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Abstract
The present study
describes the isolation, identification and screening of fungal
strain Rhizopus oligosporus var. microsporus (Saito) Schipper &
Stalpers, (1984) for the production of extracellular lipases. One
hundred and sixty seven cultures of fungi were isolated from
different environments such as soil, air, milk, pickle, oily bread,
destroyed fruits and vegetables by serial dilution method. The
strains were initially selected qualitatively on Tween 80-Agar
plates and were shifted to the slants of PDA (Potato Dextrose Agar)
for maintenance and storage at 4oC. Quantitative screening for
extracellular lipase production by isolated strains was carried out
in shake flasks and the most potent strain, IIB-63 producing 3.20 ±
0.003 U mL-1 of enzyme was selected. The strain was then identified
on the basis of standard morphological measurements and was assigned
the code IIB-63.
The selected strain was then subjected to physical (UV and Gamma
radiations) and chemical mutagenic (NA, EtBr, MNNG/NTG) treatments
in order to improve its lipolytic potential. During the treatment
mutants were qualitatively and quantitatively selected and
IIB-63NTG-7 was found to be the mutant showing highest lipases
production (10.37 ± 0.06a U mL-1) with a zone size of 12.3 mm on
Luria-Bertanitributyrin agar plates. This mutant showed an overall
325% increase in activity over its parent strain for the production
of extracellular lipase.
The fermentation experiments for the production of extracellular
lipases by wild and mutant strains were carried out in 250 mL
Erlenmeyer flasks and laboratory scale 5L stirred fermenter. The
cultural conditions were optimized for both wild and mutant strains
of R. oligosporus.
Seven different culture media were tested for the production of
extracellular lipase by both wild and mutant strain of R.
oligosporus in shake flask fermentation. Of all the media evaluated
M5 (gL-1 Peptone: 20, Glucose: 10, K2HPO4:2.0, MgSO4.7H2O: 0.12,
NH4Cl: 1.0, Yeast Extract: 2.5, pH: 7.0) gave highest units of
extracellular lipases 3.16 ± 0.02a U mL-1 (W) and 10.99±0.02a U mL-1
(M). Other culture media gave lesser production of enzyme by both
wild (M2>M7>M3>M4>M1>M6) and mutant (M1>M2>M3>M4>M7>M6) strains. The
production of enzyme was found to be highly significant (P≤0.05) in
media M5.
The effect of incubation temperature (15 -45oC), initial pH
(4.0.-10.0), inoculum size (0.5-3.5 mL) and volume of the medium
(25-150 mL) on the production of extracellular lipase by both wild
and mutant strains was investigated in shake flask. The rate of
fermentation was also studied and found maximum extracellular lipase
was obtained after an incubation of 48 h by both wild and mutant
strains.
Various agro industrial by-products (CSM: cotton seed meal, SBM:
Soybean meal, WB: Wheat Bran, WF: Wheat Flour, SFM: Sunflower meal,
AM: Almond meal, RB: Rice Bran) were tested for their effect on
lipases production. In the presence of SBM (0.4%) the maximum
lipolytic activity was 5.09 ± 0.008a U mL-1 (W) & 16.43 ± 0.005a U
mL-1 (M) which was approximately 1.27 (W) & 1.42 (M) times higher
than that in the absence of additive.
Different additional carbon sources was added to basal medium with
the aim of improving extracellular lipases production. In the
present study effect of different carbon sources such as lactose,
maltose, sucrose, xylose, dextrose, glucose, starch and Tween 80
were evaluated for the production of extracellular lipases by wild
and mutant strains of Rhizopus oligosporus. Of all the carbon
sources tested, Tween 80 showed considerable increase in lipases
production by both wild (5.52 ± 0.005a U mL-1) & mutant (19.13 ±
0.005a U mL-1) strains as compared to others.
Marked increase in the productivity of the enzyme has been observed
upon addition of some nitrogen additives compared with the
non-supplemented medium. Different organic nitrogen sources such as
peptone, p-nitrophenol, casein, nutrient broth, urea, yeast extract
and corn steep liquor were independently added to the fermentation
medium. Maximum extracellular activity of lipase 5.85 ± 0.01a U mL-1
(W) & 28.32 ± 0.01a U mL-1 (M) was obtained when 0.8 % of casein was
added in the fermentation medium as an organic nitrogen source.
Different Inorgaic salts used are ammonium chloride [NH4Cl],
ammonium sulfate [(NH4)2SO4], ammonium nitrate [NH4NO3], ammonium
acetate [NH4CH3COO], ammonium ferro (II) sulfate 12- hydrate [(NH4)2
Fe (SO4)2.12H2O], hydroxyl ammonium chloride [HONH3Cl], Ammonium
oxalate [(NH4)2C2O4] and ammonium molybdate [(NH4)6MoO24]. Maximum
extracellular production of lipases 8.31 ± 0.01a U mL-1 (W) and
34.34 ± 0.01a U mL-1 (M) were observed when 0.8 % (NH4)2C2O4 was
added in the substrate as an additional inorganic
nitrogen source.
The rate of fermentation by extracellular lipases by both wild and
mutant strains of R. oligosporus var. microsporus was investigated
in stirred fermenter. It was found that the growth and lipases
production was increased gradually and reached its maximum 9.07±
0.42a U mL-1 (W) & 42.49 ± 3.91a U mL-1 (M) after 30 h of
fermentation for both wild and mutant strain. There is overall
increase of 109% (W) and 124% (M) in the production of extracellular
lipases as compared to shake flask. Another significant finding of
the present experiment is that the fermentation period is reduced to
30 h in case of wild and 23 h in case of mutant from 48 h in shake
flask studies.
Effect of different sizes of inoculum was investigated for
extracellular lipases production by both wild & mutant strains of R.
oligosporus var. microsporus IIB-63 in stirred fermenter. The size
of the vegetative inoculum was varied from 1-5% and fermentation was
carried out. It was observed that lipases activity of the both wild
(13.76 ± 0.99a U mL-1) and mutant (46.34 ± 3.05a U mL-1) strains was
gradually increased with the increase of inoculum size and reached
its maximum at 3% of inoculum.
The initial pH of the fermentation medium was varied from 7.0 to 9.0
and was controlled throughout the fermentation process however the
experiment with uncontrolled pH was also carried out. The production
of extracellular lipases was found maximum 30.39 ± 2.58 U mL-1 (W) &
50.42 ± 4.37 U mL-1 (M) when the pH of the medium was maintained at
8.0. However in the experiment with uncontrolled pH there is no
remarkable increase in the production of enzyme.
The rate of the agitation was varied from 150-300 rpm. Maximum
enzyme production by both wild (27.30± 1.98 U mL-1) and mutant
(54.01± 4.54 U mL-1) strains was obtained when the agitation speed
was maintained at 250 rpm. Change in the rate of agitation resulted
in decreased enzyme production. The rate of aeration was varied from
0.2-1.0 vvm. Production of enzyme by mutant strain was found maximum
i.e., 59± 4.88 U mL-1 when the aeration rate was set at 0.8 vvm
while wild strain gave maximum lipase units (28± 1.97 U mL-1) at 1.0
vvm.
The enzyme produced after optimization of the cultural conditions
was subjected to ammonium sulfate precipitation for salting out the
proteins. 60% ammonium sulfate showed the enzyme activity of 11.45 U
mL-1 by wild and 28.2 U mL-1 by mutant strain of R. oligosporus var.
microsporus. While in 80% ammonium sulfate the enzyme activity by
both the wild (13.14 U mL-1) and mutant (29.5 U mL-1) strains
increased which indicates the partial purification of enzyme.
Desalted enzyme was subjected to DEAE-cellulose column for ion
exchange chromatography. Wild strain shows 206.73 fold purification
and 82.56% recovery while the mutant strain shows 407.34 fold
purification with 62.83% recovery. Sephadex G-100, is a cross-linked
polymer used for gel filtration chromatography, which is used for
differentiating the molecular size. There is 446.19 fold (W) and
710.02 fold (M) purification of enzyme which shows that most of the
contamination proteins are removed.
The effect of pH, temperature and metal ions was also investigated
on the activity of purified lipases. It is evident from the results
that the maximum residual activity by both strains 81% (W) and 100%
(M) was observed in the reaction mixture of pH 8.0. The results
showed that lipases retained 80% of its activity at 25oC-30oC by
wild and 100% of its activity at 20oC-50oC by mutant strain of R.
oligosporus var. microsporus. Mn++ stimulated the activity of
lipases by the wild while it has inhibitory effect on lipases
activity of mutant strain. Other ions Like Ca++, K+, Mg++, Cu++ and
Na+ stimulated the activity of lipases by both wild and mutant
strains. Both wild and mutant strains showed the same response of
inhibition of enzyme activity in the presence of Hg++ and Fe++.
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