Optimization of
process parameters for the production of L-asparaginase
from an isolated fungus
V Sreenivasulu1*,
KN Jayaveera2 and P Mallikarjuna Rao3
ABSTRACT
The extracellular L-asparaginase production by fungi isolated from soil samples
by using pH and dye based method. Various physical and chemical parameters were
optimized under submerged fermentation for L-asparaginase
production. Maximum productivity of L-asparaginase
(19.5 U/ml) was achieved by employing medium containing 2% (w/v) L-asparagine as substrate concentration, 1%(w/v) glucose as
carbon source, 1% (w/v) ammonium sulphate as an
additional nitrogen source with the incubation period of 96 h and incubation
temperature at 30oC, initial pH 6.5 at an inoculum
level 20% (v/v) with 48 h old inoculum was found to be
optimum for maximum yield.
Key words: L-asparaginase,
optimization and submerged fermentation
INTRODUCTION
L-Asparaginase is an enzyme present in a wide range of
organisms including animals, microbes, plants and in the serum of rodents but
not in human beings1. This enzyme acquired some clinical importance
in 1961, when the antitumour effect of guinea pig
serum, originally discovered by Kidd (1953) 2, was traced by Broome (1961) 3 to the presence of
this enzyme.
L-Asparaginase (L-asparagine amido hydrolase, E. C. 3.5.1.1)
catalyses the hydrolysis of L-asparagine into L-aspartate and ammonia. This catalytic reaction is
essentially irreversible under physiological conditions4.
Supplementation of L-asparaginase results in
continuous depletion of L-asparagine. Under such an
environment, cancerous cells do not survive. This phenomenal behaviour of cancer cells was exploited by the scientific
community to treat neoplasias using L-asparaginase5,
6,7. This enzyme is also a choice
for acute lymphoblastic leukemia, lymphosarcoma and
in many other clinical experiments relating to tumour
therapy in combination with chemotherapy. This treatment brought a major break
through in modern oncology, as it induces complete remission in over 90% of
children within 4 weeks8. L-asparaginase is also used for the treatment of pancreatic
carcinoma9 and bovine lymphomosarcoma 10.
Moreover, L-asparaginase preparation from food grade Aspergillus
A wide
variety of microbial strains also produce L-asparaginase
13,14, 15. The enzyme from Escherichia
coli and Erwinia carotovora
was clinically used to treat patients
suffering from acute lymphoblastic leukemia and lymphomas16. However, L-asparaginase
from bacterial origin can cause hypersensitivity in the long-term used, leading
to allergic reactions and anaphylaxis17. For example, Erwinia asparaginase is
considered less toxic and is frequently employed when compared with allergic
reactions to E. coli asparaginase. However, Erwinia
asparaginase had a shorter half-life than E. coli18,
suggesting the need to discover new L-asparaginases
that are serologically different but have similar therapeutic effects. The
search for the other asparaginase sources like
eukaryotes can lead to an enzyme with less adverse effects.
It has been
observed that eukaryote microorganisms like yeast and filamentous fungi have a
potential for asparaginase production 19,20.
This requires screening of soil samples from various sources for isolation of
potential microbes which have the ability to produce the desired enzyme.
The increasing importance of L-asparaginase in
recent years for its anticarcinogenic
applications promoted us to screen for newer L-asparaginase
producing organisms. Hence, in the present work, an attempt was made to isolate
L-asparaginase producing fungi and in this paper, we
report the factors that influence the maximization of L-asparaginase
production by using submerged fermentation. To the best of our knowledge, very
little work has been carried out on L-asparaginase
from fungal source.
All chemicals
used in this study were of analytical grade.
Different soil samples were collected locally, near
The soil
samples were inoculated to the isolation medium and poured into sterile Petri
dishes. The plates were incubated at 28 ˚C and control media were also included in which the
substrate L-asparagine was omitted. The single discrete colonies which have
exhibited clear pink zone surrounding
microbial colonies after 48 h incubation indicate L-asparaginese
producing cultures. L-asparaginase converts L-asparagine into aspartic acid and ammonia. This can easily
be detected by the change in pH of the medium using phenol red. These
colonies(L-asparaginase positive cultures) were
picked up and grown in the modified Czapeck Dox agar and potato dextrose agar slants.
Secondary screening
Secondary
screening for isolated colonies was further performed by streaking on modified Czapeck Dox agar medium with
phenol red as an indicator for the detection and confirmation of colonies for
L-asparaginase
production. Control was also maintained without L-asparagine.
Among 50 isolates tested, one isolate was designated as
VS-26. It was identified as Aspergillus
sp. on the basis of its morphological
characteristics. This isolate was used for subsequent studies and was
maintained on modified Czapeck Dox
agar slants at 4 ˚C and subcultured at every 4
weeks.
Inoculum preparation:
The organism was grown on modified Czapeck
Dox agar slants at 28 ˚C for 7 days for complete
sporulation.
5 ml of sterile water was added to the slant. The spores were scrapped
off and transferred into a 250-ml Erlenmeyer flask containing 45 ml of inoculum medium that is modified Czapeck
Dox medium15 containtining (gl-1): Glucose, 2.0; L- Asparagine,
10.0; KH2PO4, 1.52; KCl,
0.52; MgSO4. 7H2O,
0.52; CuNO3.3H2O, trace; ZnSO4.7H2O,
trace; FeSO4.7H2O.trace;
pH 6.2. The flasks were incubated at 28 ˚C in rotary shaker at 150
rpm for 48 hours. The contents of the flasks were harvested and washed with
sterile deionised
water and the cells were resuspended in sterile deionised water. This cell suspesion
was used as inoculum for subsequent experiments.
5ml (10%) of inoculum (4x107
spores/ml) was inoculated into 45ml of basal production medium (modified Czapeck Dox medium) contained in
250ml Erlenmeyer flasks and incubated at 28 ˚C on rotary shaker at 150 rpm
for 96 h. At the end of fermentation,
broth was harvested by filtration
through Whatman no.1 filter paper, the
clear filtrate was used as crude enzyme source and used for the enzyme
activity.
Optimization studies
This includes
optimization of various physico-chemical parameters
required for maximum L-asparaginase production by Aspergillus sp. VS-26 under submerged
fermentation. Study of various parameters included incubation time (48-168 h), different initial
pH values (4 - 9), various temperatures (20, 25, 30, 35, 40, 45 and 50˚C),
various inoculum levels (5, 10, 20, 30, 40 and 50 %
of inoculum volume), concentration of L-asparagine (0.5 – 4% w/v), effect of carbon sources
(sucrose, glucose, dextrose, fructose, lactose, potato starch, maltose, starch
soluble, galactose, and cellulose at 1%w/v), effect
of additional nitrogen source (beef
extract, peptone, casein, soybean meal, urea, malt extract, yeast extract,
gelatin, NH4 H2PO4, NH4 Cl, KNO3 and NH4SO4
at 1% w/v).
The
procedure adopted for optimization of various parameters influencing L-asparaginase production was to evaluate the effect of
independent parameters keeping others constant and to incorporate it at the
optimized level in the next experiment while optimizing other parameter. All experiments
were in triplicate and the mean values are presented.
The enzyme activity was measured in culture filtrates
by Nesselerisataion method according to Imada et al. (1973)13 . The reaction mixture
containing 0.5 ml of L-asparagine (0.04 M), 0. 5ml of
phosphate buffer (0.1M) pH 8.0, 0.2 ml of enzyme solution in 0.8 ml deionised water was incubated at 37 0 C for
30min. The reaction was terminated by
addition of 0.5 ml of 1.5 M trichloroacetic acid
(TCA). Then to 3.4 ml deionised water, 0.1 ml of above mixture and 0.5ml of Nesselers reagent were added and colour
developed was read at 450 nm in Elico double beam UV
visible spectrophotometer (SL164).
Enzyme blanks were used as control.
The L-asparaginase activity
was calculated from the standard graph
prepared with ammonium sulphate and enzyme
activity expressed as U/ml.
One unit of L-asparaginase
activity is defined as that amount of enzyme which catalyses the formation of 1
µ mol of ammonia per minute under the conditions of the assay.
RESULTS AND DISCUSSION
The isolated colonies are streaked on modified Czapeck Dox agar medium with
phenol red as an indicator for further confirmation of colonies for L-asparaginase production.The
profound growth was observed in association with clear pink zone suggesting
microbial strains are producers of L-asparaginase
enzyme. Among them 10 isolates showed good L-asparaginase
activity (data not shown), a strain designated as VS-26 gave maximum enzyme
activity (3.64 U/ml) 22 and shown clear pink zone within and surrounding the colony as shown in fig.1 (a).
The pink zone was not observed in the control medium that is not containing L-asparagine, shown in fig.1(b). And this strain (VS-26),
identified as Aspergillus sp. was selected for optimization
studies.
Fig.1.(a ) - A clear
pink zone was produced within and
around the fungal colony (strain no. VS-26) on modified Czapek Dox agar medium with 1% w/v L-asparagine
(sole nitrogen source) with phenol red.
Fig.1.(b) - No pink zone was observed around the
fungal colony (Strain no.VS-26) on modified Czapek Dox agar medium
containing phenol red without L-asparagine.
OPTIMIZATION STUDIES
Effect of incubation period
To study the optimum incubation time for maximum L-asparaginase production, the fermentation samples were
withdrawn periodically at every 24 h up to 168 h and assayed. The results are
indicated in fig.2. The organism grew
well in the medium and maximum L-asparaginase
production (3.73 U/ml) was achieved at 96h. After that the L-asparaginase production gradually decreased with increased
incubation periods. The same incubation period was reported by Mishra, (2007)23 for L-asparaginase
production by Aspegillus
Effect of initial pH
It is known that pH plays an important role on the
growth of organism as well as their metabolite. The effect of initial pH of the medium on L-asparaginase
production was studied and the results are presented in fig.3. The
results indicated that a gradual increase in L-asparaginase
production was observed from pH 5.0 to 6.5 followed by a gradual decrease of
enzyme yield beyond pH 6.5. Higher or
lower than this pH (6.5) resulted in lower yield of L-asparaginase.
The optimum quantity of L-asparaginase
production was 5. 1U/ml. The same pH was observed on the production
of L-asparaginase from Aspergillus
Effect of incubation
temperature
The higher temperatures
had some adverse effect on the metabolic activities of
the micro-organism and it has been reported by various scientists that the
metabolic activities of the micro organisms become slow at lower temperature 24.
The effect of temperature was studied by keeping the isolate for the production at 20, 25, 30,
35, and 40 ˚C. The results
indicated at 30˚C was optimum temperature for L-asparaginase
production (5. 4U/ml). The results are
indicated in fig.4. The decrease in the yield of enzyme was observed when
incubation temperature was higher
or lower than optimum incubation temperature.
Effect of inoculum level
It is also
an important factor for the production of
L-asparaginase. A lower inoculum
density may give insufficient biomass causing induced product formation, where
as a higher inoculum may produce too much biomass
leading to the poor production25. Various inoculum
levels were tried for study of their effect on L-asparaginase
production. The results are indicated in fig.5.
The enzyme production was increased with increase in level of inoculum up to 20% (10 U/ml) and further increase in inoculum level did not increase the enzyme production.
Effect of L-asparagine concentration
L-asparagine act as sole
nitrogen source and also as an inducer to L-asparaginase
production. So, its concentration variation will show some impact on enzyme
production. The L-asparagine 2% (w/v) gave the
optimum concentration for L-asparaginase
production(16.5U/ml), the results are
indicated in fig.6. Sarquis et al. (2007)26 reported
L-asparaginase
production by filamentous fungi
is under nitrogen regulation.
Effect of different carbon sources
Effect of different carbon sources (1 % w/v) were
studied on L-asparaginase enzyme production. In this
isolate (VS-26), it was investigated and observed that glucose is the best
carbon source which gave maximum yield (18.8 U/ml), followed by soluble starch
and sucrose which were also found to enhance biocatalyst production, results
indicated in fig.7. The role of glucose in the synthesis of L-asparaginase is controversial. It is generally accepted as catabolic
repression in the case of E. coli and Erwinia aeroideae
at higher concentrations was reported
by Jeffries, (1976)27 ; Liu
and Zajic, (1972)28 . Glucose was best carbon source under aerobic conditions for
synthesis of L-asparaginase by Serratia marcescens
(Nima) was
reported by Sukumaran et al. (1979)29. The increased production of L-asparaginase in the presence of glucose was observed in the
present study.
Effect of additional
nitrogen source
Additional nitrogen source (1%w/v), different organic
and inorganic nitrogen sources were tried. Among various additives ammonium sulphate exhibited marginal increase of enzyme
production(19.5 U/ml). Results are presented in fig.8. Praksham et al.
(2006) 12 reported ammonium
chloride as best additional nitrogen source for L-asparaginase
production by Staphylococcus sp.-6A.
With the above-optimized conditions the productivity of
L-asparaginase was 19.5 U/ml for isolated fungal
strain VS-26. Initial level of L-asparaginase production in Czapeck
Dox medium was 3.64 U/ml .
All
optimized factors in submerged fermentation
showed impact on L-asparaginase enzyme
production by this isolated microbial strain. A significant improvement (5.4
times) in production by this microbial isolate was noted under optimized environment.
The results of the present study indicated a scope for exploring terrestrial
fungi as a sole source for extracellular L-asparaginase,
an enzyme that has gained industrial and pharmaceutical significance recently.
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Received on 06.04.2009
Accepted on 28.05.2009
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Research Journal of Pharmacognosy and Phytochemistry. 1(1): July.-Aug. 2009, 30-34