Letters in Applied Microbiology 1997, 24, 2326

A rapid plate assay for screening L-asparaginase producing

micro-organisms
R. Gulati, R.K. Saxena and R. Gupta
Department of Microbiology, University of Delhi South Campus, New Delhi, India
1067/96: received 11 March 1996 and accepted 3 June 1996
R. GULATI , R. K. SAXENA AND R. GUPTA. 1997. A pH and dye-based fast procedure for
screening L-asparaginase producing micro-organisms is reported. The procedure
is suitable for bacterial and fungal screening. The results are obtained within 24 and 48
h for bacteria and fungi, respectively. The results correlate with quantitative estimations in
culture broths.
I NTRODUCTI ON Twenty-ve fungal species and nine bacterial species used
in the study were obtained from our departmental stock
Microbial L-asparaginases have attracted considerable atten-
culture collection. Modied Czapek Doxs medium (Saxena
tion since the demonstration that L-asparaginase from Escher-
and Sinha 1981), pH 62, used for fungi contained (g l
1
of
ichia coli has anti-tumour activity (Broome 1961 ; Mashburn
distilled water) : glucose, 20; L-asparagine, 100; KH
2
PO
4
,
and Wriston 1964). This enzyme has been the subject of
152; KCl, 052; MgSO
4
.7H
2
O, 052; CuNO
3
.3H
2
O, trace;
several reviews (Cooney and Handschumacher 1970 ; Capizzi
ZnSO
4
.7H
2
O, trace; FeSO
4
.7H
2
O, trace; agar, 200. Modied
et al. 1970 ; Wriston and Yellin 1973) on its distribution,
M-9 medium (pH 70) used for bacteria contained (per 1000
biochemical and immunological properties. Since then a large
ml of distilled water) : Na
2
HPO
4
.2H
2
O, 60 g; KH
2
PO
4
, 30
number of bacteria and fungi have been screened for L-
g; NaCl, 05 g; L-asparagine, 50 g; 1 mol l
1
MgSO
4
.7H
2
O,
asparaginase producing potential (Wade et al. 1971 ; Arima et
20 ml; 01 mol l
1
CaCl
2
.2H
2
O, 10 ml; 20% glucose stock,
al. 1972 ; Imada et al. 1973 ; Curran et al. 1985). The enzyme
100 ml; agar 200 g.
is routinely screened in culture ltrates using
Modied Czapek Doxs medium was supplemented with
Nesslers reagent (Imada et al. 1973). The procedure is lengthy
different concentrations of the dye. A 25% stock of the dye
and time consuming, hence there is still a need to develop a
was prepared in ethanol and the pH was adjusted to 70 using
sensitive and rapid procedure that may directly give us poten-
1 mol l
1
NaOH. The stock solution of the dye, ranging from
tial asparaginase producers.
004 ml to 03 ml, was added to 100 ml of modied Czepek
In the present investigation, a novel and semi-quantitative
Doxs medium, giving nal dye concentrations of 0001
plate assay for screening L-asparaginase-producing micro-
0009%, respectively. The media were autoclaved and plates
organisms is reported. It is generally observed that L-aspara-
prepared. Control plates were of modied Czapek Doxs
ginase production is accompanied by an increase in pH of the
medium (i) without dye and (ii) without asparagine (instead
culture ltrates (De Jong 1972). The plate assay was devised
containing NaNO
3
as nitrogen source). The plates were
using this principle by incorporating the pH indicator phenol
inoculated with a 96-h culture of Fusarium oxysporum as a
red in medium containing asparagine (sole nitrogen source).
test organism. The zone and colony diameters were measured
Phenol red at acidic pH is yellow and at alkaline pH turns
after 48 h. After standardizing the dye concentration, all
pink, thus a pink zone is formed around microbial colonies
other fungi and bacteria were tested by the plate assay on
producing L-asparaginase.
appropriate media at 37C (or 30C). Zone diameters were
measured after 18 h and 48 h for bacteria and fungi, respec-
tively. Broth studies were also carried out in order to compare
MATERI ALS AND METHODS
the results obtained with those of plate assay.
L-Asparaginase was purchased from Hi-Media Laboratories
Quantitative estimations of enzyme activities were carried
(India). All other chemicals of analytical or equivalent grade
out using modied Czapek Doxs and M-9 liquid media for
were purchased locally.
fungi and bacteria, respectively. Erlenmeyer asks (250 ml)
containing 50 ml of the appropriate medium were inoculated
Correspondence to: Dr R. K. Saxena, Department of Microbiology, University
of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. with each of the test organisms. The asks were incubated at
1997 The Society for Applied Bacteriology
24 R. GULATI ET AL.
Fig. 1 Effect of varying phenol red
concentrations on clarity of zones (t)
produced by Fusarium oxysporum on
modied Czapek Doxs medium. c,
Control without phenol red dye ; 15,
0001%, 0003%, 0005%, 0007% and
0009% v/v phenol red, respectively
37C (or 30C) at 250 rev min
1
for 48 h and 96 h for cient, r (Lewis 1984), between the zone size (cm) and enzyme
activities (IU ml
1
) in culture broths. bacteria and fungi, respectively, in a controlled environment
incubator-shaker (model G25R; New Brunswick Scientic
Co. Inc., Edison, NJ). Uninoculated media served as controls.
RESULTS AND DI SCUSSI ON
The fungal cultures were harvested by ltration through
Whatman No. 1 lter paper and the bacterial cultures by Studies with different concentrations of the dye revealed that
as the concentration of the dye increased, the clarity and centrifugation at 6000 rev min
1
for 15 min. The enzyme
activities were estimated in culture ltrates by Nesselerization visibility of the pink zone increased (Fig. 1). Furthermore,
the dye did not inhibit the growth of the test fungus F. (Imada et al. 1973) and expressed as IU ml
1
. One inter-
national unit of L-asparaginase activity is dened as that oxysporum (Table 1). The most appropriate dye concentration
was found to be 0009%. Among the various fungi and bac- amount of enzyme which catalyses the formation of 1 mmol
of ammonia per min under the conditions of the assay. Results teria tested by the plate assay and broth studies, Aspergillus
oryzae, Penicillium nelicum, P. granulatum and Bacillus licheni- were statistically evaluated by calculating the correlation coef-
Table 1 Effect of phenol red
concentrations incorporated
in modied Czapek Doxs medium
on growth and zone size of
Fusarium oxysporium in 30C after 48
h of incubation

Plates with asparagine Plates with NaNO

3

Concentration of Colony Zone Colony Zone
phenol red diameter diameter diameter diameter
(%v/v) (cm) (cm) (cm) (cm)

0001 219 209 Nil

0003 200 117 226 Nil
0005 213 103 237 Nil
0007 253 106 243 Nil
0009 257 106 243 Nil
Control (without dye) 262 Nil 237 Nil

, No demarcable zone.
1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 2326
PLATE ASSAY FOR L- ASPARAGI NASE 25
Table 2 Colony diameter and zone
diameter after 48 h, and L-asparaginase
activity with change in pH of culture
ltrate after 96 h of various
fungi screened at the requisite
temperature*

Colony Zone Enzyme pH of the

diameter diameter actvity culture
Fungus (cm) (cm) (IU ml
1
) ltrate

Aspergillus tamarii 160 083 009 832

A. terreus 168 097 010 860
A. acolumnaris 182 150 010 850
A. alliaceus 103 090 008 860
A. niveus 137 100 009 870
A. oryzae 160 190 014 895
A. amstelodami 100 021 001 762
A. nidulans 110 014 001 701
A. awamori 182 025 001 790
A. japonicus 191 072 006 843
A. striatus 165 087 003 828
A. wentii 170 060 005 861
A. niger 157 086 007 870
A. penicilliformis 172 060 007 872
A. puniceus 192 091 008 868
A. giganteus No growth 000 640
A. sunderbanii 142 061 005 820
Pencillium nigricans 132 051 004 810
P. digitatum 201 170 012 800
P. chrysogenum 100 057 003 800
P. citrinum 123 110 011 890
P. nelicum 210 215 015 912
P. granulatum 232 198 013 905
Fusarium solani 210 062 004 863
F. oxysporum 253 107 008 883

Correlation coefcient r between 1 and 2 097.

*Incubation temperature : 37C for Aspergillus spp. ; 30C for Penicillium and Fusarium spp.
Initial pH of the medium, 62.
, No zone produced.
Table 3 Colony diameter and zone
diameter after 18 h and L-
asparaginase activity with change in
pH of culture ltrate after 48 h of various
bacteria screened at 37C

Colony Zone Enzyme pH of the

diameter diameter activity culture
Bacterium (cm) (cm) (IU ml
1
) ltrate*

Bacillus subtilis 600 087 011 862

B. licheniformis 533 097 015 899
B. circulans 500 097 010 870
Escherichia coli JP3301 733 120 012 910
E. coli Y9010 () 000 690
Erwinia herbicola () 000 680
Enterobacter aerogenes () 000 690
Vibrio scheri () 000 700
Zymomonas mobilis () 000 680

* Initial pH of the medium 70. Correlation coefcient (r) between 1 and 2 was 108.
(), No growth ; , no zone produced.
1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 2326
26 R. GULATI ET AL.
Fig. 2 Zone (t) produced by L-
asparaginase activity of Bacillus licheniformis
using 0009% v/v phenol red. (a)
Control : modied M-9 medium without
asparagine (NaNO
3
as sole nitrogen
source). (b) Modied M-9 medium with 1%
w/v asparagine (sole nitrogen source)
duction of extracellular L-asparaginases from microorganisms.
formis (Fig. 2) were found to be good producers of L-aspa-
Agricultural and Biological Chemistry 36, 356361.
raginase (Tables 2 and 3). Adirect correlation existed between
Broome, J.D. (1961) Evidence that the L-asparaginase activity of
zone diameters and enzyme activities in culture broths. A
guinea pig serum is responsible for its antilymphoma effects.
high correlation coefcient (r) of 097 and 108 was found for
Nature 191, 11141115.
fungi and bacteria, respectively.
Capizzi, R.L., Bertino, J.R. and Handschumacher, R.E. (1970) L-
The present plate assay is advantageous as the method is
Asparaginase. Annual Review of Medicine 21, 433445.
quick and L-asparaginase production can be visualized
Cooney, D. and Handschumacher, R.E. (1970) L-Asparaginase and
directly from the plates without performing time-consuming
L-asparagine metabolism. Annual Review of Pharmacology 10,
assays (Wade et al. 1971 ; Arima et al. 1972 ; Imada et al. 421440.
Curran, M.P., Daniel, R.M., Guy, G.R. and Morgan, H.W. (1985)
1973). The incubation period required prior to selection of
A specic L-asparaginase from Thermus aquaticus. Archives in
an L-asparaginase producer is signicantly reduced. The
Biochemistry and Biophysics 241, 571576.
incubation period for bacteria and fungi was 18 h and 48 h,
De Jong, P. (1972) L-Asparaginase production by Streptomyces
while in broth studies it is 2448 h for bacteria and often
griseus. Applied Microbiology 23, 11631164.
exceeds 96 h for fungi (Imada et al. 1973). Thus, the plate
Imada, A., Igarasi, S., Nakahama, K. and Isono, M. (1973) Aspara-
assay is rapid, reliable and results correlate directly with those
ginase and glutaminase activities of microorganisms. Journal of
from broth studies.
General Microbiology 76, 8599.
Lewis, A.E. (1984) Biostatistics. pp. 8494, New York : Van Nos-
trand Reinhold Company Inc.
Mashburn, L.T. and Wriston, J.C. (1964) Tumor inhibitory L- ACKNOWLEDGEMENTS
asparaginase from Escherichia coli. Archives in Biochemistry and
The authors acknowledge with thanks the technical assistance
Biophysics 105, 450.
of Mrs Meena Singh, Mr Mustafa Hussain and Mr Rajiv
Saxena, R.K. and Sinha, U. (1981) L-Asparaginase and glutaminase
Chawla, JTPA, UDSC, New Delhi, during the course of the activities in the culture ltrates of Aspergillus nidulans. Current
Science 50, 218219. present investigation and preparation of this manuscript.
Wade, H.E., Robinson, H.K. and Philips, B.W. (1971) Asparaginase
and glutaminase activities of bacteria. Journal of General Micro-
biology 69, 299312.
REFERENCES
Wriston, J.C. and Yellin, T.O. (1973) L-Asparaginase : a review.
Advances in Enzymology 39, 185248. Arima, K., Igarasi, S., Nakahama, K. and Isono, M. (1972) Pro-
1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 2326