
control bacteria. DNA files have a length of about 300 bp, in accordance with
the length of the PKS and NRPS sequences ie 279 base pairs (Piel, 2002). Of
the nine marine anemone bacteria E. Medusivora which has been successfully
screened, there is one bacterium which can inhibit the growth of the test
bacteria Vibrio parahaemolyticus. This bacteria is bacteria that cause disease
outbreaks in shrimp and mouse grouper (Istiqomah et al., 2004). If V.
parahaemolyticus enters the human body can cause vomiting or even
defecation to acute diarrhea (Zein, 2004).

Burgess et al., (1999) state that if two competing species are grown on
the same place, there will be competition between these microbes to find
space and nutrients,so that it will lead to various strategies forgrow.
According to Manitto (1981) this is related to bacterial self defense
mechanism. Chemical substance often plays a role in the survival of a species
in dealing with other species. The species one will produce a compound that
can poison other species, so that growth the specs will be disrupted. Chemical
interactions between different bacterial species will affect production and
secretion of antimicrobial secondary metabolites (Burgess et al., 1999).
 Absence of anti-bacterial activity other than BNL 2.1 allegedly
influenced by several factors including Sea anemone bacteria other than BNL
2.1 are not can produce antibiotic compounds. According to Bunch & Harris
(1986), for producing antibiotics structural genes that provide code are
needed for enzymes involved in metabolite biosynthesis secondary and
regulatory genes that determine active whether or not repression or induction
of structural genes for biosynthesis. If you don't have the gene,bacteria cannot
produce compounds antibiotics. Microorganisms can produce metabolism
secondary in its genes for self defense and resistance. Polyketide synthase
(PKS) and Non Ribosomal Peptide Synthetase (NRPS) is known as part of a
type of secondary metabolite produced by microorganisms, including it is a
very anti-tumor agent and antibiotic medically valuable. To find out the type
Secondary metabolites produced, can be done by detecting the presence of a
gene fragment affect the production of secondary metabolites of isolates
selected bacteria. This gene fragment includes genes structural, that is the
gene that gives the code for enzymes involved in metabolite biosynthesis
secondary.

The results of the detection analysis of the existence of PKS and genes
NRPS shows that the selected bacteria have PKS coding genes and no genes
encoding NRPS. PCR analysis results show that secondary metabolites are
produced. It is believed that the PKS enzyme bioactive ingredient. Polyketide
is a bioactive compound synthesized by multifunctional enzymes (Otsuka et
al., 2004). Some polyket can be obtainedfrom macrolided and polyaromatic
compounds which is produced by bacteria. Polyketided syntheses from
catalyst reactions sequenced by enzyme activity which is called polyketide
synthases (PKSs). PKS is a complex multimenzime protein consists of a
group of coordinate cells which active. Biosynthesis occurs by means of
carbon chains simple 2-, 3-, 4-, like acetyl-CoA, propionyl CoA, butyyl-CoA
and its malonyl-derivatives, methilmalonil- and ethilmalonil-CoA. PKS
consists of a series of modules which are active sites for catalyzing each
condensation and elongation cycle chain. Each module in the MCC consists
of a core domain for the addition of each unit of peptide or ketide and a
number of optional domains that are responsible for modified clusters
(Ansari, 1992). Wrongone example of a product containing a compound
Polyketide is an erythromycy that is used as an antibiotic ingredient, a drug
for tract infections breath, gonorrhea and syphilis.

3.3 Selection of Green Anemone Symbiotic Bacteria with Extraction

Of several green Anemone symbionts bacterial isolates, obtained 1

yellow isolate. Morphology of bacteria the yellow one indicates that bacteria
have potential as sources pigment.To strengthen the statement, pigment
isolation using the method solvent extraction. This process is also carried out
sonication to break down bacterial cell membranes. Pigment screening
(screening) in the next stage performed using an ultraviolet
spectrophotometer visible at a wavelength of 300-800 nm.

The spectrum extracts coarse pigments in acetone solvents (Figure

6.A) shows that bacteria produce isolation contains carotenoid type pigments.
Results this is in accordance with Jeffry et al. (1997) which states that the
carotenoid spectra pattern has a peak absorption at a wavelength of 300-500
nm (at visible area of light). Carotenoid absorption peaks at wavelengths
visible light is caused by chromophore, that is a covalent unsaturated group
that can absorb radiation in UV areas and visible. In this system overlaps π
orbitals energy separation between the base level and the level reduced
excitement and the system absorbs at longer wavelength with large intensity
increase (Fessenden & Fessenden, 1986). The uptake is mainly caused by a
conjugated system C = C -C = C - C in carotenoids.

3.4 High Performance Water Chromatography Analysis (HPLC)

Different compounds have a polarity different too. By liquid

chromatography analysis High performance (HPLC) compounds will separate
according to polarity based on time retention. The results of the crude extract
analysis using HPLC is presented in Figure 6.B. Chromatogram observed at a
wavelength of 475 nm because at this wavelength, a separation pattern occurs
which is good compared to separation patterns at other wavelengths. On
picture 6 there are 32 peaks. Each peak is indication of the presence of
different compounds. High and low peaks vary, show difference in
concentration of these compounds. This explanation shows that Anemone
bacteria Green is able to synthesize the types of carotenoids different from
different concentrations. Carotenoids in bacterial cell membranes have a
function photoprotection is by trapping species Reactive oxygen produced by
light radiation the sun, so it can't damage the membrane cell (Kopsell &
Kopsell, 2006).
 Photoprotection function in the ecology of tropical oceans where
sunlight is so strong all year long, demanding adaptation for tropical marine
organisms to survive from exposure to damaging sunlight. Between this form
of adaptation is to synthesize carotenoids are responsible for the red color to
yellow, so that the color variation of the organism from tropical oceans are
richer when compared to non-tropical seas. Carotenoids consist of two main
groups, namely carotene (carotenoids which only consist of elements carbon
and hydrogen) and xanthophyll (carotenoids oxygenated, consisting of
elements of carbon, hydrogen and oxygen). To find out the type of
carotenoids contained in this sea anemone bacteria was tested epoxidation
using SbCl3 dan H2SO4concentrated.

The test results showed no color changes to blue (the pigment solution
remains yellow),thus indicating that carotenoids are produced by bacterial
isolates are Xantophils. This result is confirmed by the emergence of many
ribbons HPLC chromatogram at a long mooring time (> 15 minutes) by the
method of Maeda et al. (2005) which is a characteristic of carotene group
pigments. Further identification needs to be done using NMR and mass
spectrometry.

In the field of fisheries, carotenoids are pigments naturally can be

used as feed additives to increase the immunity of shrimp. Enhancement
immunity power will affect numbers survival and growth rate of shrimp.
Besides that carotenoids also function to protect shrimp from UV radiation,
and as a provider provitamin A. Carotenoids can also increase tolerance to
ammonia levels and low conditions oxygen. Carotenoids can stimulate
growth,maturation rates, increased fertility, and improve egg quality in the
process of reproduction (Mahendra & Semangun, 2008).

In industry, natural pigments can be used on food and medicine. Food

and pharmaceuticals that use natural pigments too has added value offered to
consumer. Food with carotenoid dyes for example, having added value
derived from the nature of carotenoids in the body that is as a source vitamin
A, anticancer and anti-obesity (Gross, 1991; Maeda et al., 2005).
3.5 Polymerase Chain Reaction 16S rDNA

The results of checking the 16S rDNA PCR show positive results with
the presence of files DNA isolates of BNL 2.1 with a suitable base length that
is around 1,500 bp (Figure 8). Sequencing The results of partial sequences of
16S rRNA gene from BNL isolates

3.5.1 isolated from E. medusivora is as the following:

3.6 BLAST Homology Data Analysis

Analysis of DNA sequences from bacterial banding isolates with

DNA sequences in the database (database) DNA (Altschul et al., 1997).
BLAST analysis results shows that bacteria are associated with E.
medusivore has as much homology 99% with Bacillus cereus and IK-B1
bacteria isolated from Green Anemone has a homology of 97% with
Xanthomonas sp.
3.7 Phylogenetic Analysis

Bacterial phylogenetic analysis is carried out with compare the closest

bacterial sequence to sequence of 16S rDNA target bacteria in the data base
Gen Bank. It is processed with the ClustalX program (Isnansetyo & Kamei,
2003).
CHAPTER IV. CONCLUSIONS AND RECOMMENDATIONS

4.1 Conclusions

Bioactive compounds produced by bacteria which is associated with

sea anemones including them antibacterial and biopigment compounds.
Compound antibacterial produced by Bacillus cereus, namely bacteria
associated with E. medusivora. These anti-bacterial compounds are
compounds bioactive synthesized by polyketide enzyme. While bio-pigments
are produced by Xanthomonas sp. which is associated with Green Anemone.
Biopigments produced by this bacteria can classified into carotenoids. The
bioactive compounds have a lot of potential to be used in the field of
fisheries, industry and health.

4.2 Recommendations

After this research, we hope that the bioactive compound and bio
pigment that we found on 2 sample can be processed to a new prouducts.
 REFERENCES

Ansari, M.Z. 1992. NRPS-PKS: A KnowledgeBased Resource for Analysis of

NRPS/PKS Megasynthetases.Nucleic Acids Res. 32:405413.

Atschul, S.F., T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller &
D.J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleid Acid Res. 25:3389-3402.

Britton, G., L. Jensen & H. Pfander. 1995.Carotenoid. Birkäuser Verlag Basel.

Boston. Berlin.

Bunch, A.W. & R.G. Harris. 1986. The Manipulation of Organism for the
production of secondary metabolites. In: Russel, G.G. (ed).
Biotechnology and Genetic Engineering Review. Vol 4. Intercept Ltd.
New Castle. 117-144 pp.

Burgess, J.G., E.M. Jordan, M. Bregu, A.M.Spragg & K.G. Boyd. 1999.
Microbial antagonism: a neglected avenue of natural products
research. J. Biotechnol 70:27–32.

Friese, E.U. 1993. Sea Anemones as a Hobby. TFH Publications. New Jersey.
365 p.

Gross, J. 1991. Pigments in Vegetables: chlorophylls and carotenoids. Van

Nostrand Reinhold. New York.

Isnansetyo, A. & Y. Kamei. 2003. Pseudoalteromonas phenolica sp.: a novel

marine bacterium that produces phenolic anti-methicillin-
resistantStaphylococcus aureus substances. Int. J. Syst. Evol. Micr. 53:
583-588.

Istiqomah, I., Triyanto., A. Isnansetyo., H.N. Kamiso & M. Murdjani. 2004.

Patogenisitas Vibrio fl uvialis yang diisolasi Dari Kerapu Tikus
(Crimileptes altivelis) dalam A. Irianto (Ed) Prosiding Seminar
Nasional Penyakit Ikan dan Udang IV. Purwokerto. 155-160.
Jeffrey, S.W., R.F.C. Mantoura & S.W. Wright. 1997. Phytoplankton pigments
in oceanography. United Nations Educational. Scientific and Cultural
Organization, Paris.

Kopsell, D.A. & D.E. Kopsell. 2006. Accumulation and Bioavaibility of

Dietary Carotenoids in Vegetable Crops. Plant Sci. 11: 499-507.

Kuki, M., H. Nagae, R.J. Cogdell, K. Shimada & Y. Koyama. 1994. Solvent
Effect on Spheroidene in Non Polar and Polar Solutions and The
Environtment of Spheroidene in The Light-Harvesting Complexes of
Rhodobacter sphaeroides 2.4.1 as Revealed by Energy of the 1Ag-
→1Bu+Absorbstion and The Frequencies of The Vibronically Coupled
C=CStretching Raman Lines in The 1Ag- and 2 Ag- State. Photochem.
Photobiol. 59:116-124.

Madigan, M.T., J.M. Martinko & J. Parker. 2000. Brock Biology of

Microorganisms. Prentice Hall. New Jersey.

Maeda, H., H. Masashi, S. Tokute, F. Katsura & M. Kazuo. 2005. Fucoxanthin

from Edible Seaweed, Undaniria Pinnatifi da, Shows Antiobesity Effect
Trough UCP1 Expression in White Adipose Tissues. Biochem. Biophys.
Res. Communications 332:392-397.

Mahendra, E & H. Semangun. 2008. Karotenoid Dalam Budidaya

Udang.Prosiding Back to Nature