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ORIGINAL ARTICLE
Isolation and screening of L-asparaginase free of glutaminase
and urease from fungal sp.
Kruthi Doriya
1
•Devarai Santhosh Kumar
1
Received: 22 August 2016 / Accepted: 7 October 2016
ÓThe Author(s) 2016. This article is published with open access at Springerlink.com
Abstract L-Asparaginase is a chemotherapeutic drug used
in the treatment of acute lymphoblastic leukaemia (ALL), a
malignant disorder in children. L-Asparaginase helps in
removing acrylamide found in fried and baked foods that is
carcinogenic in nature. L-Asparaginase is present in plants,
animals and microbes. Various microorganisms such as
bacteria, yeast and fungi are generally used for the pro-
duction of L-asparaginase as it is difficult to obtain the
same from plants and animals. L-Asparaginase from bac-
teria causes anaphylaxis and other abnormal sensitive
reactions due to low specificity to asparagine. Toxicity and
repression caused by bacterial L-asparaginase shifted focus
to eukaryotic microorganisms such as fungi to improve the
efficacy of L-asparaginase. Clinically available L-asparag-
inase has glutaminase and urease that may lead to side
effects during treatment of ALL. Current work tested 45
fungal strains isolated from soil and agricultural residues.
Isolated fungi were tested using conventional plate assay
method with two indicator dyes, phenol red and bromoth-
ymol blue (BTB), and results were compared. L-Asparag-
inase activity was measured by cultivating in modified
Czapek–Dox medium. Four strains have shown positive
result for L-asparaginase production with no urease or
glutaminase activity, among these C
7
has high enzyme
index of 1.57 and L-asparaginase activity of 33.59 U/mL.
L-Asparaginase production by C
7
was higher with glucose
as carbon source and asparagine as nitrogen source. This is
the first report focussing on fungi that can synthesize L-
asparaginase of the desired specificity. Since the clinical
toxicity of L-asparaginase is attributed to glutaminase and
urease activity, available evidence indicates variants neg-
ative for glutaminase and urease would provide higher
therapeutic index than variants positive for glutaminase
and urease.
Keywords L-Asparaginase L-Glutaminase Urease
Glutaminase-free L-asparaginase Urease and glutaminase-
free L-asparaginase
Abbreviations
ALL Acute lymphoblastic leukaemia
BTB Bromothymol blue
L-Asn L-Asparagine
L-Gln L-Glutamine
MCD Modified Czapek–Dox
Introduction
L-Asparaginase is an amidohydrolase that catalyses L-as-
paragine to L-aspartate and ammonia. L-Asparaginase is
found to have tumour inhibitory properties. It is mainly
used in the treatment of acute lymphoblastic leukaemia
(ALL). Normal cells can synthesize L-asparagine with the
help of asparagine synthetase, whereas certain sensitive
malignant cells cannot synthesize it by itself and require an
external source of L-asparagine for growth. During the
treatment of ALL with L-asparaginase, all the circulating
asparagine in the body of the patient get hydrolysed to
aspartic acid and ammonia preventing the absorption of
&Devarai Santhosh Kumar
devarai@iith.ac.in
1
Department of Chemical Engineering, Industrial Bioprocess
and Bioprospecting Laboratory, Indian Institute of
Technology Hyderabad, Room No: 530, Kandi Campus,
Kandi, Medak Dist, Hyderabad, Telangana State 502285,
India
123
3 Biotech (2016) 6:239
DOI 10.1007/s13205-016-0544-1
asparagine by tumour cells thereby depriving the tumour
cells of their extracellular source of L-asparagine (Broome
1961). L-Asparaginase is commonly used as a combination
chemotherapy drug for the treatment of acute lym-
phoblastic leukaemia (ALL) in adults and children and
non-Hodgkin’s lymphoma in children (Mashburn and
Wriston 1964). L-Asparaginase also reduces acrylamide
formation in food by selectively hydrolysing asparagine to
aspartic acid and ammonia without affecting other amino
acids, retaining food quality. Application of L-asparaginase
enzyme (2 U/g) successfully reduced acrylamide content
by 90 % in potato products that have high asparagine
content (Friedman 2003; Ciesarova
´et al. 2006).
L-Asparaginase is widely present in plants, animals and
microbes but not in humans. Microbes are a better source
for the production of enzyme as they are easy to cultivate
and manipulate (Kumar and Sobha 2012). Clinically three
asparaginase formulations are available, two from bacterial
sources Escherichia coli (E. coli asparaginase) and Erwinia
chrysanthemi (Erwinia asparaginase) and PEGylated form
of E. coli asparaginase. L-Asparaginase therapy has side
effects such as anaphylaxis, coagulation abnormality,
thrombosis, liver dysfunction, pancreatitis, hyperglycaemia
and cerebral dysfunction, etc.These side effects are either
due to the production of anti-asparaginase antibody in the
body or L-glutaminase activity of L-asparaginase enzyme
(Haskell et al. 1969; Mahajan et al. 2012). Toxicity of L-
asparaginase is mainly due to the fact that the enzyme
preparations are amidohydrolase, not L-asparaginase.
Clinically available L-asparaginase shows notable hydroly-
sis of L-glutamine and D-asparagine, signifying multiple
enzyme activities contaminating enzyme preparation and
difficult to eliminate other enzymes (Campbell and Mash-
burn 1969). Notwithstanding numerous studies on bacterial
L-asparaginase, treatment with it sometimes results in
hypersensitive reactions such as anaphylactic shock. L-
Asparaginase isolated from filamentous fungi, Aspergillus
terreus showed a greater carcinostatic effect on static
tumour (De-Angeli et al. 1970). Similar effect was
observed when L-asparaginase from deuteromycetes
Fusarium tricinctum was purified which regressed lym-
phosarcoma in mice (Scheetz et al. 1971). Later purified
extracellular L-asparaginase from A. terreus was conju-
gated with polyethylene glycol and it did not indicate any
glutaminase activity (Loureiro 2012). Sarquis et al. exam-
ined Aspergillus tamari and A. terreus for L-asparaginase
production and found that asparaginase activity is reduced
in the presence of urea and glutamine (Sarquis et al. 2004).
On the other hand, Bano and Sivaramakrishnan discovered
that purified L-asparaginase from green chillies showed
presence of glutaminase and urease. Further studies
revealed that urease is present in E. coli enzyme prepara-
tion, which may result in toxic effects by hydrolysis of
blood urea (Bano and Sivaramakrishnan 1980). Manna
et al. produced and purified L-asparaginase from Pseu-
domonas stutzeri MB-405 which showed high specificity
towards asparagine but did not hydrolyze glutamine, also
asparaginase activity was lacking at 2 M urea (Manna et al.
1995). Therefore, current study is an effort to isolate fungi
that can produce asparaginase free of glutaminase and
urease. This process involves isolation of fungi from soil
and agricultural residue for extracellular synthesis of L-
asparaginase.
Materials and methods
L-Asparagine was procured from Sigma-Aldrich, India.
Other chemicals used were of analytical grade. Aspergillus
terreus MTCC 1782 was obtained from Microbial Type
Culture Collection Centre and Gene Bank, Institute of
Microbial Technology, Chandigarh, India.
Isolation of fungi from collected samples
Soil samples were collected from different locations of
Vizag, Kanyakumari and Kerala as mentioned in Table 1.
Soil and substrate samples were collected in air-tight
containers and kept at room temperature in laboratory.
Fungi were isolated by serial dilution of soil and agricul-
tural residues, and plated on modified Czapek–Dox (MCD)
agar plates with L-asparagine as a sole nitrogen source and
incubated at 30 °C for 96 h. Fungal strains showing change
were selected and grown on potato dextrose slants.
Screening studies
Semi-quantitative assay for L-asparaginase producing
fungi
MCD medium with composition glucose 2 g/L, L-as-
paragine 10 g/L, KH
2
PO
4
1.52 g/L, KCl 0.52 g/L,
MgSO
4
7H
2
O 0.52 g/L, FeSO
4
7H
2
O trace, ZnSO
4
7H
2-
O trace, CuNO
3
3H
2
O trace and agar 18 g/L was prepared
(Gulati et al. 1997). About 2.5 % (w/v) stock solution of
the phenol red dye was prepared and MCD medium was
supplemented with 0.009 % phenol red dye. 0.04 % (w/v)
of stock solution of the bromothymol blue dye was pre-
pared and 0.007 % BTB dye was supplemented in MCD
medium. Final pH of the media was adjusted to 5.5 using
1 M NaOH (Mahajan et al. 2013). Prepared media was
autoclaved and poured into pre-sterilized plates. Control
plates were prepared with NaNO
3
as sole nitrogen source.
MCD plates were inoculated with isolated fungi as test
organism and A. terreus MTCC 1782 as positive test.
Colony diameter and zone diameter for all the test
239 Page 2 of 10 3 Biotech (2016) 6:239
123
organisms were measured and respective zone index was
calculated after 72 h of incubation. Morphological obser-
vation of positive isolates was done by the method of
staining and observing fungal spores using lacto phenol
cotton blue staining solution.
Plate assay for L-glutaminase
L-Glutaminase activity of the fungal strains was detected
by supplementing MCD medium with L-Gln as sole nitro-
gen source. Test strains were inoculated and observed for
colour change from yellow to pink in case of phenol red
dye and yellow to blue for BTB dye.
Plate assay for urease
MCD medium without nitrogen source was autoclaved and
1 % filter-sterilized urea solution was added to MCD
media for detection of urease-producing fungi. Test strains
were inoculated and observed for change in the colour of
the medium.
Quantitative detection of L-asparaginase assay
Quantitative determination of L-asparaginase activity was
carried out using selective strains (MTCC 1782, C
3
,C
7
,W
3
and W
5
). These strains were cultivated on potato dextrose
slants at 30 °C for 96 h. From these, 1 mL of conidial
suspension was inoculated into Erlenmeyer flask contain-
ing 50 mL of MCD medium with initial pH of 6.2. Flasks
were incubated at 30 °C at 180 rpm for 96 h. Samples were
withdrawn every 24 h to determine enzyme activity.
Effect of carbon and nitrogen sources
To investigate the effect of different carbon sources on L-
asparaginase production, fructose, glucose, maltose,
sucrose, lactose and starch were added at concentration of
0.2 %(w/v) to the MCD medium. Influence of nitrogen
source on asparaginase production was obtained by
substituting asparagine of MCD medium with yeast extract,
peptone and sodium nitrate at a concentration of 1 % (w/v).
1mL of C
7
suspension (with 10 910
6
cells/mL) was
inoculated and flasks were incubated at 30 °C at 180 rpm
for 72 h. Supernatant was used to determine asparaginase
activity and protein content. The effect of inoculum vol-
ume at different levels was investigated by employing C
7
in MCD medium.
L-Asparaginase activity is obtained by measuring the
ammonia liberated using Nesslerization method by spec-
trophotometric analysis at 425 nm as described by Kumar
et al. (2011). Enzyme assay mixture consisted of 900 lLof
freshly prepared L-asparagine (40 mM) in Tris–HCl buffer
(pH 8.6) and 100 lL of enzyme filtrate, incubated at 37 °C
for 30 min and reaction was stopped by adding 100 lLof
1.5 M trichloroacetic acid (TCA). The reaction mixture was
centrifuged at 10,000 rpm for 5 min at 4 °C to remove the
precipitates. The ammonia released in the supernatant was
determined using colorimetric technique by adding 200 lL
of Nessler’s reagent into the sample containing 200 lLof
supernatant and 1.6 mL distilled water. This mixture was
vortexed and incubated at room temperature for 20 min.
Absorbance was measured at 425 nm against the blanks that
received TCA before the addition of enzyme. The ammonia
liberated in the reaction was determined based on the
standard curve obtained using ammonium sulfate. One unit
(IU) of L-asparaginase activity was defined as the amount of
the enzyme that liberates 1 lM of ammonia per min at
37 °C, using asparagine as substrate.
Extracellular protein content was determined using
Lowry method (Lowry et al. 1951). Specific activity is
expressed as unit enzyme activity per mg of protein.
Results and discussion
Isolation of fungal species
A total of 45 fungal species were isolated on the basis of
zone formation from soil, wheat bran, rice husk, cotton
Table 1 Fungal species isolated from diverse sources for the production of L-asparaginase
Source Region Description No. of isolates
Soil Kanyakumari Soil samples were collected from different locations of sea shore 10
Vizag Soil samples were collected from different locations of sea shore 8
Kerala Soil samples were collected from Western ghats, corresponding to
coordinates 9.8403°N, 77.0353°E
9
Agricultural residues Cottonseed oil cake Substrates were collected from local market 18
Rice husk
Wheat bran
Red gram animal feed
3 Biotech (2016) 6:239 Page 3 of 10 239
123
seed oil cake and red gram feed. Among isolated fungi, 34
isolates were able to grow in secondary screening with
MCD medium containing different nitrogen sources. Out of
45 fungal isolates, 27 were from soil implying that 60 % of
isolated fungi were from soil samples, rest from agricul-
tural residues. Aspergillus sp., Penicillium sp., Trichophy-
ton sp. and Onychocola sp. were predominant fungi
isolated from the soil samples. Rhizopus sp. and Fusarium
sp. were isolated from agricultural residues. Aspergillus sp.
was the most dominant species among fungi isolated from
soil and agricultural residues. These results were compa-
rable to previously reported studies (Qiao et al. 2008;
Tanc
ˇinova
´and Labuda 2009).
Screening studies
Current study involved the screening of isolated fungi for
the existence of three industrially important enzymes using
phenol red and BTB dye. For screening of L-asparaginase,
L-glutaminase and urease enzyme, MCD supplemented
with L-Asn, L-Gln and urea, respectively, as the sole
nitrogen sources are used. These amidohydrolases cleave
amine groups and liberate aspartic acid and ammonia in
case of L-asparaginase, glutamic acid and ammonia in case
of L-glutaminase and carbonic acid and ammonia if urease
is produced (as shown in Fig. 1). Ammonia liberated in the
medium further reacts with water to produce NH
4
OH
resulting in increase in the pH of the medium.
Phenol red dye is yellow at acidic pH and turns pink at
alkaline pH; presence of pink colour zone around the
colonies on MCD plates with different nitrogen sources is
due to the liberation of corresponding enzyme (Gulati et al.
1997). Thirty-four isolates showed pink zone around the
colonies indicating increase in pH. In Fig. 2, last column
shows presence of pink-coloured zone around fungal iso-
lates S
3.4
,W
3
,W
5
,C
3
and C
7
in L-Asn plates indicating L-
asparaginase activity. These isolates did not show any
colour change in plates containing L-Gln connoting the
absence of L-glutaminase. S
3.4
and MTCC 1782 isolates
produce the urease enzyme which is confirmed by the pink-
coloured zone around the colony in plates with urea as
nitrogen source. MTCC 1782 strain showed pink-coloured
zone when grown on L-Asn, L-Gln and urea indicating that
strain produces three enzymes. Strains W
3
,W
5
,C
3
and C
7
show pink colour zone only on L-asparagine plate, indi-
cating strains are free of L-glutaminase and urease. To
ensure reproducibility, all the isolates were screened with
BTB as both the dyes are formulated for screening the
hydrolysis of L-Gln, L-Asn and urea. Among phenol red
and BTB, 0.007 % of BTB dye showed sharp colour con-
trast zone, ranging from yellow at acidic pH, green at
neutral pH to blue at alkaline pH (Mahajan et al. 2013).
MCD plates with different substrates supplemented with
BTB dye is shown in Fig. 3. After 72 h of incubation,
thirty-four isolates showed blue-coloured zone around the
colonies indicating increase in pH.
Fig. 1 Amidohydrolases: urease, L-glutaminase, L-asparaginase convert urea, L-Gln, L-Asn, respectively, producing ammonia and acid resulting
in increase in the pH with product formation. Pink-coloured zone around the colony indicates enzyme activity
239 Page 4 of 10 3 Biotech (2016) 6:239
123
In comparison with phenol red, hydrolysed and unhy-
drolyzed enzymes were clear and precise in MCD sup-
plemented with BTB. Methyl red was incorporated as pH
indicator in the recent study to screen L-asparaginase- and
L-glutaminase-producing microorganism (Dhale Dhale and
Mohan Kumari 2014). Enzyme activity is calculated semi-
quantitatively by relative ratio of zone diameter to colony
diameter. Level of enzyme production was indicated by
zone index. The comparison of zone index values of iso-
lates S
3.4
,W
3
,W
5
,C
3
,C
7
and Aspergillus MTCC 1782
strain using phenol red and BTB dye is given in Table 2.
Using this qualitative plate assay, rapid screening of the
fungi for the synthesis of the enzyme by direct visualiza-
tion and activity of the enzyme can be measured (Hankin
and Anagnostakis 1975). Gulati et al. revealed that equiv-
alent relation exists between zone index and enzyme
activity measured from broth. In the current work, enzyme
index varied from 0.8 to 4, which is in line with study
conducted by Shrivastava et al. (2010). Enzyme index of
C
7
is 1.57 with colony diameter of 3.5 cm and zone
diameter of 5.5 cm which is lower than that of MTCC 1782
strain with enzyme index of 2.40. Out of 34 isolated fungal
species, only 4 isolates showed L-asparaginase free of L-
glutaminase and urease as shown in Table 3. Isolated fungi
(S
3.4
,W
3
,W
5
,C
3
and C
7
) were cultured in PDA slants,
later morphologically identified as Curvularia sp., Rhizo-
pus sp. and Aspergillus sp., respectively (Ellis et al. 2007).
The L-asparaginase activity of the four isolated strains
with no glutaminase and urease activity is measured in
liquid broth studies along with MTCC 1782 (shown in
Fig. 4). MTCC 1782 strain is found to have the highest
activity at 72 h with L-asparaginase activity of 34.45 U/mL
and specific activity of 71.92 U/mg. Reported activity for
optimized Aspergillus terreus MTCC 1782 was 40.186 IU/
Fig. 2 Assay for screening L-
asparaginase-producing fungi
amended with different
substrates, on plate
supplemented with phenol red
dye. a–dS
3.4
isolate grown on
plates containing NaNO
3
, urea,
L-Gln and L-Asn; e–hC
3
isolate
grown on plates containing
NaNO
3
, urea, L-Gln and L-Asn;
i–lW
5
isolate grown on plates
containing NaNO
3
, urea, L-Gln
and L-Asn; m–pC
7
isolate
grown on plates containing
NaNO
3
, urea, L-Gln and L-Asn;
q–tW
3
isolate grown on plates
containing NaNO
3
, urea, L-Gln
and L-Asn; u–xAspergillus
terreus MTCC 1782 strain
grown on plates containing
NaNO
3
, urea, L-Gln and L-Asn
3 Biotech (2016) 6:239 Page 5 of 10 239
123
mL (Baskar and Renganathan 2012). Among the four iso-
lated strains C
7
has highest activity of 33.59 U/mL and
specific activity of 64.85 U/mg. Hence, medium has to be
developed and optimized for L-asparaginase production
from C
7
to enhance L-asparaginase activity. All the strains
exhibit the maximum activity at 72 h.
Table 2 L-Asparaginase enzyme index measurement using phenol red and bromothymol blue amended in MCD medium after 72 h incubation
and species observed under light microscope
Isolate Phenol red Bromothymol blue Species
Colony diameter (cm) Zone diameter
(cm)
Zone index Colony diameter (cm) Zone diameter (cm) Zone index
S
3.4
4.30 6.70 1.56 3.30 6.80 2.06 Curvularia sp.
W
3
2.40 2.40 1.00 3.70 4.70 1.27 Rhizopus sp.
W
5
8.80 8.80 1.00 2.20 2.60 1.18 Rhizopus sp.
C
3
3.00 3.00 1.00 3.80 5.50 1.45 Aspergillus sp.
C
7
3.00 4.60 1.53 3.50 5.50 1.57 Aspergillus sp.
MTCC 1782 2.50 6.00 2.40 2.50 6.00 2.40 Aspergillus sp.
Fig. 3 Assay for screening L-
asparaginase-producing fungi
amended with different
substrates, on plate
supplemented with BTB dye. a–
dS
3.4
isolate grown on plates
containing NaNO
3
, urea, L-Gln
and L-Asn; e–hC
3
isolate grown
on plates containing NaNO
3
,
urea, L-Gln and L-Asn; i–lW
5
isolate grown on plates
containing NaNO
3
, urea, L-Gln
and L-Asn; m–pC
7
isolate
grown on plates containing
NaNO
3
, urea, L-Gln and L-Asn;
q–tW
3
isolate grown on plates
containing NaNO
3
, urea, L-Gln
and L-Asn u–x;Aspergillus
terreus MTCC 1782 strain
grown on plates containing
NaNO
3
, urea, L-Gln and L-Asn;
1S
3.4
,2C
3
,3W
5
,4C
7
,5W
3
,6
MTCC 1782: microscopic
images of isolates using light
microscope 940 magnification
239 Page 6 of 10 3 Biotech (2016) 6:239
123
Effect of carbon and nitrogen sources
Six different carbon sources (fructose, glucose, maltose,
sucrose, lactose and starch) were evaluated for the L-as-
paraginase production by C
7
which is free of glutaminase
and urease. Batch cultivation of C
7
in MCD medium using
different carbon sources revealed distinctive variations on
L-asparaginase production and specific activity (Fig. 5). In
comparison to other carbon sources, C
7
produced maxi-
mum asparaginase (16.2 U/mL) when glucose is used as a
carbon source; lactose, maltose and starch were the poorest
carbon sources. Sucrose and fructose also supported
Table 3 Fungal species screened for multi-enzyme production (amide)
S. no. Isolation source Isolate Control
(NaNO
3
)
Urea L-
Asn
L-
Gln
L-Asparaginase enzyme index
Colony diameter
(cm)
Zone diameter
(cm)
Zone
index
1 Soil from Vizag V
1
––??2.7 6.6 2.44
2V
2
–?? ? 2.2 5 2.27
3V
3
–?? – 1 3 3.00
4V
4
–?? ? 3 6 2.00
5V
5
–?? ? 3.1 6 1.94
6V
6
–?? – 1 3.1 3.10
7 Soil from Kanyakumari K
1
–?? ? 1 2.1 2.10
8K
2
––??2.4 5.8 2.42
9K
3
–?? ? 2.5 5.2 2.08
10 K
4
––??2.1 4 1.90
11 K
5
––??1.7 4.7 2.76
12 K
6
––??2.5 6.8 2.72
13 K
7
–?? ? 0.7 1.8 2.57
14 K
8
––??1.25 4.4 3.52
15 Soil from Western Ghats S
1.1
–?? ? 2 6.5 3.25
16 S
1.4
–?? ? 2.1 3.9 1.86
17 S
2.1
–?? ? 3.2 8.5 2.66
18 S
3.4
–?? – 3.3 6.8 2.06
19 S
4.1
–?? ? 2 8.5 4.25
20 Red gram husk P
2
–?? ? 3.5 5.5 1.57
21 P
3
–?? ? 2.5 3 1.20
22 Rice husk R
1
–?? ? 2.6 6 2.31
23 R
3
–?? ? 3.7 6.3 1.70
24 Wheat bran W
1
–?? – 6 6.5 1.08
25 W
2
–?? ? 4 7 1.75
26 W
3
––?– 3.7 4.7 1.27
27 W
4
–?? ? 2 4 2.00
28 W
5
––?– 2.2 2.6 1.18
29 Cotton seed oil cake C
1
–?? ? 2.5 6 2.40
30 C
3
––?– 3.8 5.5 1.45
31 C
4
–?? – 7.5 7 0.93
32 C
5
–?? – 7.5 7 0.93
33 C
6
–?? ? 8.5 7 0.82
34 C
7
––?– 3.5 5.5 1.57
35 MTCC
1782
–?? ? 2.5 6.0 2.4
V (1–6) soil from Visakhapatnam (Vizag), K (1–8) soil from Kanyakumari, S (1.1, 1.4, 2.1, 3.4 and 4.1) soil from Western Ghats, P (2–4) red
gram husk, R (1 and 3) rice husk, W (1–5) wheat bran, C (1, 3, 4, 5, 6 and 7) cotton seed oil cake
3 Biotech (2016) 6:239 Page 7 of 10 239
123
L-asparaginase production to a significant degree but glu-
cose acted as good inducer and primary source of carbon
for biosynthesis of L-asparaginase using C
7
. Several reports
suggest that glucose serves as a best carbon source for L-
asparaginase production and a similar effect was observed
for L-asparaginase production using Aspergillus and
Fusarium strains (Baskar and Renganathan 2012; Hosa-
mani and Kaliwal 2011). Effect of nitrogen compounds on
L-asparaginase by C
7
was studied by supplementing nitro-
gen sources (asparagine, yeast extract, peptone and sodium
nitrate) to MCD medium. C
7
amended with asparagine
favoured maximum enzyme production indicating L-as-
paragine itself acts as a nitrogen source and influence L-
asparaginase production (Fig. 6). Peptone also supported
the production of L-asparaginase to a substantial quantity.
A considerable decrease in enzyme activity was observed
when C
7
was amended with yeast extract. Lower enzyme
activity was detected in the media supplemented with
sodium nitrate; on the contrary, a study in which Fusarium
oxysporum has shown higher enzyme production with
sodium nitrate as a nitrogen source (Tippani and Sivade-
vuni 2012). Effect of inoculum volume on L-asparaginase
production by C
7
is shown in Fig. 7. At low inoculum
concentration, the L-asparaginase production was less, and
the enzyme activity increased with increase in inoculum
volume. At inoculum volume of 5 910
7
cells/mL, enzyme
activity is 33.59 U/mL. With further increase in inoculum
concentration, the biosynthetic activity decreased due to
nutrient depletion.
Most of the L-asparaginase purified from various sources
such as chillies and E. coli shows specificity towards both
L-Gln and urea. Several studies reveal that specificity of L-
asparaginase is important in selective depletion of aspar-
agine-dependent tumour cells (Hill et al. 1967; Durden and
Distasio 1981; Distasio et al. 1982). To reduce the toxic
effects associated with bacterial L-asparaginase, fungi is
preferred as being eukaryotic and evolutionarily closer to
human. It can minimize the chances of immunological
reactions (Shrivastava et al. 2012). Several fungal endo-
phytes were isolated from various sources and tested for
their ability to synthesize L-glutaminase-free L-asparagi-
nase. L-Glutaminase-free L-asparaginase produced by
endophytic fungi from seaweed was isolated, later identi-
fied as Fusarium, Alternaria sp., Aspergillus sp. and Col-
letotrichum sp. (Thangavel et al. 2013). Alternaria sp.
endophytic fungi isolated from the leaf of Withania som-
nifera of Western Ghats is reported to show maximum L-
asparaginase activity that is free of L-glutaminase (Na-
garajan et al. 2014). In the current study, 45 fungi isolates
were subjected to screening, with a view to assess the
isolates for their ability to utilize different substrates as a
nitrogen source. Twenty fungi isolates have shown the
presence of urease, L-glutaminase and L-asparaginase
0
20
40
60
80
Specific activity (U/mg)
Time (h)
W3
W5
MTCC 1782
C3
C7
0 204060801000 20 40 60 80 100
0
5
10
15
20
25
30
35
40
L-asparaginase activity (U/mL)
Time (h)
W3
W5
MTCC 1782
C3
C7
Fig. 4 L-Asparaginase activity and specific activity of isolated strains
Glucose Fructose Starch Sucrose Lactose Maltose
0
5
10
15
20
L-asparaginase activity (U/mL)
Carbon source
L-asparaginase activity
0
10
20
30
40
50
60
Specific activity
Specific activity (U/mg)
Fig. 5 L-Asparaginase activity and specific activity of C
7
strain with
different carbon sources
239 Page 8 of 10 3 Biotech (2016) 6:239
123
enzyme. Four isolates have shown the presence of L-as-
paraginase free of urease and L-glutaminase, and six iso-
lates presented L-asparaginase free of L-glutaminase with
presence of urease using plate assay. Fungal isolates were
selected on the basis of zone formation around the colo-
nies, when grown on MCD with phenol red or BTB as a pH
indicator. The change in colour stated the accumulation of
ammonia which resulted due to the hydrolysis of amido-
hydrolase (Singh and Srivastava 2012). Fungi secrete
numerous enzymes into the medium and regulation of other
contaminating enzymes would make it possibly the pre-
ferred drug in the treatment of cancer.
Current preparation of asparaginase used in treatment
protocols are E.coli asparaginase, its PEGylated form and
Erwinia asparaginase; several studies on different other
sources of asparaginase have yielded encouraging
outcomes. Further studies and regulatory supports will
allow the introduction of new asparaginase drugs with
potential benefits to patients. Fungal strain, namely C
7
,
which is an L-asparaginase (free of L-glutaminase and
urease)-producing strain has shown highest enzyme activ-
ity of 33.59 U/mL with carbon source as glucose; aspar-
agine as nitrogen source at inoculum volume of 5 910
7
cells/mL is to be considered for further study on purifica-
tion and characterization of L-asparaginase enzyme.
Acknowledgments The authors sincerely thank the Director of
Indian Institute of Technology Hyderabad for his continued encour-
agement and support. DSK thanks Science and Engineering Research
Board-Department of Science and Technology (SB-EMEQ-048/2014)
for financial support. We extend our sincere thanks to Dr. Then-
malarchelvi Rathinavelan, Department of Biotechnology, and Dr.
Debraj Bhattacharya and Dr. K. B. V. N Phanindra, Department of
Civil Engineering, IIT Hyderabad for allowing us to do spectropho-
tometric analysis.
Compliance with ethical standards
Conflict of interest We hereby declare that there has not been any
conflict of interest at any point of time during the preparation of this
manuscript.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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