1. RESEARCH POSTER PRESENTATION DESIGN © 2012
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ABSTRACT
METHODOLOGY
CONCLUSION
REFERENCES
Isolation and Characterization of Prodigiosin from Soil Bacterium
Aditya Sharma, Aditya Rishi, Ashley Abraham Antony, K.Kumanan, G.Dhanavathy
Department of Biotechnology, School of Bioengineering, SRM University.
INTRODUCTION
1
Pigment production and extraction
2
Biochemical analysis- preliminary confirmation
3
Estimation of prodigiosin by UV-Spec
Invitro determination of anti-microbial activity
- Well Diffusion Method
6
Invitro determination of anti-neoplastic activity
- MTT Assay
OBJECTIVES
RESULTS
4
4
Serratia marcescens is a gram negative soil
bacterium which secretes a light sensitive pink
pigment called prodigiosin. The pigment
prodigiosin was isolated from Serratia marcescens.
It was characterized for its chemical composition
and biological activity. The pigment was also
tested for its anti-microbial activity on a few
bacterial strains. The pigment was then tested for
its anti-neoplastic activity against cell-lines like A-
72 and Hep-2 under in-vitro conditions. Further,
the pigment was tested for its DNA staining ability
by agarose gel electrophoresis
1) To isolate the pigment prodigiosin from soil
bacterium Serratia marcescens.
2) To characterize prodigiosin.
3) To test prodigiosin for its anti-microbial activity.
4) To test prodigiosin for its DNA staining ability.
5) To test prodigiosin for its anti-neoplastic activity.
Characterisation of pigment- TLC & HPLC
4
5
Determination of prodigiosin’s DNA staining
- Agarose gel electrophoresis7
•Nutrient broth was found to be the optimum medium
to produce prodigiosin. (1516.9 prodigiosin units/cell
in nutrient broth , greater than that produced in
triolein , maltose containing medium and dextrose).
•The natural red pigment produced by S. marcescens
demonstrated a retention factor and retention time
corresponding to prodigiosin reported in the literature
in addition to a positive presumptive test.
•The clinical isolates evaluated in our study-
Streptococcus aureus, Staphylococcus agalactiae,
Salmonella enterica and Eschirechia coli, exhibited
relevant sensitivity to prodigiosin as significant
inhibition zones were achieved.
•A dose-dependent decrease in the number of viable
cells was observed in all the cell lines studied .
However, we did not observe a significant decrease in
the viability of the non-malignant Vero cells. Hence,
it can be inferred that prodigiosin possesses specific
neoplasm directed cytotoxicity .
• It was observed that prodigiosin starts staining DNA
at a concentration as low as 3 µg. However, effective
staining was observed at a range between 10 µg and
40 µg. At 50 µg concentration, agarose gel
electrophoresis showed DNA fragmentation, due to
the positive correlation between cytotoxicity and
DNA cleavage.
•Casullo HW, Fukushima K and Takaki GMC. (2010).
Prodigiosin production by Serratia marcescens UCP 1549
using renewable-resources as a low cost substrate.
Molecules. Vol. 15, p6931-6940
•Darshan N and Manonmani HK. (2015) Prodigiosin and
its potential applications. Journal of Food Science and
Technology. Vol. 52, p5397-5407.
•Lapenda JC, Silva PA, Vicalvi MC , Sena K X and
Nascimento S C. (2015). Antimicrobial activity of
prodigiosin isolated from Serratia marcescens UFPEDA
398. World journal of Microbiology and Biotechnology.
Vol31,p399–406
•Melvin M, Tomlinson J, Saluta G, Kucera G, Lindquist N
and Manderville R. (2000). Double-strand DNA cleavage
by copper prodigiosin. Journal of American Chemical
Society. Vol.122,p6333-6334
•Song MJ, Bae J, Lee DS, Kim CH, Kim JS, Kim SW and
Hong SI. (2006). Purification and characterization of
prodigiosin produced by integrated bioreactor from
Serratia sp. KH-95. Journal of Bioscience and
Bioengineering, Vol. 101, p157–161.
Serratia marcescens is a rod-shaped, motile, gram
negative bacterium which belongs to the family
Enterobacteriaceae. It can grow in temperatures ranging
from 5–40°C and in pH levels ranging from 5 to 9.
Pigment production is highly variable among species and
depends on many factors such as species type incubation
time, pH, carbon and nitrogen sources and inorganic salts.
Prodigiosin is a red pigment produced in Serratia
marcescens and belongs to a family of compounds termed
"prodiginines“.These have different compound structures
such as linear and cyclic derivatives, such as
undecylprodigiosin, cycloprodigiosin and
cyclononylprodigiosin etc. Prodigiosin is an alkaloid with
a unique tripyrrole chemical structure. It has three rings
forming a pyrrolylpyrromethane skeleton with a C-4
methoxy group, a molecular formula C20H25N3O and a
molecular weight of 323.44 Da. Prodigiosin is sensitive to
light and insoluble in water.
Prodigiosin has no known defined role in the physiology
of the strains in which it is produced, it exhibits a wide
range of biological activities such as antifungal,
antibacterial antiprotozoal, antineoplastic and antimalarial
activities. It has potent and specific immunosuppressive
activity best known for its capacity to trigger apoptosis of
malignant cancer cells. The exact mechanism is highly
complex but could involve multiple processes like
phosphatase inhibition, copper mediated cleavage of
double stranded DNA, or disrupting the pH gradient
through transmembrane transport of H+ and Cl- ions.
Evaluation of cytotoxic properties like chemically inert,
non-irritating, non-toxic and non-allergic has potential for
use in commercial sunscreens. Thus it holds vast potential
in its industrial utility and its exploitation as a wonder.
1. Isolation of pigment prodigiosin from soil
bacterium Serratia marcescens
Plate.1: Serratia
marcescens streaked
nutrient agar plates showing
discrete colonies
Plate.2: 2 ml of lyophilized
prodigiosin pigment isolated in
4% acidified ethanol
2. Characterisation of prodigiosin
Fig.1: HPLC analysis of prodigiosin
showing a retention time of 10.757
min.
Plate.3: Thin layer chromatography of
pigment prodigiosin exhibiting an Rf
of 0.81
According to Rahul et al. (2015), the substance
produced by S. marcescens, which has a retention
factor of 0.8 in hexane:methanol (1:2) mobile
phase is characterized as prodigiosin.
Moreover, Chaudhari et al. (2014) describes a red
pigment produced by S. marcescens, the retention
time of which is 10.424 minutes in 0.05%
orthophosphoric acid: acetonitirile solvent system,
as being prodigiosin. The above present findings
corresponds with this data.
3. In-vitro determination of anti-microbial activity
Fig.2: Antimicrobial activity of varying prodigiosin concentration
- A comparison
Significant inhibition zones were found for certain
strains which were in agreement with Lee et al. (2011).
The present findings are in agreement with this data.
4. Prodigiosin induced cell cytotoxicity on A-72
cells, Hep-2 cells and Vero cells- A comparison
Fig.3: A
comparison
showing specific
neoplasm
directed
cytotoxicity of
prodigiosin.
A dose-dependent decrease in the number of viable cells was
observed in all the cell lines studied . However, we did not observe a
significant decrease in the viability of the non-malignant Vero
cells.Hence, it can be inferred that prodigiosin possesses specific
neoplasm directed cytotoxicity
5. Determination of DNA staining ability
Plate.4: Electrophoresis of DNA samples in
0.8% agarose gel stained with 30 µg of
prodigiosin, showing positive staining of DNA
samples
It was observed that prodigiosin starts
staining DNA at a concentration as low as 3
μg.However, effective staining was observed
at a range between 10 μg and 40 μg.
At 50 μg concentration, agarose gel
electrophoresis showed DNA fragmentation.