Biochimie 121 (2016) 38e51

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Biochimie
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Research paper

Purication and characterization of glutaminase free asparaginase

from Pseudomonas otitidis: Induce apoptosis in human leukemia
MOLT-4 cells
Islam Husain a, Anjana Sharma a, *, Suresh Kumar b, Fayaz Malik b
a
b

Bacteriology Laboratory, Department of P. G. Studies and Research in Biological Science, Rani Durgavati University, Madhya Pradesh, India
Cancer Pharmacology Division, Indian Institute of Integrative Medicine (IIIM-CSIR), Jammu & Kashmir, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 5 March 2015
Accepted 12 November 2015
Available online 17 November 2015

Asparaginase is an important antineoplastic drug extensively used for the treatment of acute lymphoblastic leukemia, but the intrinsic glutaminase activity of this enzymatic drug is responsible for several
life threatening side effects. This study describes the purication and characterization of glutaminase free
asparaginase from Pseudomonas otitidis. The puried enzyme exhibited molecular mass of
approximately 2053 kDa on native-PAGE and341 kDa on SDS-PAGE, revealing that the enzyme is
homohexamer. The isoelectric point of enzyme was 5.5, calculated by 2D-PAGE. Optimum activity of
asparaginase was achieved at 40
C and pH 7.5, which is close to the internal environment of the human
body. Monovalent cations (Na and K) and reducing agents (2-mercaptoethanol and glutathione) has
enhanced asparaginase activity. Whereas, divalent (Ca2, Mg2, Zn2 and Mn2), trivalent (Fe3) cations
and thiol group blocking agent (iodoacetamide) inhibited the enzyme activity signicantly. In vitro serum
and trypsin half life of asparaginase is almost 2 and 1.5 fold respectively, which is higher than commercial asparaginase. MTT assay results showed that the anticancer activity of puried asparaginase was
comparable or higher than commercial E. coli asparaginase. Microscopic studies and cell cycle analysis
suggested that puried enzyme induced apoptotic cell death in dose-dependent manner. Loss of mitochondrial membrane potential suggests that enzyme induces cell death via activation of intrinsic
apoptotic pathway. Puried asparaginase was found to be nontoxic for human noncancerous FR-2 cells
and human blood lymphocytes, which is a remarkable therapeutic feature.
 te
 Franaise de Biochimie et Biologie Mole
culaire (SFBBM). All rights
2015 Elsevier B.V. and Socie
reserved.

Keywords:
Asparaginase
Glutaminase
Purication
MTT assay
Cell cycle analysis
MMP loss

1. Introduction
Acute lymphoblastic leukemia is a malignant disorder of
lymphoid progenitor cells affecting a signicant segment of the
children and adults with peak prevalence between the age group of
2e5 years [1]. Bacterial asparaginase (L-Asparaginase amidohydrolase, E.C. 3.5.1.1) is an enzymatic drug used for the treatment of
children with acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML) and other lymphoid malignancies [2]. In the
recent past, scientists and clinicians have harnessed asparaginase
against sarcomas [3], ovarian cancer [4], brain cancer [5], and breast
cancer [6]. The anti-neoplastic action of asparaginase is explained

* Corresponding author.
E-mail address: anjoo1999@gmail.com (A. Sharma).

based on the fact that certain tumor cells, more specically ALL
tumor cells are decient in their ability to synthesize the nonessential amino acid asparagine de-novo due to absence of asparagine synthetase [7] and requires huge amount of asparagine to
keep up their rapid malignant growth. To fulll their nutritional
requirement they use serum asparagine. Asparaginase, as a
chemotherapeutic drug rapidly hydrolyzes serum asparagine into
aspartate and ammonia [8]. The nutritional stress induced by
asparaginase due to the depletion of serum asparagine inhibits
DNA, RNA and protein biosynthesis in ALL and other asparagine
dependent tumor cells, resulting in subsequent apoptosis due to
cell cycle arrest in G1 phase [9]. However, normal cells remains
unaffected due to presence of asparagine synthetase [10].
Asparaginase has been reported in several microbial species but
the biochemical, kinetic and anticancer activities of the enzyme
varies with genetic nature of the organisms [11]. Today

http://dx.doi.org/10.1016/j.biochi.2015.11.012
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culaire (SFBBM). All rights reserved.
0300-9084/ 2015 Elsevier B.V. and Socie

I. Husain et al. / Biochimie 121 (2016) 38e51

asparaginases puried from Escherichia coli and Erwinia chrysanthemi are being used for clinical purposes [12]. Recent studies
have shown that asparaginase from both these sources have curative potential but are often associated with serious side effects that
can be life threatening. The intrinsic glutaminase activity is one of
the major drawbacks of both asparaginases (E. coli and
E. chrysanthemi) that cause serious side effects such as leucopenia,
immunosuppression, acute pancreatitis, thromboembolysis, hyperglycemia and neurological seizures [13]. Additionally, short
serum half life and low tolerance to proteolytic enzymes are some
other limitations that necessitate repetitive administration of
asparaginase [14]. However, multiple administrations of serologically same asparaginase in induction and consolidation chemotherapy has led to the formation of anti-asparaginase antibodies,
resulting in neutralizing asparaginase activity by the hostile
immunological system [15]. Therefore, for the event-free, longterm, survival rates of patients; glutaminase free asparaginase from
new sources is urgently required possessing similar or more
effective antitumor activity than the previously reported ones. In
our earlier reports, we had isolated glutaminase free asparaginase
producing indigenous bacterial strains [16] and fermentation process parameters were also standardized for maximum biosynthesis
of glutaminase free asparaginase from Enterobacter cloacae [17].
Subsequently, enzyme was puried and characterized from this
bacterium (Husain and Sharma, unpublished data). In the current
study, we have reported purication and characterization of a
glutaminase free asparaginase from Pseudomonas otitidis and
anticancer activity was evaluated against a panel of human cancer
cell lines. Furthermore, anticancer activity of the enzyme was
validated against human leukemia MOLT-4 cells.
2. Materials and methods

39

inoculated in 50 ml of semi-synthetic broth medium and incubated

at 37
C and 180 rpm for 24 h. The culture was harvested at
10,000 rpm for 5 min at 4
C and supernatant was used as crude
enzyme.
2.3. Asparaginase and glutaminase assays
The asparaginase activity was measured using Nessler's method
[19]. The reaction mixture containing 0.1 ml enzyme preparation
and 0.9 ml of pre-warmed 0.01 M asparagine prepared in 0.05 M
TriseHCl buffer (pH 8.6). The content of tube was mixed by vortexing and incubated at 37
C for 30 min. The reaction was terminated by the addition of 0.1 ml of 1.5 M trichloroacetic acid (TCA),
centrifuged at 10,000 rpm for 5 min at room temperature to
remove the precipitate. Nessler's reagent (0.25 ml) was added in
tubes containing 0.5 ml supernatant and 1.75 ml distilled water.
The content of the tubes were vortexed and incubated at room
temperature for 10 min. The absorbance A480 values were
measured against the control prepared by addition of TCA before
enzyme addition. The ammonia produced in the reaction was
calculated on the basis of standard curve prepared with ammonium
sulphate. One asparaginase unit (IU) is dened as the amount of
enzyme that liberates 1 mmol of ammonia min1 at 37
C. Specic
activity of asparaginase is expressed as U mg1 protein. Glutaminase activity was determined by following Nessler's method as
described by Imada et al. [20]. Total protein concentration was
determined by the method of Lowry et al. [21], using bovine serum
albumin as the standard.
2.4. Enzyme purication and quantication
All the purication steps were performed at 4
C and chromatographic runs were monitored for protein at 280 nm.

2.1. Reagents
Anhydrous L-asparagine, L-glutamine, D-glutamine, L-glutamic
acid, DL-aspartic acid, L-histidine, L-ornithine, BOC-L-asparagine, Na-acetyl-L-asparagine, urea, acrylamide, D-asparagine, DL-asparagine, EDTA, dithiothreitol, SDS, 2-mercaptoethanol, glutathione, Lcysteine, thiourea, trichloroacetic acid (TCA), dimethylsulphoxide
(DMSO) and Folin-Ciocalteu's phenol reagent, were purchased from
Himedia, Mumbai, India. Chromatographic matrix, RPMI-1640,
fetal bovine serum (FBS), 3-(4,5-dimethylthiazole-2-yl)-2, 5diphenyltetrazolium bromide (MTT), penicillin, streptomycin, propidium iodide, Rhodamine-123, 40 ,6-diamidino-2-phenylindole
dihydrochloride (DAPI), DNase-free RNase, proteinase-K, phenylmethanesulfonyl uoride (PMSF), eukaryotic protease inhibitor
cocktail, and triton X-100 were purchased from Sigma chemical Co.,
USA. IPG strips were purchased from Bio-Rad Lab., USA. All the
chemicals used were of analytical grade and purchased from
standard sources.
2.2. Bacterial strain, culture condition and enzyme production
The glutaminase free asparaginase producing strain P. otitidis
(NCBI accession no. KF607097) was obtained from Bacterial
Germplasm Collection Centre (BGCC no. 2388), Rani Durgavati
University, Jabalpur (M.P.), India, which was previously isolated in
our Lab [16]. The strain was maintained on Luria-Bertani (LB) slant
(pH 7) with regular sub-culturing after every four weeks and stored
at 4
C. Semi-synthetic broth medium [18] was used to produce
asparaginase. The primary inoculum was prepared by adding a
loopfull of 24 h old pure culture from slant into 20 ml of aforementioned medium. The ask was incubated overnight at 37
C and
180 rpm. The 2% inoculum (A600 0.6e0.8) of this culture was

2.4.1. Ammonium sulphate precipitation

The extracellular asparaginase produced by P. otitidis was fractionated by adding powdered ammonium sulphate. The maximum
asparaginase activity was observed with the fraction precipitated at
60e90% saturation. The precipitate was collected by centrifugation
at 10,000 rpm for 30 min and dissolved in minimal volume of
0.05 M TriseHCl buffer (pH 8.6). The enzyme solution was dialyzed
for 24 h against the same buffer.
2.4.2. DEAE-cellulose chromatography
The dialysate obtained from the previous step was loaded on
diethylaminoethyl (DEAE) cellulose column (2  15 cm2, Sigma),
pre-equilibrated with 0.05 M TriseHCl buffer, pH 9.6 at a ow rate
of 1 ml min1. The absorbed protein was eluted with a linear
gradient of KCl (0e200 mM) prepared in 0.05 M TriseHCl buffer, pH
8.6. The fractions containing asparaginase activity were pooled,
dialyzed against 0.05 M TriseHCl buffer, pH 8.6.
2.4.3. Sephadex G-100 chromatography
The dialyzed sample from DEAE-cellulose column was loaded on
pre-equilibrated Sephadex G-100 column with 0.05 M TriseHCl
buffer, pH 8.6. The protein elution was done with the same buffer at
a ow rate of 0.2 ml min1 until no protein was seen in elutes. All
the active fractions were pooled, concentrated and used as puried
enzyme.
2.5. Determination of molecular weight and homogeneity of
asparaginase
Molecular mass and homogeneity of puried asparaginase was
determined by native-PAGE following the method of Laemmli [22].

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I. Husain et al. / Biochimie 121 (2016) 38e51

The protein samples were mixed with 5X native-PAGE sample

buffer and electrophoresed in 10% polyacrylamide gel (stacking gel
was omitted) in Tris-glycine buffer (pH 8.5) at 4
C. The subunit
molecular weight of enzyme was determined by SDS-PAGE using
12% separating gel (pH 8.8) and 5% stacking gel (pH 6.8). The protein
bands were observed by staining the gel with coomassie brilliant
blue R-250 and molecular weight was determined with standard
marker proteins.
2.6. Determination of isoelectric point (pI) of asparaginase
Puried asparaginase was dissolved in rehydration buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT (DLdithiothreitol), and 1.0% IPG buffer (4e7). Before loading for 2-DE,
traces of bromophenol blue was added and centrifuged at
20,000 rpm for 10 min. A total of 300 ml of solubilization buffer
containing 250 mg protein sample was incubated with the dry IPG
(immobilized pH gradient) gel strips (pH 4e7 linear gradients
13 cm) at 20
C for 16 h. The rst dimension separation was conducted at 20
C with an Ettan IPG phor system (GE Healthcare,
USA). Three phase program was used for performing IEF as
described by Gorg et al. [23]. Focused IPG strips were then equilibrated by rst incubating them in an equilibration solution (6 M
urea, 30% v/v glycerol, 2% w/v SDS, 50 mM TriseHCl, pH 8.8) having
1% w/v DTT and a trace amount of bromophenol blue for 15 min,
followed by 15 min incubation with 2.5% w/v iodoacetamide. The
equilibrated IPG strip was placed on top of the SDS gel and overlaid
with 5 ml of hot (45
C) agarose solution. The SDS gel cassette was
inserted in the electrophoresis apparatus containing electrode
buffer and run overnight (15 h) with a voltage setting of 50 V for 1 h
and 100 V for 14 h at 4
C. After electrophoresis, the gel was stained
with coomassie brilliant blue R-250. Molecular weight of the
enzyme was measured according to the standard protein marker.
2.7. Effect of pH and temperature on activity and stability of
asparaginase
The optimum pH for asparaginase activity was determined over
a pH range of 4.5e10.5 using following buffer: 0.05 M potassium
phosphate buffer, pH 4.5e7.5 and 0.05 M TriseHCl buffer, pH
8.0e10.5. For pH stability asparaginase was incubated with same
buffer at 4
C for 24 h in the absence of substrate and residual activity was determined using standard asparaginase assay. For
determining the optimum temperature, enzyme was incubated at
different temperatures (20-70
C) for 30 min and for thermal stability enzyme was pre-incubated at desired temperature for 15 min
and residual activity was determined using standard asparaginase
assay.
2.8. Effect of various effectors, detergents and modulators on
asparaginase activity
Asparaginase activity was investigated in the presence of
various effectors, detergents and modulators [24e26] after 30 min
of pre-incubation at 37
C, separately. Enzyme activity was determined using standard asparaginase assay and relative activity was
expressed as the percentage ratio of the activity of the enzyme
incubated with compound to that of the untreated enzyme.
2.9. Substrate specicity
In order to determine the substrate specicity of the puried
asparaginase, amide bond containing various substrates including
D-asparagine, DL-asparagine, L-glutamine, D-glutamine, L-glutamic
acid, DL-aspartic acid, L-histidine, L-ornithine, BOC-L-asparagine, N-

a-acetyl-L-asparagine, urea and acrylamide were added in assay

mixture separately in place of L-asparagine at the concentration of
0.01 M. The relative activity was expressed as the percentage ratio
of the enzyme activity against different substrates to enzyme activity with asparagine.
2.10. In vitro serum and trypsin half life (t1/2)
In order to study the in vitro serum half life, puried asparaginase (50 IU, 0.5 ml volume) was mixed with 2.5 ml human
serum, homogenized vigorously and sample was incubated at 37
C
[27]. Asparaginase activity was determined in 0.1 ml reactive serum
containing asparaginase at regular intervals of 5 h till the enzyme
was found to be active.
To determine the trypsin resistance, 0.5 ml puried asparaginase (50 IU) was added to 2.5 ml of 0.05 M phosphate buffer (pH
7.4), containing 50 IU of trypsin [27]. Reaction mixture was homogenized vigorously, incubated at 37
C and the enzyme activity
was determined at regular intervals of 10 min till the enzyme was
found to be active.
2.11. Assessment of anti-cancerous activity of asparaginase
2.11.1. Cell cultures, growth conditions and treatment
Human leukemic cell lines MOLT-4 and K-562, breast cancer cell
lines MDA-MB-231 and T47D and human normal breast epithelial
cell line FR-2 were obtained from National Cancer Institute (NCI),
Bethesda, USA. Cells were grown in RPMI-1640 medium containing
10% fetal bovine serum (FBS), penicillin (100 U ml1) streptomycin
(100 mg ml1), in CO2 incubator (Thermocon Electron Corporation,
USA) at 37
C with 98% humidity and 5% CO2 gas environment. Cells
were treated with lter sterilized asparaginase during logarithmic
growth phase.
2.11.2. Cell proliferation assay
The anti-proliferation effect of puried asparaginase was
investigated by MTT assay [28]. Briey, 1.2  104 to 1.5  104
exponentially growing cells were seeded in 96-well plates, containing 200 ml RPMI-1640 medium, with 10% fetal bovine serum.
Cells were treated with different concentration of asparaginase.
After 48 h, 10 ml/well of MTT dye (5 mg ml1 stock) was added in
each well and plates were incubated at 37
C in CO2 incubator for
4 h. The plates were centrifuged at 1600 rpm for 15 min and supernatant was discarded. The formazan blue crystal, formed by
viable cells, was dissolved with 150 ml DMSO and the rate of color
production was measured at 570 nm with ELISA reader. The OD of
control samples were considered to be 100% and accordingly the
viability of other samples were calculated by using the following
formula.

% Viability O:D Treated well=O:D Control well  100

2.11.3. Soft agar colony-forming assay

Colony forming assay was performed as described previously by
Gupta et al. [29]. MOLT-4 cells were treated with different concentration of puried P. otitidis asparaginase for 24 h. After treatment, 1  103 cells were plated in 6 well culture plate in medium
containing 0.3% agar overlying a 0.5% agar layer. The cells were
subsequently incubated for 21 days at 37
C in CO2 incubator and
colonies were stained with 0.5 ml of 0.1% crystal violet solution for
1 h and counted. Colonies of 50 or more cells were counted from
three independent experiments and results were expressed as a
percentage of colonies with respected to untreated control.

I. Husain et al. / Biochimie 121 (2016) 38e51

2.11.4. Phase contrast microscopy

Phase contrast microscopy was performed to assess the
morphological changes in MOLT-4 cells after treatment with
asparaginase. 0.6  106 cells were cultured in six-well plates, after
2 h, cells were treated with different concentration of asparaginase
for 24 h. After treatment, cells were subjected to photography.
Changes in cellular morphology were assessed using a microscope
(1  70, Olympus) and photographs were taken by using DP-12
camera.
2.11.5. DAPI staining of cells for nuclear morphology
In order to study the effect of asparaginase treatment on nuclear
morphology, MOLT-4 cells (0.6  106) were cultured in six well
plates and treated with different concentration of asparaginase for
24 h. After treatment, cells were centrifuged at 1600 rpm for
5 min at 4
C and washed once with PBS buffer. Cells were xed in
methanol for 30 min at 4
C, centrifuged and suspended in 500 ml
PBS. Cells were stained with DAPI (1 mg ml1) for 10 min. The cell
pellet was resuspended in 50 ml of mounting uid (PBS:glycerol, 1/
1) and 10 ml of this cell suspension was spread on clean glass slides
and covered with coverslips [30]. The slides were then observed for
any nuclear morphological alterations and apoptotic bodies under
an inverted uorescence microscope (Olympus 1  70, magnication 30) using UV excitation.
2.11.6. DNA fragmentation assay
The DNA fragmentation assay was performed as described
earlier [31]. Briey, cells of exponential growth phase were cultured
in six well plates at a density of 2  106 cells/2 ml and treated with
different concentration of asparaginase for 24 h. After treatment,
cells were harvested and washed with PBS containing 20 mM EDTA.
The pellet was lysed in 250 ml of lysis buffer (100 mM NaCl, 5 mM
EDTA, 10 mM Tris-HCl, pH 8.0 and 5% Triton X-100) containing
100 mg ml1 of DNase-free RNase and incubated at 37
C for 90 min
followed by 1 h incubation with proteinase-K (200 mg ml1) at
50
C. The DNA was extracted with 150 ml of phenol for 1 min and
centrifuged at 15,000 rpm for 2 min. The aqueous phase was further
extracted with chloroform: isoamylalcohol (24:1) and centrifuged.
DNA was precipitated from aqueous phase with three volumes of
chilled alcohol and 0.3 M sodium acetate at 20
C overnight. The
precipitate was centrifuged at 15,000 rpm for 10 min. The DNA
pellet was washed with 80% alcohol, dried, dissolved in 50 ml TE
buffer and electrophoresed in 1.8% agarose gel at 50 V for 1.5 h.
2.11.7. Cell cycle progression analysis
In order to study the effect of asparaginase on the level of
apoptosis and on the cell cycle distribution of MOLT-4 cells, cell
cycle was assessed by propidium iodide uorescence [32]. MOLT4 cells (1  106) were seeded in 6 well plates and treated with
different concentration of asparaginase. Plates were incubated at
37
C in CO2 incubator with 5% CO2 for 12 h and 24 h. After treatment, cells were centrifuged at 1600 rpm, xed with 70% ethanol
and stored at 4
C overnight. Further, cells were centrifuged,
resuspended in 200 ml PBS and incubated with RNAse A at 37
C for
90 min. PBS was added to make its volume 500 ml and stained with
propidium iodide (100 mg ml1) for 30 min on ice in the dark. DNA
content was measured by using a BD FACS ow cytometer (Becton
Dickinson, Franklin Lakes, NJ). Data were collected in list mode, and
10,000 events were analyzed for FL2-A vs FL2-W. Modt software
was used to distinguish different phases of the cell cycle.
2.11.8. Measurement of mitochondrial membrane potential (MMPor
Djm) loss
For measurement of mitochondrial membrane potential (MMP)
loss, MOLT-4 cells (1  106) were seeded in 12-well plates, treated

41

with different concentration of asparaginase and incubated at 37
C

in CO2 incubator for 24 h. After treatment, Rhodamine-123
(200 nM ml1) was added 30 min before termination of the
experiment. Cells were harvested and washed once with PBS. The
cell pellet was resuspended into 500 ml PBS and mitochondrial
membrane potential was measured in the FL-1 channel of the ow
cytometer (Becton Dickinson, Franklin Lakes, NJ) [28].
2.12. Toxicological evaluation of puried asparaginase against
normal cells
2.12.1. Cytotoxicity assay against human normal FR-2 cells
Cell cultures, growth conditions of normal cells (FR-2) are
described in Section 2.11.1 and cytotoxic effect of asparaginase on
normal FR-2 cells was investigated using MTT assay as described in
the Section 2.11.2.
2.12.2. Cytotoxicity assay against human normal lymphocytes
Blood sample were obtained from healthy male volunteer.
Lymphocytes (peripheral blood mononuclear cells) were isolated
from blood samples by centrifugation using Ficoll-based density
gradient, washed twice with serum free RPMI-1640 medium and
treated with different concentration of puried asparaginase for
48 h. After treatment, cytotoxicity was investigated with MTT assay
as described in the Section 2.11.2.
2.12.3. In vitro hemolysis assays
Hemolysis assay was performed to investigate the hemolytic
effect of puried asparaginase on human blood (erythrocytes) according to the method of Huang et al. [33]. The blood agar plate
composed of 5% fresh debrinated human blood was prepared.
25 ml crude and puried asparaginases were separately placed into
the holes previously punched in the blood agar plate. After incubation at 37
C for 24 h, the plates were examined for transparent or
translucent halo zone. Quantitative hemolytic assay was performed
according to the method of Bulmuset al. [34]. Briey, debrinated
human blood cells (erythrocytes) were taken, washed three times
with 150 mM NaCl and cells were suspended in 100 mM sodium
phosphate buffer, pH 7.2. Cells were incubated at 37
C with
different concentration of puried and crude asparaginase for 24 h.
After incubation, cells were centrifuged at 2500 rpm for 15 min and
optical density was measured at 541 nm. Sodium phosphate buffer
as blank and untreated cells were used as a positive control.
2.12.4. Statistical analysis
All experiments were performed in triplicate and data reported
as mean SD. Statistical analysis was done by using student t-test
and p value < 0.05 was considered to be statistically signicant in
this study.
3. Results
3.1. Purication and molecular weight determination of the
asparaginase
A glutaminase free asparaginase was extracted and puried to
apparent homogeneity from cell free supernatant of P. otitidis, using
various steps as described in materials and methods section. The
crude enzyme prepared from 60 to 90% ammonium sulphate precipitation was subjected to a DEAE-cellulose ion exchange column
(Fig. 1a). The fractions (6,7,8,9) showing maximum asparaginase
activity were further subjected to gel ltration chromatography on
Sephadex G-100 column (Fig. 1b). As the results summarized in
Table 1, enzyme was puried approximately to 151.88 fold with
38.90% yield and 107.84 U mg1 specic activity. The homogeneity

42

I. Husain et al. / Biochimie 121 (2016) 38e51

Fig. 1. Purication of asparaginase from P. otitidis. (a) Cation exchange chromatography of asparaginase by DEAE-cellulose column (2  15 cm2, Sigma) pre-equilibrated with 0.05 M
TriseHCl buffer (pH 9.6) and absorbed protein was eluted with a linear gradient of KCl (0e200 mM). (b) The collected fraction of highest asparaginase activity were applied on gel
ltration chromatography with Sephadex G-100 column (2  20 cm2, Sigma), equilibrated with 0.05 M TriseHCl buffer (pH 8.6) and eluted with the same buffer at a ow rate of
0.2 ml min1 (c) Assessment of homogeneity and molecular weight analysis of puried extracellular asparaginase on Native-PAGE; Lane 1- Protein marker; Lane 2 and 3 Sephadex
G-100 puried asparaginase. (d) SDS-PAGE analysis of puried enzyme in 12% polyacrylamide gel; Lane 1- Protein marker; Lane 2-Sephadex G-100 puried asparaginase; Lane 3DEAE-cellulose puried asparaginase. (e) Two-dimensional electrophoretic resolution of puried asparaginase. Puried protein after was resolved by IEF and followed by SDS-PAGE.
Gel was visualized by coomassie brilliant blue R-250 staining. Molecular weights were calculated with standard molecular weight markers (PMWH, Bangalore Genei, India).

Table 1
Summary of various steps involved in purication of asparaginase from P. otitidis.
Purication step

Total activity (IU)a

Total protein (mg)

Specic activity (IUmg1)

Fold purication

Yield (%)

Crude extract
(NH4)2SO4 precipitation
DEAE-cellulose
Sephadex G-100

2370
1587
1269.6
922.04

3300
363
23.59
8.55

0.71
4.37
53.81
107.84

1
6.15
75.78
151.88

100
66.96
53.56
38.90

One unit of asparaginase (IU) is dened as the amount of enzyme that liberates 1 mmol of ammonia min1 at 37
C.

and molecular weight of the puried enzyme was determined by

native-PAGE. As the results presented in Fig. 1c, puried enzyme
exhibited a single band with an apparent molecular mass of
2053 kDa. However, one single band of 341 kDa appeared on
SDS-PAGE (Fig. 1d), indicating that the puried enzyme exist in
hexameric form. Puried asparaginase did not exhibit intrinsic
glutaminase activity which indicates that puried enzyme is
glutaminase free asparaginase.
3.2. Isoelectric point (pI) of asparaginase
Two-dimension polyacrylamide gel electrophoresis (2D-PAGE)
is one of the methods used for determination of isoelectric point
(pI) of protein. To determine the pI of puried asparaginase, isoelectric focusing followed by SDS-PAGE was performed. According
to the result presented in Fig. 1e, puried enzyme showed a protein
spot at 5.5 pI point on 4e7 nonlinear gradients IPG strip as well as

on SDS-PAGE gel. Thus, our result indicates that the pI of asparaginase is 5.5.
3.3. Effect of pH and temperature on activity and stability of
asparaginase
To optimize the pH and temperature, asparaginase activity was
evaluated from pH 4.5 to 10.5 and temperature in range of
20e70
C, using asparagine as a substrate. The puried asparaginase was more active at pH range 6e9. and maximum activity
was achieved at pH 7.5. However, enzyme activity decreased
abruptly when the pH was increased above 9. The enzyme showed
stability at slightly alkaline pH and retained more than 70% of its
original activity when incubated at pH 9 for 24 h (Fig. 2a and b). The
puried asparaginase exhibited maximum activity at 40
C and
decreased above 45
C. In thermal stability experiment, no significant activity was lost when the puried enzyme was pre-incubated

I. Husain et al. / Biochimie 121 (2016) 38e51

43

Fig. 2. Effect of physical conditions on asparaginase activity and stability (aeb) Effect of pH on activity and stability of puried asparaginase, enzyme activity was assayed at various
pH buffers. The buffer used as follows: pH 4.5e7.5, Potassium phosphate buffer, pH 8.0e10.5, TriseHCl buffer, (ced) effect of temperature on activity and thermal stability of puried
asparaginase. Effect of various metal ions (e) and modulators (f) on the asparaginase activity. Asparaginase was mixed with the corresponding metal salts (NaCl (50 mM), (KCl
(150 mM), (CaCl2 (150 mM), (MgCl2 (40 mM), (ZnCl2 (100 mM) (MnCl2 (100 mM), (FeCl3 (100 mM), (CdCl2 (10 mM), (NiCl2 (10 mM), (HgCl2 (100 mM) or modulators EDTA (5 mM),
DDT (5 mM), iodoacetamide (5 mM), SDS (2 mM), 2-mercaptoethanol (0.5 mM), glutathione (0.5 mM), L-cysteine (25 mM), thiourea (1 mM), and human serum (10%) in 50 mM
phosphate buffer (pH 7.5) for 30 min at 37
C and enzyme activity was measured by Nesslerization reaction. No addition was used as control. Each value represents the mean SD
for three determinations.

at 45
C and above this temperature the enzyme activity was

further decreased in temperature dependent manner (Fig. 2c and
d).

3.4. Effect of various effectors, detergents and modulators on

asparaginase activity
Asparaginase activity increased in presence of Na and K ions
while, other metalions viz. Ca2, Mg2, Zn2, Mn2, Fe3, Cd2, Ni2

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I. Husain et al. / Biochimie 121 (2016) 38e51

and Hg2 signicantly inhibited asparaginase activity (Fig. 2e).

Reducing agents, 2-mercaptoethanol and glutathione accelerated
the asparaginase activity while, in the presence of L-cysteine, EDTA
and iodoacetamide asparaginase activity slightly inhibited. However, enzyme activity was almost lost in presence of thiourea
(Fig. 2f).
3.5. Substrate specicity of asparaginase
In order to study the substrate specicity of asparaginase,
various substrates were investigated. As shown in Table 2, among
the tested substrates, asparaginase puried from P. otitidis exhibited highest activity towards its natural substrate L-asparagine and
no activity was observed towards L-glutamine, D-glutamine, Lglutamic acid, L-ornithine, BOC-L-asparagine, N-a-acetyl-L-asparagine, urea and acrylamide. These observations suggesting that puried enzyme showed high substrate specicity.
3.6. In vitro serum and trypsin half life(t1/2) of asparaginase
As illustrated in Fig. 3a, puried enzyme exhibited a reduction of
50% and 90% of initial activity after50 and 85 h of incubation,
respectively. However, on increasing the incubation time, asparaginase activity was lost in time dependent manner. As depicted in
Fig. 3b, puried asparaginase showed good trypsin resistance and
exhibited a reduction of 50% of initial activity after38 min of incubation at 37
C. These results suggest that puried enzyme can
remain active and stable over prolong time in human circulatory
system.
3.7. Anti-proliferative activity of asparaginase
To study the anti-proliferative effect of puried P. otitidis
asparaginase, human cancer cell lines, MOLT-4, K-562, MDA-MB231 and T47D were treated with 2, 5, 10, and 15 IU ml1 concentration of enzyme and cytotoxic activity was investigated by MTT
assay. The E. coli asparaginase procured from Sigma Chemical Co. St.
Louis, USA, was used as a reference preparation and untreated cells
were taken as controls. The IC50value of puried enzyme was
calculated to be 1.2, 4.1, 7.7 and 11 IU ml1 against MOLT-4, K-562,
MDA-MB-231 and T47D cells respectively after 48 h of treatment.
The E. coli asparaginase (Sigma) exhibited IC50 value
1.1, 8.2 and 11.5
IU ml1 against MOLT-4, K-562 and MDA-MB-231 cells respectively,
but this value did not achieve against T47D cells till 15 IU ml1.
These results suggest that puried enzyme induces cytotoxicity on
human cancer cells and its cytotoxic potential is comparable or

Table 2
Substrate specicities of puried asparaginase from P. otitidis.
Substrate

Concentration (mM)

Relative activity (%)a

L-Asparagine

10
10
10
10
10
10
10
10
10
10
10
10
10

100 1.6
1 0.2
3 0.4
N.D.
N.D.
N.D.
1 0.5
1.6 0.6
N.D.
N.D.
N.D.
N.D.
N.D.

D-Asparagine
DL-Asparagine
L-Glutamine
D-Glutamine
L-Glutamic

acid
DL-Aspartic acid
L-Histidine
L-Ornithine
BOC-L-Asparagine
N-a-Acetyl-L-Asparagine
Urea
Acrylamide

N.D. Not detected.

a
Relative activity shown in the table. Each value represents the mean SD for
three determinations.

Fig. 3. Physiological properties of asparaginase puried from P. otitidis. In vitro (a)

serum and (b) trypsin half life of asparaginase. Asparaginase was incubated with human serum and trypsin, separately and asparaginase activity was measured by standard asparaginase assay. The procedure is discussed in Materials and Methods. Each
value represents the mean SD for three determinations.

higher to commercial asparaginase (Fig. 4a, b, c, and d). Puried

asparaginase showed highest anticancer activity against MOLT-4
cells hence, we selected MOLT-4 cell line for further investigations.
3.8. Asparaginase inhibits colony formation potential
Single cell have the ability to grow and form colony therefore,
cell survival after asparaginase treatment was measured by using
colony forming assay. For this, MOLT-4 cells were treated with 1, 2,
and 5 IU ml1 concentration of asparaginase for 24 h and used for
colony forming assay. The result represented in Fig. 5, indicated
that 72%, 56% and 40% colony formation were recorded at 1, 2, and
5 IU ml1 treatments, respectively. These results suggest that the
puried P. otitidis asparaginase inhibits the proliferation of MOLT4 cells in dose dependent manner.
3.9. Phase contrast microscopy
Phase contrast microscopy was performed to observe changes in
cell morphology. MOLT-4 cells were treated with 1, 2 and 5 IU ml1
concentration of puried enzyme and after 24 h of treatment,
phase contrast microscopy was performed. We observed that
asparaginase had destroyed integrity of cell membrane and
increased apoptotic effects such as plasma membrane blebbing and
cell shrinkage in dose dependent manner. However, morphological
changes was not observed with untreated cells. These results

I. Husain et al. / Biochimie 121 (2016) 38e51

45

Fig. 4. Antiproliferative effects of puried P. otitidis and E. coli asparaginases on human leukemic cell line (MOLT-4 and K-562) and breast cancer (MDA-MB-231 and T47D) cell lines.
E. coli asparaginase procured from Sigma Chemical Co. St. Louis, was used as a reference preparation. The cells were treated in triplicate with different concentration (2, 5, 10 and
15 IU ml1) of puried and reference asparaginase preparation for 48 h and the cell viability was determined by the MTT assay.

3.10. Fluorescence microscopy for nuclear morphology

In order to test whether puried enzyme affect the integrity of
cell nucleus, we used DAPI staining. For this, MOLT-4 cells were
harvested after 24 h of treatment with puried enzyme (1, 2 and
5 IU ml1) and subjected to DAPI staining. Results showed that on
asparaginase treatment, alteration in nuclear morphology such as
shrinkage, chromatin condensation and loss of normal nuclear architecture; all are characteristics of apoptosis were increased with
increasing asparaginase concentration. However, intact nuclei in
control (untreated) cells indicated no alteration in nuclear
morphology (Fig. 7). These results suggest that puried enzyme
induces DNA damage which led to cells death.
3.11. Asparaginase induces DNA fragmentation

Fig. 5. P. otitidis asparaginase inhibits the colony formation of MOLT-4 cells. Cells
treated with indicated concentration of puried asparaginase and colony forming
assay was performed. Data are Mean S.D. of calculated colonies percentages from
three similar experiments.

further suggest that the puried enzyme affect the cell viability and
induces apoptotic cell death (Fig. 6).

By performing DAPI staining we observed that puried P. otitidis

asparaginase induces alteration in nuclear morphology. Further,
DNA fragmentation assay was performed to analyze the effect of
asparaginase on genetic material of MOLT-4 cells. For this, MOLT4 cells were treated with 1, 2, and 5 IU ml1 enzyme for 24 h,
genomic DNA was isolated and electrophoresed on agarose gel.
According to the results that are presented in Fig. 8, at low concentration, DNA ladder was barely visible and this became
increasingly prominent in cells treated with higher concentration
of asparaginase. However, DNA isolated from untreated cells did
not show any DNA ladder. These results suggest that puried

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I. Husain et al. / Biochimie 121 (2016) 38e51

Fig. 6. Asparaginase induced cell death in MOLT-4 cell line. Cells were treated with the indicated concentration of asparaginase puried from P. otitidis for 24 h and observed for
morphological changes under microscope (1  81, Olympus).

Fig. 7. Alteration in nuclear morphology by treatment with puried P. otitidis asparaginase. MOLT-4 cells were incubated with indicated concentration of asparaginase, collected
after centrifugation at 1600 rpm, washed once with PBS and then stained with DAPI for 10 min. The procedure is discussed in Materials and Methods.

asparaginase severely damaged MOLT-4 cells by DNA fragmentation which leads to apoptosis.
3.12. Cell cycle phase distribution
MTT results showed that puried enzyme affects the viability of
cells. Microscopic studies suggested that enzyme induced
apoptosis. Further, FACS analysis was performed to analyze the
effect of asparaginase on cell cycle progression. For this, MOLT4 cells were harvested after 24 h of treatment with puried
asparaginase, stained with propidium iodide and subjected to ow

cytometry. According to the results presented in Fig. 9, the histogram of control (untreated) cells showed standard cell cycle
pattern, in which G1 and G2 peaks were separated by S phase. It is
very interesting to note that at all the three (1, 2, and 5 IU ml1)
concentration of enzyme we could not nd any cell cycle arrest at
G0/G1, S, and G2/M phase. In contrast, on comparison with control
(untreated) cells, 4.97%, 16.92% and 35.80% apoptotic cell death
were found at 1, 2, and 5 IU ml1 treatment of enzyme, respectively.
Hence, the results of this experiment suggest that P. otitidis asparaginase induced apoptotic cell death without G0/G1, S, and G2/M
phase arrest, which could be transient arrest that comes before

I. Husain et al. / Biochimie 121 (2016) 38e51

47

3.14. Cytotoxic effect asparaginase on human noncancerous FR2 cells

Since the anticancer activity data indicates that asparaginase
puried from P. otitidis was potentially inhibited the proliferation
and induce apoptosis in human leukemia cells, MOLT-4. Further, we
also evaluated cytotoxicity of enzyme against normal breast
epithelial cell line FR-2,. For this, FR-2 cells were treated with 2, 5,
10, and 15 IU ml1 concentration of enzyme, harvested after 48 h
and were subjected to MTT assay. According to the results that are
summarized in Fig. 11a, puried asparaginase did not exhibit
cytotoxic effect on noncancerous (normal) cells. These results
suggest that the puried asparaginase was selectively and potently
cytotoxic for cancer cells and nontoxic for normal cells.
3.15. Cytotoxic effect of puried asparaginase on human normal
lymphocytes
Cytotoxicity of puried asparaginase was also investigated
against human normal lymphocytes. For this, cells were treated
with 2, 5, 10, and 15 IU ml1 concentration of enzyme for 48 h and
subjected to MTT assay. The results are presented in Fig. 11b, puried asparaginase did not inhibit viability of human normal lymphocytes. These results further suggest that puried asparaginase
was nontoxic for normal blood cells.
3.16. In vitro hemolysis

Fig. 8. P. otitidis asparaginase induced DNA fragmentation in human leukemia cell line
MOLT-4. Genomic DNA was extracted from cells treated with different concentrations
of puried asparaginase for 24 h. Other conditions are described in Materials and
Methods.

completion of treatment time (24 h). Hence, further cell cycle

progression analysis was performed after 12 h of asparaginase
treatment and it was noted that puried asparaginase induce cell
cycle arrest in S phase (Supplementary data Fig. S1).

3.13. Asparaginase induces mitochondrial membrane potential

(MMP or Djm) loss
Mitochondrial integrity is required for cells to be functional and
measurement of MMP is one of the methods used for investigation
of apoptosis. To observe the effect of puried asparaginase on MMP,
MOLT-4 cells were cultured in the presence of different concentration (1, 2 and 5 IU ml1) of asparaginase, harvested after 24 h and
used for ow cytometric analysis following staining with
Rodamine-123. Results showed that, MMP loss (27.1%) was significant at 1 IU ml1 and further increased upto 31.9%, and 40.9% at 2
and 5 IU ml1 of enzyme treatment, respectively (Fig. 10). In
contrast, control (untreated) cells showed only 6.7% loss in MMP.
Thus, it can be concluded that disruption of mitochondrial membrane in MOLT-4 cells occurred by asparaginase treatments. These
results suggest that asparaginase induced cell death probably via
activation of mitochondrial mediated pathway of apoptosis but
further studies are required to understand the exact mechanism of
cell death.

Hemolytic activity is an important indicator of cytotoxicity and

is one of the methods used for toxicological evaluation of drug.
Hence, the effect of the crude and puried asparaginase on human
blood (erythrocytes) was investigated. The results presented in
Fig. 11c shows that crude enzyme formed a translucent zone on the
blood agar plate this could be due to presence of various proteins in
crude extract that toxic for erythrocytes. In contrast, puried
enzyme did not exhibit hemolytic effect, indicating that puried
asparaginase was nontoxic for erythrocytes. The results of quantitative hemolytic assay also showed that puried enzyme had no
hemolytic effects even at highest concentration (15 IU ml1)
(Fig. 11d). These results collectively suggested that the asparaginase
puried from P. otitidis is nontoxic for erythrocytes.
4. Discussion
Since more than last four decades [35] asparaginase is an
important component of multidrug chemotherapy in children and
adults with ALL, AML and non-Hodgkin lymphoma [10], and researchers have continuously paid much attention because of its
antineoplastic nature [36]. Currently, commercially available
asparaginase possesses 2e10% intrinsic glutaminase activity due to
which it hydrolyzes L-glutamine of circulating system. The product
(sodium glutamate) of L-glutamine hydrolysis is responsible for
several cytotoxic reactions [18]. In this study, glutaminase free
asparaginase from P. otitidis was puried to electrophoretic homogeneity with 151.88 fold purication, which is comparable to the
purity of chemotherapeutic proteins. The subunit and native molecular weight of enzyme was 341 kDa and 2053 kDa, respectively hence, native protein appeared as ahomohexamer. These
results are in accordance with the earlier observations which stated
that microbial asparaginases are occurs in various forms viz,
monomeric [37], dimeric [12,38], trimeric [25], tetrameric [39] and
hexameric [40,41].The isoelectric point (pI) of enzyme was found to
be 5.5 which is slightly higher and lower than asparaginases puried from Pseudomonas uorescens AG (4.5) [42] and Pseudomonas
stutzeri MB-405 (6.38) [37], respectively. Most of the asparaginases

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I. Husain et al. / Biochimie 121 (2016) 38e51

Fig. 9. Effect of puried asparaginase on cell cycle progression in leukemia cells. MOLT-4 cells (1  106) were seeded in 12-well plates and treated with different concentration (1, 2
and 5 IU ml1) of asparaginase for 24 h. After incubation, cells were collected at 1600 rpm, washed once with PBS, and xed in 70% ethanol overnight. Cells were then washed once
with PBS and stained with 100 mg of propidium iodide for 30 min. Modt software was used to differentiate between phases and to determine the amount of apoptotic population.

Fig. 10. Asparaginase induced concentration dependent mitochondrial membrane potential (MMP or Djm) loss in human leukemia cells. MOLT-4 cells (1  106) were treated with
indicated concentration of puried asparaginase for 24 h, washed once with PBS and stained with Rhodamine-123. MMP was measured as discussed in Materials and Methods.

act optimally at pH range 7-9 [41] and temperature range of

30e40
C [24,26,39,43]. The puried asparaginase showed

maximum activity at pH 7.5 and temperature 40
C, which is close

to the pH of human circulating system (pH 7.4) and temperature of

I. Husain et al. / Biochimie 121 (2016) 38e51

49

Fig. 11. Toxicological evaluation of asparaginase puried from P. otitidis against human normal cells. (a) Cytotoxic effects of asparaginase on noncancerous human breast epithelial
cell line FR-2 and (b) blood lymphocytes. Cells were treated in triplicate with different concentration (2, 5, 10 and 15 IU ml1) of puried asparaginase for 48 h and the cell viability
was determined by the MTT assay. (c) Hemolytic effect of puried and crude asparaginase on human erythrocytes. (d) Quantitative measurement of the hemolytic activity. A: blank
(Sodium phosphate buffer); B: positive control (without asparaginase); C: crude asparaginase; D: 15 IU ml1; E: 7.5 IU ml1; F: 3.75 IU ml1 concentration of asparaginase.

human body (37
C). This property appraises the medicinal

importance of P. otitidis asparaginase. Inhibition studies of enzyme
activity could provide a rst insight into the nature of the enzyme,
its cofactor requirements and the nature of the active center
[33,44]. Among the various metal ions tested, monovalent cations,
Na and K were found to enhanced asparaginase activity, indicating that the enzyme might contain Na and K ions. Whereas,
divalent and trivalent cations, Mg2, Zn2, Cd2, Ni2, Hg2, and
Fe3, respectively inhibited asparaginase activity, indicating that
the active site of asparaginase may contain Sulfhydryl (-SH) moieties. Reducing agents such as 2-mercaptoethanol, glutathione
were found to enhance enzyme activity. While, the presence of eSH
inhibitors, such as iodoacetamide decreased asparaginase activity,
conrming the presence of eSH group at the active site of the
enzyme. Asparaginase from different sources possesses poor substrate specicity and hydrolyzes amide bond of blood urea [45] and
L-glutamine [46] which leads to ammonia toxicity [15]. By performing substrate specicity assay, we noted that puried asparaginase showed great substrate specicity towards L-asparagine
and does not hydrolyzes, urea,L-glutamine, D-glutamine, L-glutamic
acid, BOC-L-asparagine, N-a-acetyl-L-asparagine and acrylamide
etc. Therefore, it could be at herapeutically remarkable feature of
P. otitidis asparaginase. Serum and trypsin are the major constituents of human circulating system that inactivate or hydrolyze
clinically administrated asparaginase and reduce its bioavailability.
However, for continuous depletion of blood asparagine, frequent

asparaginases administrations are required [14,47]. Therefore,

asparaginase exhibiting longer plasmatic and trypsin half life could
contribute in the upgradation of therapeutic index of asparaginase
therapy. We have noted that the in vitro serum and trypsin half life
of puried enzyme was 50 h and 38 min respectively, which is 2
and 1.5 fold higher than commercial E. coli asparaginase (native)
[27,48]. These observations collectively suggest that puried
asparaginase could be consider as drug with longer plasmatic half
life.
To explore the anticancer activity, we have performed MTT assay
with panel of human cancer cell lines, MOLT-4, K-562, MDA-MB231 and T47D and noted that puried enzyme potently and
signicantly inhibited the proliferation of tested cancer cell lines.
The cytotoxic efcacy of puried asparaginase against these cell
lines is comparable or higher from commercial E. coli asparaginase
(sigma) which suggest that asparaginase puried from P. otitidis
could be consider as more potent and effective chemotherapeutic
agent as compared to the commercially available asparaginase
preparations. In order to assess, whether the puried asparaginase
possesses a long-term cytotoxic effect on the ability of a single cell
to proliferate into a viable colony, we have performed clonogenic
assay and found that our puried asparaginase had signicantly
decreased the clonogenic potential of MOLT-4 cells. Plasma membrane blebbing, cell shrinkage, chromatin condensation, nucleosomal DNA fragmentation and formation of apoptotic and scattered
apoptotic bodies are the major morphological characteristics of

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I. Husain et al. / Biochimie 121 (2016) 38e51

apoptosis [49]. By performing the phase contrast microscopy,

uorescence microscopy and DNA fragmentation assay, we noted
that asparaginase treated cells showed all aforementioned
morphological changes, which increases in dose dependent
manner. Previously, similar observations were also recorded with
asparaginase puried from Bacillus circulans [36]. Cell cycle control
is the major regulatory mechanism in the cell growth process. Most
of the anticancer drugs arrest cell cycle at G0/G1, S, and G2/M phase
and induced apoptosis [50]. Therefore, further we investigated the
effect of puried enzyme on cell cycle progression and noted that
on 24 h of treatment with asparaginase there was no cell cycle
arrest in the G0/G1, S, and G2/M phase. In contrast, the percent of
apoptosis increased on increasing the concentration of puried
asparaginase. However, after 12 h of treatment cell cycle was
arrested in S phase. Hence, authors suggest that the apoptosis
induced by puried asparaginase might be due to transient arrest
which appears at early time points of 24 h of enzyme treatment.
This type of transient arrest of cell cycle has been also reported with
some other compounds recently tested as potential anticancer drug
[51,52]. Mitochondrial integrity is required for cells to be functional
and loss of mitochondrial membrane potential (MMP or Djm) is an
essential event in mitochondrial pathway of apoptosis [53]. Hence,
further we attempted to understand the effect of puried enzyme
on mitochondrial membrane permeability in MOLT-4 cells. We
have noted that upon treatment with asparaginase the MMP loss
was increased in concentration dependent manner, which leads to
apoptotic cell death. Previous studies suggested that cellular
apoptosis has been mediated via mitochondrial pathway of
apoptosis which was dominated by up-regulation of pro-apoptotic
proteins (Bax) and down-regulation of antiapoptotic proteins (Bcl2) [50,52,53]. Here, we suggests that similar mechanism might be
occur after treatment of asparaginase but further studies are
required to understand the pathway of apoptotic cell death of
tested human leukemic MOLT-4 cells, which is included in our
future research.
Today, several drugs have been used in ALL therapy and
numerous are under clinical trials, but they have low selectivity and
causes toxicity in noncancerous (normal) cells. However, one of the
major goal of cancer chemotherapy is to explore and develop new
drugs that can selectively induced apoptosis in cancer cells and
should be nontoxic for normal cells [1,54]. In order to check cytotoxic effects of puried asparaginase on human noncancerous cells,
we treated breast epithelial cell line FR-2 and blood lymphocytes
with same concentration of enzyme that was used against cancer
cells. We found that puried enzyme did not affect cells viability of
FR-2 and blood lymphocytes at highest concentration (15 IU ml1)
of enzyme used in this study. In contrast, at this concentration
approximately 85%, 77%, 80% and 65% cells viability of MOLT-4, K562, MDA-MB-231 and T47D cells respectively were reduced which
revealed that asparaginase puried from P. otitidis is selectively
cytotoxic for cancer cells and nontoxic for normal cells. Hemolysis
is one of the major problems associated with chemotherapeutic
drugs [55]. According to the ISO/TR 7405-1984(f), the drug
considered as hemolytic if the hemolytic percentage was above 5%
[56]. By performing qualitative and quantitative hemolysis assay
we noted that puried asparaginase did not show any observational
hemolytic toxicity on the red blood cell (erythrocytes) while, in
both hemolysis assays crude enzyme showed hemolytic toxicity.
These observations suggest that puried enzyme is hemocompatible and could be safe to use as chemotherapeutic agent.
5. Conclusion
Glutaminase free asparaginase from P. otitidis was puried to
electrophoretic homogeneity. The characterization studies showed

that the enzyme possesses optimum activity and stability near to

the pH of human circulating system and temperature of human
body. High substrate specicity, prolonged serum and trypsin half
life are therapeutically remarkable features of puried asparaginase. The cytotoxic potential of puried asparaginase is comparable or higher to commercial asparaginase. Mechanistically,
puried enzyme induces apoptosis probably via activation of
mitochondrial mediated pathway of apoptosis. Noteworthy, puried enzyme was nontoxic for human normal cells FR-2 and blood
lymphocytes. Therefore, the present study raises the possibility to
use P. otitidis asparaginase as apotent antileukemic agent.
Conict of interest
The authors declare that they have no conict of interest.
Author contributions
Conceived and designed the experiments: IH, AS designed the
experiment related to purication and characterization of protein
and performed by IH. IH, FM and SK designed the experiment
related to antitumor activity of enzyme and performed by IH and
SK. Analyzed the data and wrote the paper: IH; proof reading by AS.
Acknowledgment
Authors are thankful to Madhya Pradesh Biotechnology Council
(MPBC), Bhopal, India, for providing nancial support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biochi.2015.11.012.
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