IJRET-OPTIMIZATION OF L-ASPARAGINASE PRODUCTION BY ASPERGILLUS TERREUS MTCC 1782 USING BAJRA SEED FLOUR UNDER SOLID STATE FERMENTATION

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IJRET: International Journal of Research in Engineering and Technology eISSN: 2319-1163 | pISSN: 2321-7308

OPTIMIZATION OF L-ASPARAGINASE PRODUCTION BY
ASPERGILLUS TERREUS MTCC 1782 USING BAJRA SEED FLOUR
UNDER SOLID STATE FERMENTATION

V.Varalakshmi1*, K. Jaya Raju2
1,2Center for Biotechnology, Department of Chemical Engineering, A.U. College of Engineering, Andhra University,
Visakhapatnam- 530003, Andhra Pradesh, India, [email protected]
*Corresponding Author

Abstract
Enzymes are the biocatalysts synthesized by living cells. They are Complex protein molecules that bring about chemical reactions
concerned with life. They are protein in nature, colloidal and thermolabile in character, and specific in their action. L-asparaginase
(L-asparagine amido hydrolase, E.C.3.5.1.1) is an extra cellular enzyme that has received considerable attention since it is used as an
anticancer agent. The present work deals with production of extracellular L-asparaginase from Aspergillus terreus MTCC 1782 using
Bajra seed flour under solid state fermentation Process parameters like Incubation time(96 h), Temperature (300 C), Moisture content
(70% v/w), pH of the medium(8.0), Inoculum Age (5 days), Inoculum volume (1 ml), carbon source (1.5% w/v glucose), nitrogen
source ( 2% w/v ammonium sulphate), and metal salts ( 0.1% w/v Magnesium sulphate) were optimized and giving an overall yield of
273.3 U/gds of maximum L-asparaginase activity after optimization. The observation made in this study hold great promise for scale
up production of L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour as substrate under solid state
fermentation.

Index terms: L-asparaginase, Aspergillus terreus, Bajra seed flour, Solid state fermentation, Optimization

-----------------------------------------------------------------------***----------------------------------------------------------------------
1. INTRODUCTION
L-asparaginase is relatively wide spread enzyme found in
many tissues, bacteria, plant and in the serum of certain
Many enzymes have been used as drugs like wise L-
rodents, not of man. The microbial sources are very common
asparaginase (L-asparagine amidohydrolase, E.C.3.5.1.1)
for L-asparaginase, because they can be easily cultured and
attracted much attention because of its anticarcinogenic
extraction, purification of L-asparaginase from them is also
potential. The important application of the L-asparaginase
convenient, facilitating for the Industrial scale production. The
enzyme is in the treatment of acute lymphoblastic leukemia
most commonly used microorganism to produce L-
(mainly in children), Hodgkin disease, acute myelocytic
asparaginase
are
Erwinia
caratovora,
Bacillus
sp.
leukemia,
acute
myelomonocytic
leukemia,
chronic
Corynebacterium glutamicum, Pseudomonas stutzeri and E.
lymphocytic
leukemia,
lymphosarcoma
treatment,
coli.[4] L-asparaginase from E .coli has excellent power to
reticulosarcoma and melanosarcoma. [1]. L-asparaginase
inhibit the activity of tumour cells, and that from E.
belongs to an amidase group that hydrolyses the amide bond in
chrysanthemi is also pharmacologically active[5]. However,
L-asparagine to aspartic acid and ammonia. L-asparaginase is
L-Asparaginase from bacterial sources causes hypersensitivity
very essential amino acid for the growth of tumor cells
in the long term leading to allergic reactions and anaphylaxis
whereas the growth of normal cell is independent of its
[6]. The search for other L-Asparaginase sources, like
requirement [2]. It can be produced within the cell by an
eukaryotic microorganisms, can lead to an enzyme with less
enzyme called Asparagine synthetase. Most of the normal
adverse effects. It has been observed that eukaryotic
tissue synthesizes L-asparagine in amounts for their metabolic
microorganisms like yeast and filamentous fungi have a
needs but the tumour cells (especially Malignant and
potential for L-Asparaginase production .Yeast sources such
Carcinoma Cell) require external source of L-asparaginase for
as Rhodatorula sp., Rhodosporidium toruloides, actinomycetes
their growth and multiplication [3]. In the presence of L-
such as Nocardio sp Streptomyces longsporusflavus and
Asparaginase, the tumor cells deprived of an important growth
fungal sources such as Aspergillus tamari and Aspergillus
factor and they may failure to survive. Thus this enzyme can
terreus, Aspergillus nidulans, Aspergillus niger have been
be used as a chemotherapeutic agent.
found to produce L-Asparaginase.


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Solid state fermentation (SSF) is a very effective technique as
reaction mixture contains 0.5ml of L-asparagine (0.04M ,
the yield of the product is many times higher than in
0.5ml of Tris-HCl buffer 0.1M (pH 8.0), 0.5ml of enzyme
submerged fermentation [7].SSF offers many advantages over
solution and distilled water to a total volume of 2.0ml was
submerged fermentation such as lower energy requirements,
incubated at 37C for 30 min. The reaction was stopped by
less risk of bacterial contamination, less waste water
adding 0.5ml of 1.5 M Trichloro-acetic acid (TCA). Then to
generation and less environmental concerns regarding the
3.7ml distilled water, 0.1 ml of the above mixture and 0.2ml of
disposal of solid waste [8]. Other advantages include ease of
Nessler's reagent was added and colour developed was read
product extraction that does not require complicated methods
after 10-15 min at 450nm in a UV-Visible spectrophotometer.
of treating the fermented residues. In comparison with SmF,
The ammonium concentration of the reaction was determined
SSF offers better opportunity for the biosynthesis of low-
by the reference from the standard curve of ammonium
volume-high cost products [9]. The present research work was
sulphate.
carried out for the production of L-asparaginase by

Aspergillus terreus MTCC 1782 under solid state fermentation
One unit (U) of L-asparaginase was defined as the amount of
using bajra seed flour as substrate and optimized the
enzyme that liberates 1mole of ammonia under optimal assay
production conditions.
conditions. Enzyme yield was expressed as the activity of L-

asparaginase per gram dry substrate (U/gds).
2. MATERIALS AND METHODS

3. RESULTS AND DISCUSSIONS
2.1 Microorganism and Inoculum Preparation
3.1 Screening of Substrates
The fungal strain Aspergillus terreus MTCC 1782 was
procured
from
Institute
of
Microbial
Technology,
In SSF, the selection of a suitable substrate for a fermentation
Chandigargh, India. It was maintained on Potato Dextrose
process is a critical factor as they play a dual role of supply of
Agar (PDA) slants. The microbial strain was grown at 30C
nutrients to the microbial culture growth and anchorage for the
for 4 days after which, it was stored at 4C until further use
growing cells. In the present study, seven substrates, viz. bajra
and sub-cultured after every four weeks. For preparing a spore
flour, ragi seed flour, cassava, sugarcane baggase, groundnut
suspension, to a well-sporulated slant of A.terreus, 10 ml of
shell powder, tamarind seeds, and corn cob were screened
sterilized 0.1% Tween 80 solution was added. The surfaces
with Aspergillus terreus and the results were shown in fig.1
were scrapped with an inoculating loop to suspend the spores

and the spore suspension was taken as inoculum.
All the substrates promoted enzyme production with A.terreus.

The maximum L-asparaginase activity of 160.38 U/gds was
2.2 Solid-State Fermentation:
achieved in a medium containing bajra seed flour as the
substrate followed by ragi seed flour and lowest activity of
Fermentations were initiated from spores in 250mL
55.66 U/gds was observed in case of corn cob.
Erlenmeyer flasks that contained 5 g of bajra seed flour. The

media was moistened to 40% (v/w) with distilled water,
Abha Mishra et al., 2006 reported production of L-
autoclaved at 1210C for 15 min at 15 lb pressure and cooled to
asparaginase from Aspergillus Niger using agricultural
room temperature and then inoculated with 1 ml of 96 h old
substrates like bran of Cajanus Cajan, Phaseolus mungo and
A.terreus spore suspension under aseptic conditions. The
Glycine max.
contents of the inoculated flasks were mixed thoroughly and

incubated at 300C temperature in an incubator for 96 h of
Hymavathi et al., 2009 reported asparaginase production by
period. All experiments were carried out in duplicate [10].
isolated Bacillus circulans MTCC 8752 under solid state

fermentation using different agricultural materials like red
2.3 Crude Enzyme Extraction
gram husk, Bengal gram husk, coconut, and groundnut cake.
After the incubation period, the crude enzyme from the
fermented substrate was extracted using 0.1M phosphate
buffer (pH 8). After mixing the fermented substrate with 41 ml
of buffer, the flasks were kept on a rotary shaker at 150 rpm
for 30 min. The slurry was centrifuged at 10,000 rpm for about
10 min at 4C in a cooling centrifuge. Supernatant was
collected and used for enzyme assay.

2.4 Assay of L-Asparaginase
The activity of L-asparaginase was determined by estimating
the amount of ammonia liberated from L-asparagine. The
method of Imada et al., 1973 was followed. The enzymatic
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IJRET: International Journal of Research in Engineering and Technology eISSN: 2319-1163 | pISSN: 2321-7308

decreased which might be due to depletion of nutrients,
180
accumulation of toxic end products.
160

140
Abha Mishra et al., 2006 reported 96 h as optimized
120
s
)

100
incubation period for the production of L-asparaginase by
d
/
g

80
Aspergillus niger.
60


(
U

40
Soniyamby et al., 2011 reported 96 h as optimum incubation
i
t
y

20
period in case of Penicillium sp.
c
t
i
v

0

e

A

m
180
z
y
n

160
E
s
)

140
d
/
g

120

(
U

i
t
y
100
Substrates screened
c
t
i
v

80

e

a


60
m
Fig-1: Screening of different substrates for L-asparaginase
z
y

production by A.terreus
n
40
E

20
3.2 Optimization of Fermentation Process:
0
Fermentation parameters that influence the L-Asparaginase
production during SSF were optimized over a wide range. The
0
24
48
72
96 120 144 168 192 216
strategy adopted
for standardization of
fermentation
parameters to evaluate the effect of an individual parameter
Fermentation time (h)
and incorporate it at standard level before standardizing the

next parameter. The Process parameters optimized were

incubation time, incubation temperature, initial moisture
Fig-2: Effect of Incubation time on L-asparaginase production
content of the substrate, initial pH adjusted with 1N HCl or 1N

NaOH, inoculum age, inoculum Volume. And also the effect
3.2.2 Effect of Temperature
of additional supplements like carbon sources, nitrogen
Fermentation was carried out at different temperatures such as
sources and metal salts were also studied. All the experiments
20, 25, 30, 35, 40, 45, 50, 55, 600C to study their effect on
were conducted in duplicate and the mean values were
enzyme production. Incubation temperature has a profound
reported.
effect on L-asparaginase production by A.terreus under solid

cultural conditions. The maximum enzyme yield of 174.9
3.2.1 Effect of Fermentation Time:
U/gds was obtained when SSF was carried out at 30C.
Optimum fermentation time for asparaginase production was

determined by conducting experiments with the bajra seed
The significance of the incubation temperature in the
flour as substrate using different time intervals from 24 h to
development of fermentation process is such that it could
216 h with a variation of 24 h. From fig.2 it can be concluded
determine the effects of inhibition, cell viability and death.
that there were variations in enzyme level produced in culture
However, the enzyme production reduced gradually with
filtrates with period of incubation. Analysis of culture
further increase in incubation temperature. This may be due to
supernatant showed enzyme activity rise from an initial of
heat that accumulates in the medium during mesophilic
49.2 U/gds at 24th h giving its peak activity of 169.46 U/gds at
aerobic SSF, because of poor heat dissipation which could
96 h of fermentation. Fermentation beyond 96h showed a
lead to a further drop in the oxygen level and there by
decrease in enzyme production, which could be either due to
reducing the growth of the test organism.
the inactivation of the enzyme because of the presence of

some kind of proteolytic activity or the growth of the organism
Sarqius et al.,(2004) have reported 30C is suitable for L-
might have reached a stage from which it could no longer
asparaginase production through submerged fermentation by
balance its steady growth with the availability of nutrient
using A. terreus and A. tamarii.
resources. At longer incubation periods, the enzyme activity
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IJRET: International Journal of Research in Engineering and Technology eISSN: 2319-1163 | pISSN: 2321-7308

K.G. Siddalingeshwara (2010) reported optimized temperature
210
as 30OC by Emericella nidulans.

190
Yogendra singh et al., (2012) observed the maximum activity
at 30C by Bacillus aryabhattai strain ITBHU02.
s
)
d
170

/
g

200

(
U
150
i
t
y

180
130
s
)
160
c
t
i
v

d
/
g

110
e

A

140
m

(
U

120
z
y

i
t
y

90
n
100
E
c
t
i
v

70
80
e

A

m
60
50
z
y
n

10
20
30
40
50
60
70
80
90 100
40
E
20
Moisture Content (% v/w)

0

15
20
25
30
35
40
45
50
55
60
Fig-4: Effect of initial moisture content on L-asparaginase
production
Temperature (0C)


3.2.4 Effect of Initial pH:

Fig-3: Effect of temperature on L-asparaginase production
Growth and metabolism along with enzyme production is

governed by an important factor called pH. Different
3.2.3 Effect of Initial Moisture Content
organisms have different pH optima and any modification in
their pH optima could result in a decrease in their enzyme
Moisture content of fermentation medium is the most critical
activity.
factor in SSF is determined for L-Asparaginase production by

maintaining the medium with moisture content range of 10 to
Experiments were carried out to find the optimum pH in order
100 %(v/w) with a variation of 10%(v/w) .The highest enzyme
to maintain the favourable conditions for increased L-
production of 185.8 U/gds was achieved at 70% initial
asparaginase production. This was established by carrying out
moisture content. A further increase in the initial moisture
the fermentation by varying the pH from 2-10 (adjusted with
content beyond 70% resulted in a significant reduction in the
1N HCl or 1N NaOH. The maximum L-asparaginase
enzyme production. Moisture optimization can be used to
production of 191.3 (U/gds) was obtained at pH-8.0. This may
regulate and to modify the metabolic activity of the
be attributed to the balance of ionic strength of plasma
microorganism. High moisture level of substrate leads to
membrane.
decreased porosity, lower oxygen diffusion, increased risk of

bacterial contaminations, enhanced aerial mycelial formation,
G.Thirumurugan et al., 2011 reported an optimum
reduction in gas volume, decreased gaseous exchange and
asparaginase production at pH 8.0 by Aspergillus Terreus.
change in the degradation of the lignin. Likewise low moisture

levels might lead to reduction in solubility of nutrients of the
Selvakumar 2011 observed peak activity of asparaginase at pH
solid substrate, lower degree of swelling and higher water
8.0 by Streptomyces noursei MTCC 10469.
tension. Therefore, initial moisture content plays an important

role in enzyme production during SSF.

Abha Mishra (2006) reported 70% moisture content to be
optimum by Aspergillus Niger.

Kaliwal B.B et al(2011) reported 70% moisture content to be
optimum by Fusarium equiseti.
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IJRET: International Journal of Research in Engineering and Technology eISSN: 2319-1163 | pISSN: 2321-7308

3.2.6 Effect of Inoculum Volume
200
Inoculum volume is also an important factor that influences
s
)

180
d
the production of metabolites under SSF. Adequate inoculum
/
g

can initiate fast mycelium growth and product formation, there
160

(
U

by reducing other organism contamination. Quantity of
i
t
y

inoculum had a definite effect on enzyme titers. Optimization
140
of inoculum volume is necessary in SSF because low density
c
t
i
v

of spores leads to insufficient biomass and end product
e

A

120
synthesis as well as permit the growth of undesirable
m
contamination and too high densities of spores may cause a
z
y

100
n
quick and too much biomass production thereby leading to fast
E
nutrient depletion and ultimately reduction in the end product
80
quality. In general lower level of inoculum may not be
sufficient to initial growth whereas a higher level may cause
0
1
2
3
4
5
6
7
8
9
10
competitive inhibition.

pH
The effect of inoculum level on L-Asparaginase production


was studied by conduction of the fermentation with different
Fig-5: Effect of initial pH on L-asparaginase production.
inoculum levels. The substrate was inoculated with culture of

1-10ml of inoculum level in different flasks. The substrate was
3.2.5 Effect of Inoculum Age
incubated at 30C for 5days. After completion of fermentation,
the enzyme was extracted and analyzed for the L-asparaginase
The effect of inoculum age on L-asparaginase production was
activity. 1 ml inoculum level gave maximum production of L-
studied by conducting the fermentation with different
asparaginase 207.7U/gds.
inoculum ages. The substrate was inoculated with 1-day old

culture to 10-day old day culture in different flasks. The
Jayaramu et al., 2010 obtained maximum titre values of
substrate was incubated at 30C for 4 days. After the
asparaginase with inoculum size of 0.75 ml using Emerricelan
completion of fermentation, the enzyme was extracted and
nidulans.
analyzed for the L-asparaginase activity. The five day old

culture gave maximum production of asparaginase 202.2
Chankya Pallem et al.,(2011) have reported the maximum
U/gds. The biosynthetic activity declines as the age of the
production of L-asaparaginase with inoculum volume of 1.5
culture increases.
ml of 7 days old Fusarium oxysporum through SSF.


Optimum production values were obtained for Pseudomonas
210
aeruginosa by Manikandan et al., 2010 with 0.5 ml of
inoculum on soya bean meal.
190
s
)


d
/
g

170

(
U

150
i
t
y

c
t
i
v

130
e

A

m
110
z
y
n
E

90
70
0
1
2
3
4
5
6
7
8
9
10
Inoculum Age(Days)


Fig-6: Effect of inoculum age on L-asparaginase production

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Chankya Pallem et al.,(2011) have reported Fusaium
220
oxysporum exhibited the maximum production of L-
200
asparaginase production by using glucose as the carbon
source.
180
s
)


d
/
g

250
160
(
U

i
t
y

140
s
)
200
d
c
t
i
v

120
/
g

e

A


(
U

m
100
150
i
t
y

z
y
n

80
E
c
t
i
v

100
e

A

60
m
z
y

40
n
50
E
0
1
2
3
4
5
6
7
8
9
10
Inoculum Volume (ml)
0


Fig-7: Effect of inoculum volume on L-asparaginase
production

Carbon Sources
3.2.7 Effect of Carbon Source:


Generally carbohydrates are used as carbon sources in the
Fig-8: Effect of carbon source on L-asparaginase production
microbial fermentation processes. The energy for the growth

of desired microorganism during industrial fermentation
3.2.7(a) Effect of Glucose Concentration:
derived either from the oxidation of medium components or
from light .Carbon sources in media formulation are used to
In order to investigate the effect of glucose concentration on
enhance growth and subsequently resulted in higher enzyme
the fermentation medium, SSF was carried out with different
production, which is normally observed in the synthesis of
glucose concentrations varying from 0.5-3.5 % (w/v). From
primary metabolites, such as enzymes. Poor growth in SSF
the data obtained, it can be concluded that the maximum
system is associated with poor nutritional level in solid
production of L-asparaginase (235.0 U/gds) was obtained with
substrates. The carbon concentration had a positive effect on
the optimum glucose concentration of 1.5% (w/v). Further
L-asparaginase production and high titres can be obtained in a
increase in glucose concentration resulted in the decrease of
medium rich of carbon source.
enzyme production it may be due to inhibitory affect at higher

concentrations.
To determine the effect of carbon sources on L-asparaginase

yield, different carbon sources were tested which include
Baskar and Renganathan 2011 reported that 0.6% glucose was
glucose, lactose, fructose, maltose, sucrose, galactose, and
found to be best carbon source for maximum L-asparaginase
soluble starch. Each of them at a concentration of 0.5% w/v
production using modified Czapek-dox media containing soya
with other optimized conditions was supplemented to the
bean flour as substrate by Aspergillus terrus MTCC 1782.
production medium of A.terreus and they have exerted a

considerable effect on the biosynthesis of L-asparaginase.
Chankya Pallem et al., (2011) have reported Fusaium
The maximum enzyme production was promoted by glucose
oxysporum exhibited the maximum production of L-
with a yield of 218.6 U/gds.
asparaginase production by using 0.3%glucose as the carbon

source.
Baskar and Renganathan (2011) reported that glucose was
found to be best carbon source for maximum L-asparaginase
production using modified Czapek-dox media containing soya
bean flour as substrate by Aspergillus terrus MTCC 1782.
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IJRET: International Journal of Research in Engineering and Technology eISSN: 2319-1163 | pISSN: 2321-7308

240
300
230
250
s
)

220
s
)

d
d
/
g

/
g

200
210
(
U

(
U

i
t
y

i
t
y
200
150
c
t
i
v

c
t
i
v

190
e

A

e

A

100
m
m 180
z
y

z
y

n
n
E
170
E
50
160
0
150
0
0.5
1
1.5
2
2.5
3
3.5
Glucose concentration%(w/v)


Fig-9: Effect of glucose concentration size on L-asparaginase
Nitrogen Sources
production



3.2.8. Effect of nitrogen source:
Fig- 10: Effect of nitrogen source on L-asparaginase
In microorganisms, nitrogen (both organic and inorganic
production
forms) is metabolized to produce amino acids, nucleic acids,

proteins and cell wall components. Nitrogen sources have
3.2.8 (a) Effect of ammonium sulphate concentration:
been preferred for enhancing the production of L-
In order to evaluate the effect of ammonium sulphate
asparaginase. Most of the industrial enzymes utilize nitrogen
concentration on the fermentation medium, SSF was carried
source either in organic form or inorganic form sometimes
out with different concentrations of ammonium sulphate
both. In many instances growth will be faster with supply of
varying from 0.5-3.5% (w/v).
organic and inorganic nitrogen source.


The results revealed that the maximum L-asparaginase
The supplementation of additional nitrogen sources (either
production (267.8 U/gds) was obtained with ammonium
organic or inorganic) such as ammonium nitrate, ammonium
sulphate concentration of 2% (w/v). Further increase in
sulphate, sodium nitrate, malt extract, yeast extract, beef
ammonium sulphate concentration resulted in the decrease of
extract, tryptone, urea and peptone to the production medium
enzyme production due to the repressor effect of ammonium
had shown a profound impact on the production of L-
sulphate at higher concentrations.
asparaginase by A.terreus under SSF. Among the various

nitrogen sources tested, ammonium sulphate in the medium
Gaffar and Shethna, (1977) and Sreenivasulu et al.,(2009)
promoted
enhanced
growth
of
microorganism
and
have reported maximum production of L-asparaginase with
consequently the L-asparaginase production with an yield of
1.0% of ammonium sulphate.
246.0 U/gds.



Gaffar and Shethna, (1977) observed the positive effect of

supplementation of ammonium sulphate in the production of

L-asparaginase.

Sreenivasulu et al.,(2009) have reported ammonium sulphate
exhibited maximum enzyme production by the isolated fungus
VS-26.

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IJRET: International Journal of Research in Engineering and Technology eISSN: 2319-1163 | pISSN: 2321-7308

280
280
s
)
d

270
/
g

260
260
s
)


(
U

d
250
i
t
y

/
g

240

(
u

c
t
i
v

240
230
i
t
y

e

A

220
m
c
t
i
v

z
y

210
n
220
e

A

E
m
z
y
n
200
E
180
0
1
2
3
4
Ammonium Sulphate concentrtion%
(w/v)
Metal Salts




Fig-11: Effect of ammonium sulphate concentration on L-
Fig-12: Effect of metal salts on L-asparaginase production
asparaginase production.


CONCLUSIONS
3.2.9 Effect of metal salts on L-Asparaginase
The observations made in this work hold great promise for
production:
maximum production of L-Asparaginase enzyme (273.3
U/gds) after optimization of fermentation parameters such as
The effect of metal salts on L-asparaginase production was
fermentation time, temperature, initial moisture content, pH,
determined by adding different metal salts to fermentation
inoculum age and inoculum volume, carbon source such as
medium. Metal salts provide metal ions that are essential for
glucose, nitrogen source such as ammonium sulphate and
cell mass formation and also act as cofactor for several
metal salt such as magnesium sulphate by Aspergillus terreus
biosynthetic enzymes. The metal salts selected were
MTCC 1782 using bajra seed flour as substrate under solid
MgSO4.7H2O, CaCl2, NaCl, KH2PO4, at 0.1% (w/v)
state fermentation. This clearly indicates that the Aspergillus
concentration. Addition of magnesium sulphate to the
terreus MTCC 1782 is a potential strain for L-Asparaginase
fermentation medium showed high L-asparaginase activity of
production under solid-state fermentation. These studies also
273.3 U/gds.
indicates that the bajra seed flour is an effective substrate for

the production of L-Asparaginase enzyme. As the bajra seed
Yasser et al 2002 reported that maximum activity was
flour has the good nutritive value, low cost, and easily
observed with MgCl2 by Pseudomonas aeruginosa using corn
available substrate has paved a way for the large scale
steep liquor.
production of L-Asparaginase enzyme, a potential antitumor
agent, which has vast applications in health care and food
industries.

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