This document describes a study that evaluated a direct screening method for fungal microorganisms that produce the enzyme periplasmic amidohydrolase (L-asparaginase). Five fungal strains were grown in modified Czapex Dox medium containing the pH indicator phenol red at different concentrations. After overnight incubation, zones of pink color formation around the fungal colonies indicated L-asparaginase production due to increased pH. Strain F1 exhibited the largest zone of 1.62 cm in the 0.1% phenol red concentration, demonstrating it as the most potent producer of the enzyme. This simple and rapid screening method within 18 hours can help identify fungal strains for their ability

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Available Online at: www.ijrpp.com Print ISSN: 2278 - 2648
_________________________________
* Corresponding author:
R.D.Shailima Vardhini,
Head, Department of Biochemistry,
St. Mary’s College, Yousufguda,
Hyderabad - 500045,
Andhra Pradesh, India.
Email address: shailima.rampogu@gmail.com
Online ISSN: 2278 - 2656
International Journal of
Research in Pharmacology and
Pharmacotherapeutics
(Research article)
EVALUATION OF THE FUNGAL MICROORGANISMS FOR
PERIPLASMIC AMIDOHYDROLASE
*1R.D.Shalima Vardhini, 2Prasad Ekke
*1 Department of Biochemistry, St. Mary’s College, Yousufguda, Hyderabad-500045, Andhra
Pradesh, India.
2Department of Biochemistry, Celesta Research Lab, Hyderabad, 500038, A.P. India.
_________________________________________________________________________
ABSTRACT
The periplasmic amidohydrolase was found to have a host of medical applications, especially against Acute
Lymphoblastic Leukemia (ALL). The application of this enzyme was also seen in the food processing industries
by reducing the formation of acrylamide, a cancer agent. The present study evaluates a direct one step method of
screening the fungal L-Asparaginase producers using the modified Czapex Dox medium.
KEY WORDS: Periplasmic Amidohydrolase, Antitumor, fungal screening method, L-Asparaginase.
______________________________________________________________________________________________
INTRODUCTION
L-Asparaginase (L-Asparagine amidohydrolase
E.C.3.5.1.1)1 constitutes one of the most
biomedically important groups of therapeutic
enzymes that catalyzes the hydrolysis of L-Asparagine
into Aspartic acid and Ammonia 2. The
need of anti leukemia and anti lymphoma agents is
far greater than any other therapeutic enzymes of
which L-Asparaginase contributes one third 3. The
enzyme L-Asparaginase gained importance for its
remarkable properties in pharmaceutical and
commercial industries. Different types of L-Asparginases
were screened and isolated from
different microbial species and were studied for
their different applications, particularly as
therapeutic agents in treatment of certain types of
human cancers, in particular, with the
lymphoblastic leukemia 4,5.
L-Asparaginase is used as an immunosuppressive
agent as it was observed in some immunological
responses 6. Some of the L-Asparaginase
preparations showed remarkable anti tumor
properties. Guinea pig serum showed to inhibit
certain spontaneous and radiation induced leukemia
in mice as reported by Broome 7. Kidd was the first
to report this in 1953 8. L-Asparaginase is also
known as Kidrolase, Elspar, Crasnitin, Leunase,
and Colaspase 9 by its trade name.L- Asparagine is
a nutritional requirement of both normal cells and
cancer cells10.
Lymphatic cells require the large amount
Asparagines to keep up with their rapid, malignant
growth for which they use both Asparagine from
the diet as well as what they can make themselves,
to a very limited extent, to satisfy their large
asparagine demand. Normal cells do not require

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more Asparagine to survive as they can make the
required asparagine internally by the enzyme
asparagine synthetases. Unlike the normal cells, the
lymphatic cells die rapidly if the dietary asparagine
is cut off as these cells cannot synthesize enough to
meet their abundant demand as the enzyme
asparagine synthetases is present at low levels 11.
The drug acts by catalysis or degrades the
asparagine into aspartic acid and ammonia. The
aspartic acid thus formed is converted to fumaric
acid in the urea cycle. This can also be converted
into one of the intermediate products of the TCA
cycle, the oxaloacetic acid. L-asparaginase hence is
very useful in maintaining the levels of amino acids
and nitrogen balance within the cells12.
The important enzyme L-Asparaginase is not only
used as therapeutic drug/agent but also as a food
processing agent13. It is used to prevent the
formation of acrylic amide, which is a carcinogen
present in starchy food items. Asparagine, present
in starchy food items like snacks and biscuits will
convert into acrylic amides upon baking [JECFA]
above 120 o C by Millard’s reaction. On adding L-Asparaginase
to food items before baking,
asparagine is converted into another amino acid,
aspartic acid and ammonia leading to the depletion
of asparagines resulting in the reduced formation of
acrylic amide in food items 14. L-Asparaginase
isolated from two bacterial sources (E.Coli and
Erwinia Caratovara) are widely used for the
treatment of acute lymphoblastic leukemia 15, 16.
Many microorganisms, fungi and yeast had proven
to be the best producers of L-Asparaginase. There
are only a few studies on L-Asparaginase
production by fungi 17. It was observed that
eukaryotic microorganisms like yeast and fungi had
a potential for L-Asparaginase production 18, 19.
The enzyme is routinely screened using Nessler’s
reagent 20. As this procedure is lengthy and time
consuming, there is still a need to develop a rapid
procedure. This obstacle was overcome by Gulati’s
rapid plate assay method 21. It was generally
observed that L-Asparaginase production was
accompanied by an increase in pH of the culture
filters 22. This was devised by incorporating a pH
indicator, phenol red in medium containing
asparagine as the sole nitrogen source. Phenol red
at acidic pH is yellow and at alkaline pH turned
pink around the L-Asparaginase producers. By
keeping this as a standard, the aim of the present
experiment is to screen the fungal microorganisms,
by a fast, single, and one step method using basic
nutrients (modified Czapex Dox media) and to
observe the results within 18 hours.
The mechanism of the enzyme23 action can be
explained by the following figure.
MECHANISM OF THE ENZYME ACTION
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MATERIALS AND METHODS
Five fungi (F1, F2, F3, F4, F5) used for the study
were isolated from soil samples. The dilution plate-method
was employed for the isolation of fungal
strains 24. The media used is the modified Czapex
Dox media which consist of minimum salts and the
pH is adjusted to 6.8. 50ml of each of the medium
was taken in three different petri plates.
The dye was prepared by using 2% of the phenol
red dye stock in ethanol with the pH was adjusted
to 7.0 with standard 1 mol-1 NaOH. The stock
ranged from 0.05ml to 0. 5ml. Different
concentration of the dye i.e. 0.001%, 0.01% and
0.1% were added to 50ml of the media taken in 3
different petri dishes. One control plate devoid of
the phenol red dye was also maintained to evaluate
the simple screening method to identify the fungi
capable of producing L-Asparaginase. After
standardization, the plates were incubated at 37o C
overnight.
Table: 1 Media Composition
S.No Chemicals Concentration g/lit
1 Glycerol 6
2 L-Asparagine 4
3 KCl 1
4 MgSo4 2
5 KH2Po4 3
6 Agar 20
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Results
After overnight incubation the plates were
analyzed for pink zone formation against the
control. The five fungal organisms showed
different dimensions of pink zone corresponding to
different concentration of the dye.
Fig-1: Control Fig-2: Pink Zone

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Table- 2: Diameter of pink zone.
S.No Phenol Red Concentration Diameter of Pink Zone in Cm
F1 F2 F3 F4 F5
1. 0.001% 0.86 0.68 0.90 0.3 0.45
2. 0.01% 1.03 0.83 0.96 0.69 0.80
3. 0.1% 1.62 0.98 1.09 0.92 0.98
Discussions
In the present study, we could successfully evaluate
a method for screening the fungal organisms that
produce L-Asparaginase in much less time and
using minimum nutrients. This process hence
would serve as a direct screening method for fungal
L-Asparaginase producers.
Acknowledgements
I thank the Management and the Principle St.
Mary’s College, Yousufguda, Hyderabad for their
continuous support and encouragement. I also
would like to thank Mr. K. Onesmies Elbert and
Celesta Research Lab, Hyderabad.
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