Extracellular Protease Produced By Bacillus Ntegaterium Ibrl Ms 8.2 Via Submerged Fermentation And Its Application
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Date
2010-01
Authors
MANG LING, CHOO
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Abstract
The work carried out was on the development and production of extracellular
protease from microorganism. Isolate MS 8.2 was isolated from a local soil sample
and showed positive results on both primary and secondary screening for
extracellular protease. Based on the secondary screening results, isolate MS 8.2 was
selected for further studies. Biochemical tests, API 50 CHB kit and 16S rRNA test
results confinned that isolate MS 8.2 was identified as Bacillus megaterium IBRL
MS 8.2. Studies on the optimization of cultivation conditions and medium
compositions for protease production were perfonned using 250 mL Erlenmeyer
flask. Optimization of cultivation conditions resulted in the maximum protease and
biomass production of 93.42 U/mL and 2.05 giL respectively. The optimum
cultivation conditions were pH 7.5, 7.5 % (v/v; 2.25 X 107 cells/mL) inoculum size,
150 rpm agitation rate and temperature 30°C. Optimization of medium composition
resulted in the maximum protease and biomass production of 155.38 U/mL and 2.15
giL, respectively. The medium composition after optimization were 0.5 % (w/v)
sucrose, 1.0 % (w/v) beef extract, 0.1 % (w/v) KH2P04, 0.01 % (w/v) calcium
acetate, 0.01 % (w/v) CaCh and 0.01 % (w/v) MgS04·7H20. After the cultivation
conditions and medium composition optimization, the optimum cultivation time was
shortened from 48 to 30 hrs. The protease production was carried out in 5 L stirred
tank fennenter using optimized medium composition. Effect of agitation rate in
stirred tank fennenter revealed that the highest protease production of 264.53 U/mL
was obtained at 450 rpm, while the maximum protease production of 301.69 U/mL
was obtained for aeration at 1.0 vvm. The characterization of protease crude enzyme
revealed that the enzyme was active at the pH range of 5 - 10 and stable at the range
of 5 - 9, while it was active at the temperature range of 35 - 50°C and stable at the
range of 25 - 45°C. Protease produced by B. megaterium IBRL MS 8.2 was
suspected as aspartate and metallo protease. Its enzyme activity was enhanced by
various metal ions. The protease also showed its substrate specificity towards casein
with optimum concentration of 8 % (w/v). Studies on the optimization of degradation
of protein serum latex were perfonned using the optimized crude protease produced
by B. megateril!m IBRL MS 8.2. The optimum degradation reaction conditions were
2 mL protease enzyme, 3 rnL phosphate buffer pH 7.0, 5 mL rubber latex serum with
concentration of 8928.70 )lg/mL, 200 rpm of agitation speed and reaction
temperature at 40°C. An increase of 30 % in the degradation of latex protein was
detected after optimization of degradation reaction.
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Extracellular Protease Produced By Bacillus Ntegaterium Ibrl Ms 8.2 , Via Submerged Fermentation And Its Application