full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 7 (1) 518-526 January 2013, ISSN 0973-8916 (Print), 2230-7303 (Online)
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Media screening: The selected one was isolated
and used for L-asparaginase production in five
different mediums i.e. Tryptone Glucose Yeast
extract (TGY) broth, Tryptone Fructose east
extract (TFY), Yeast malt glucose (YMG) broth,
Yeast malt (YM) broth and 4% peptone broth in
37°C and at 200 rpm. They are analyzed for the
L-asparaginase production potentiality for 24 hrs
incubation period. After incubation period they are
transferred to 2 ml centrifuged tubes &
centrifuged and supernatant liquid was taken, L-
asparaginase assay was prepared from the
supernatant liquid (14). The obtained results
noted in the Fig. 6.
L-asparaginase activity (IU/ml)
5
4
3
2
1
0
TGY TFY YMG YM Peptone
Different media
Fig. 6. Media screening for L-asparaginase activity
Optimization of parameters
Effect of incubation time on production of L-
asparaginase: Experiments were conducted
with the selected media viz. 4% peptone for a
period of 54 hrs at regular intervals of 6 hrs to
evaluate the optimum time of fermentation for
the enzyme production and the results were
shown in Fig. 7. It can be concluded that the
accumulation of maximal L-asparaginase
production (4.8 IU/ml) is observed at 36 hrs.
Fermentation beyond 36 hrs showed a decrease
in enzyme production, which could be either due
to the inactivation of the enzyme because of the
presence of some kind of proteolytic activity or
the growth of the organism might have reached
a stage from which it could no longer balances
its steady growth with the availability of nutrient
resources.
Effect of temperature on L-asparaginase
production: Fermentation carried out at various
temperatures such as 32, 35, 36, 37 and 39°C
to study their effect on enzyme production.
Results were presented in Fig.7 indicating that
maximum enzyme production (4.833 IU/ml) was
obtained when SMF was carried out at 36°C.
However, the enzyme production reduced
gradually with further incubation temperature
above 37°C. This may be due to the denaturation
of microbial strain at high temperatures.
Effect of inoculum volume on production of
L-asparaginase: Fermentation carried out with
different inoculum volumes (1-6 ml) for a period
of 48 hrs to study its effect on L-asparaginase
production. The maximum enzyme production
(5.366 IU/ml) was obtained with 2 ml of 1 day
seed culture of Streptomyces species. as shown
in Fig. 7. If inoculum volume is further increased
then gradually there is a decrease in the
production of enzyme and microbial activity which
might be attributed to the nutrient limitations
necessary for product formation or accumulation
of some non-volatile self inhibiting substances.
Effect of pH on L-asparaginase production:
Experiments were performed to optimize pH in
order to maintain the favourable conditions for
the increase in the production of L-asparaginase.
This is established by carrying out the
fermentation by varying the pH from 5-8.5. The
profound effect of initial pH of the fermentation
on L-asparaginase production was shown in Fig.
7 and the maximum L-asparaginase activity (5.73
IU/ml) was recorded at pH 7.5. A further increase
in pH results the gradual decrease of L-
asparaginase activity due to denaturation or
inactivation of the microbial strain.
Effects of yeast extract concentration on
production of L-asparaginase: Since Yeast
extract is the substrate of L-asparaginase, the
addition to fermentation medium might stimulate
enzyme production. Hence, fermentation was
carried out with different concentrations of yeast
extract ranging from 0.4-1.8 (% w/v) for a period
Rao et al