Keywords (Extracted from title, table of contents and
abstract of thesis)
enzyme, production, alpha, amylase, Aspergillus, oryzae, submerged, fermentation,
amylolytic, potential |
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Abstract
The present study,
deals with the isolation, screening and selection of Aspergillus
oryzae for the alpha amylase production. Seventy eight isolates of
A. oryzae were isolated from different soil samples. The strains
were initially selected qualitatively on starch agar medium and
screened quantitatively for enzyme production in shake flasks and a
strain producing 130 ±0.1U/ml of enzyme was selected which was
assigned the code IIB-30. The selected strain was subjected to
physical and chemical mutagenic treatments in order to improve its
amylolytic potential. During the treatments, isolates were
qualitatively and quantitatively screened. Among these, EMS-18
exhibited the highest enzyme activity (347±1.2 U/ml). This mutant
showed 2.6 fold increased activity over the parental strain in terms
of enzyme production. The cultural conditions and nutritional
requirements of the selected strains (both wild and mutant) were
optimized in 250 ml Erlenmeyer flasks prior to scale up studies in a fermenter.
Six different fermentation media were evaluated for the alpha
amylase production by both wild and mutant strains of A. oryzae in
shake flasks fermentation. Of all the media, M4 containing (g/l);
starch 20, yeast extract 8.5, NH4Cl 1.3, MgSO4.7H2O 0.12, CaCl2 0.06
gave maximal enzyme production i.e., 168±2 (wild) and 385±2 (mutant)
which was highly significant (p≤0.05). The effect of incubation
temperature, initial pH, volume of media and inoculum size was
investigated on the enzyme production. The optimal enzyme production
was obtained at 30°C, pH 5, volume, 10 % and inoculum size 4 %, by
both wild and mutant strains. The rate of fermentation was also
studied and the highest yield of enzyme was obtained 72 h after
inoculation.
Corn starch (2 %) and lactose (1.5 %) as carbon sources while,
ammonium sulfate (0.3 %) and peptone (0.2 %) as nitrogen sources
were also optimized. Different surfactants were added to the
fermentation media and Tween 80 at the level of 0.1% was found to be
the best for enzyme production.
The scale up studies for alpha amylase production was carried out in
a 7.5 L stirred fermenter. The rate of fermentation for enzyme
production by both wild and mutant strains was investigated.) It was
found that the enzyme production increased gradually and reached
maximum (335 U/ml and 608 U/ml) after 64 h (wild) and 48 h (mutant).
The kinetic depiction of results showed optimal fermentation period
for enzyme production to be 64 h and 48 h, respectively. The other
cultural conditions such as initial pH (5), aeration level (1.5 vvm),
dissolved oxygen (15 %), inoculum size (10 %) and agitation
intensity (200 rpm) were optimized for enzyme production.
The fermented broth was subjected to ammonium sulfate precipitation
at different saturation levels (20-90 %). The optimum level of
ammonium sulfate saturation was found to be 70 % that gave 1.3 fold
purification. By using Sephadex- DEAE column, the active fractions
were eluted using 0.05 M Tris-HCl buffer containing 0.30 M NaCl at
pH 7.5. The molecular weight of alpha amylase was found to be 48 kDa
on SDS-PAGE after gel filtration. A total of 9.5 fold enzyme
purification was accomplished. The effect of time, temperature, pH
and metal ions on purified enzyme was also investigated and maximum
activity was achieved after 30 min at 40ºC and pH 5 in the presence
of Ca+2 ion.
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