PENGHASILAN SEBATIAN AKTIF BERSIFAT ANTIBIOTIK DARIP ADA BAKTERIA MARIN PSEUDOMONAS DOUDOROFFI ABK37-MM
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Date
2003-07
Authors
SYED MUTHALIB, SYED EZANEE ZULFAKHRI
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Abstract
A total of 450 bacterial and 7 fungal isolates were obtained from seawater, marine mud,
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~beach sand, floating material and marine macroorganisms collected from 11 sampling
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sites around the Penang Island. These marine microorganisms isolates were screened for
·the production of antimicrobially active compound. From 450 bacterial isolates, 71.1%
. were pigmented and sample from marine mud produced the highest marine bacterial
isolates by 55.6% compared to other samples. Screening for the production of
antimicrobial active compound from these bacterial isolates was performed by streaking
and submerged culture fermentation techniques using yeast extract medium with half
strength artificial seawater produced 3.3% and 47.1% positive isolates respectively, with
various strength of antimicrobial activities. From these 47.1% marine bacterial isolates,
sample from marine mud and yellow or yellowish pigmented isolates produced the
highest potential isolates by 73.6% and 66.0%, respectively. Of the 47.1%, 87.3%
isolates produced antibacterial compound, 19.3% isolates produced antifungal compound
and 6.1% isolates produced antiyeast compound. Despite this, only 1.9% marine bacterial
isolates showed antimicrobial activities against all the 8 test microorganisms.
One potential marine bacterial isolate which produced the highest antibiotically active
compound was chosen for the optimization of medium and cultural conditions in a shake
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nask system and was then identified as Pseudomonas duodoroffi ABK37-MM. The
optimized medium for the production of antibiotically active compound against the test
bacteria, Staphylococcus aureus was 1/8 diluted yeast extract medium ~~th half strength
artificial seawater. The optimized medium compositions (%; w/v) were 0.06 maltose,
. 0.06 yeast ext-act, 0.145 malt extract and 0.4 L-histidine. The optimized artificial
,. seawater compositions (%; w/v) were 1.8 NaCl , 0.5 MgCb.6H20, 0.1 CaCb,2H20, 0.1
r,KCI, 0.1 KBr, 0.2 Na2S04 and 0.05 NaHC03. The optimized cultural conditions for the
·production of antibiotically active compound were at an initial medium pH of 7.4,
agitation rate of 140 rpm, incubation at room temperature (30±2°C), inoculum size of 6%
and the volume of medium to the volume of flask ratio at 0.5. The production of
antibiotically active compound increased by 37.0% with 14.3% faster than before
optimization in a shake flask system .. Medium compositions, buffer concentration and a
few physical parameters were reoptimized in the tubular air lift fermenter. The optimized
medium (%; w/v) were 0.08 maltose, 0.08 yeast extract, 0.145 malt extract and 0.4 Lhistidine
with 0.1 NaHC03. The optimized physical parameters were 1.5 vvm rate of
aeration, inoculum size of 8% and the volume of medium to the volume of fermenter
ratio of 0.75. Optimization at the fermenter level increased the production of
antibiotically active compound by 26.0% at the same production time after optimization
at the shake flask level, that was at 144 hours of cultivation. In general, there was an
increase of about 72.6% in the production of antibiotically compound after optimization
at a fermenter level compared to shake flask system.
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Partial purification of the antibiotically active compound with sephadex G-1 00 gel
filtration chromatography produced 9.2 folds of purity with specific antibiotically active
compound of 1148.2 U/mg protein. Determination of molecular weigh!_ was done using
SDS-PAGE and was estimated about 39 kD compared to known low molecular weight
protein. Determination of protein compound and reducing sugar in the compound were
4.9 mg/ml and 0.02 mg/ml, respectively. The antibiotically active compound was found
stable at and below of room temperature (30±2°C) and was fully deactivated at the
temperature of 40°C. This compound was also found stable at pH range of 6 to 8 and
fully deactivated by proteolytic enzyme suggesting that the major part of the compound
was protein or protein moiety. TEM studies on the effects of the antibacterial compound
on the test bacterial cell, Staphylococcus aureus revealed a chain reaction that occurred at
the cell wall and cell membrane leading to cell lysis compared to untreated cell. The
frequency and magnitude of the cell wall and cell membrane bleb bing and fragmentation
of the test bacterial cell increased with the prolonged of exposure time to antibacterial
compound, and it seems to produced pores in the bilayer cell wall leading to cell lysis.
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PENGHASILAN SEBATIAN AKTIF BERSIFAT ANTIBIOTIK DARIP ADA BAKTERIA MARIN