Isolation of Serratia Marcescens (Isolate 30) and Partial Purification of Its Acrylamide-Degrading Amidase
A total of 217 bacterial isolates were isolated by an enrichment procedure from Malaysian soils which had been treated and non-treated with acrylamide. From the preliminary screening using MTT (3-[4,5-dimethylthiazol-2-yl]-,5- diphenyltetrazoliumbromide) assay, 10 bacterial isolates were found to...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2005
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/7033/1/FBSB_2005_24%281-24%29.pdf |
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| Summary: | A total of 217 bacterial isolates were isolated by an enrichment procedure from
Malaysian soils which had been treated and non-treated with acrylamide. From
the preliminary screening using MTT (3-[4,5-dimethylthiazol-2-yl]-,5-
diphenyltetrazoliumbromide) assay, 10 bacterial isolates were found to be
capable of utilizing acrylamide as a nitrogen source. Formation of the blue
colour of formazan and high absorbance were the criteria in the selection of
bacteria. These ten isolates were labeled as Isolate 5, 9, 10, 15, 30, 45, 60, 110,
115 and 145. The isolates were subjected to secondary screening using MTT
assay, Direct Plate Count and High Perfomance Liquid Chromatography (HPLC).
From the results obtained, five bacterial isolates, Isolate 9, 10, 15,30 and 60 gave
the highest colony count in CFUI mL and absorbance reading from the MlT
assay. These five bacterial isolates were then selected for further of examined for
their ability to degrade acrylamide by HPLC. Isolate 30 showed the best
degradation of acrylamide with 99.84% degradation after 48 hours incubation in enrichment culture with acrylamide as a substrate at a final concentration of 100
m a . Isolate 60 showed the second highest degradation of acrylamide at
70.53%, followed by Isolate 10 at 50.8% degradation and isolates 15 and 9 at
16.24% and 14.58% respectively. Control without bacteria accounted only 0.16%
acrylamide degradation.
Isolate 30 was selected for optimization of growth media with six different
parameters. These parameters were temperature, pH, different types of carbon
sources, different concentrations of carbon source, different amides as the
substrate and effect of different concentrations of selected amide substrate. From
the results obtained, Isolate 30 showed an optimum growth temperature at 27 " C.
Its highest colony count loglo cfulml was directly proportional to the growth of
bacteria. This isolate showed the highest growth at pH 7.5 where this type of
bacteria grew well near neutrality and slightly alkaline media. For the carbon
sources optimization, glucose was selected due to the high colony count. Isolate
30 was found to grow best at the glucose concentration of 1%. Among the amide
substrates, acrylamide was found to be the best sole nitrogen source which can
support bacterial growth in enrichment cultures and the bacteria showed the
highest colony count when 400 mg/L acrylamide was provided.
From the macroscopic and microscopic observations of Isolate 30, the bacteria
was gram-negative rod, might become differentiated into a convex, pigmented
and relatively opaque centre and an effuse, colourless, almost transparent
periphery with an irregular crenated edge. Biochemical tests using Microbact 24E (12A + 12B) showed that the Isolate 30, which had been isolated from Tanjung
Karang, Selangor gave 99.83% similarity to Serratia marcescens.
Amidase (EC 3.5.1.4) produced by Serratia marcescens strain 30 was partially
purified and characterized. The purification procedure used, including ionexchange
chromatography, M O ~ O - Qs~tro ng-anion exchanger, ultrafiltration and
gel filtration, yielded a partially purified amidase fraction having an overall
purification factor of 13.83 fold and a yield of 48.33%. Indophenol blue method
was used for the amidase assay and arnidase characterization studies was done to
determine the K, and Vmax value, the effect of temperature and pH on amidase
activity and the effect of heavy metals on amidase activity. K, values for
acrylamide were determined by incubating amidase at 50" C with various
concentrations of acrylamide (0.25 to 2 0 M ) in phosphate buffer (50mh4; pH
7.5). By using non-linear regression analysis, the Km and Vmax values were
0.9376mM acrylamide and 60.92 pmol ammonia disappearecUmin respectively.
The amidase exhibited maximal activity at 50" C and pH value at 7.5. The result
of effects of heavy metals on amidase activity showed that AgN03 and CuC12
inhibited amidase activity while HB03 enhanced amidase activity. The enzyme is
a monomer with an apparent molecular weight of 1 1 1 kDa. |
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