A PROJECT REPORT

ON

Isolation, characterization and production of

L-Asparaginase from Actinomycetes.

Submitted in partial fulfillment for the award of the degree

of
Bachelor of Science in Biotechnology

By
Mr. Hashmi Syed Shaheer

Under the guidance of

Ms. Syed Naaziya A.

Mahatma Gandhi Mission’s

College of Computer Science & IT,
Department of Biotechnology & Bioinformatics
MGM Campus, Nanded-431605

Affiliated to

Swami Ramanand Teerth Marathwada University,

Nanded-431605

2023-24
 Mahatma Gandhi Mission’s
College of Computer Science & IT,
MGM Campus, Nanded 431605

Certificate
This is to certify that the project entitled “Isolation, characterization and
production of L-Asparaginase from Actinomycetes” by Mr. Hashmi Syed
Shaheer (Exam Seat No. BZ15107) submitted in partial fulfillment of the
requirements for award of degree of Bachelor of Science in Biotechnology of
Swami Ramanand Teerth Marathwada University, Nanded 431605 during the
academic year 2023-24, is a bonafide record of work carried out under my
guidance and supervision.

Ms. Syed Naaziya A.

Guide

Dr. Cherekar M. N. Prof. Shirish L. Kotgire

Head Of Department Principal

Date: / /
 Declaration
I hereby declare that the Project report entitled “Isolation, characterization and
production of L-Asparaginase from Actinomycetes” has been completed in
Department of Biotechnology & Bioinformatics, MGM’s College of Computer
Science & IT, Nanded and Submitted to Swami Ramanand Teerth Marathwada
University Nanded, under the guidance of Ms. Syed Naaziya A. for the award of
degree of Bachelor of Science in Biotechnology.
This report comprises only my original work and has not been submitted for any
other degree/ diploma to any university/ institute. Due acknowledgement has been
made in the text to all other material used

Mr. Hashmi Syed Shaheer

Candidate

Date: / /
Place: Nanded
 Acknowledgement

My heartfelt thanks to college Principal Prof. Shirish L. Kotgire whose valuable

support lead me to learn much more than the curriculum. I am elevated to avail
myself of this opportunity to express my deep sense of my gratitude and
indebtedness toward Dr. Cherekar M. N. Head of Department of Biotechnology
& Bioinformatics and my guide Ms. Syed Naaziya A. whose constant
encouragement and guidance has helped throughput the course of this dissertation
work.

I am also indebted to S.R.T.M University for keeping this dissertation as a part of

syllabus, which gave me great opportunity and knowledge.

I have a special thanks to all the faculty member of department for their constant
encouragement and support throughout this duration. I am also grateful to
librarians, for their help during my project work.

Mr. Hashmi Syed Shaheer

Candidate

Date: / /
Place: Nanded
INDEX -

Sr.No Topic Page No.

1. INTRODUCTION 1-2

2. APPLICATIONS 3-4

3. REVIEW OF LITRATURE 5-6

4. AIM AND OBJECTIVES 7

5. MATERIALS AND METHODOLOGY 8-12

6. RESULT AND DISCUSSION 13-15

7. CONCLUSION 16

8. REFERENCES 17-19
LIST OF FIGURE –

Sr.No Figures Page No.

1. MEDIA PREPARATION –NUTRIENT AGAR 11

2. MODIFIED M9 MEDIUM 11

3. SERIAL DILUTION OF SOIL SAMPLE 11

4. ISOLATED COLONIES FROM SOIL SAMPLE 11

5. M9 MEDIUM PLATES 11

6. IDENTIFY L-ASPARGINASE BY USING PHENOL RED 12

INDIACTOR

7. BIOCHEMICAL TEST 12
LIST OF TABLES –

Sr.No Tables Page No.

1. MORPHOLOGICAL CHARACTERIZATION 14

2. BIOCHEMICAL CHARACTERIZATION 14
ABSTRACT

L-Asparginase is an anti-neoplastic agent used in lymphoblastic leukaemia chemotherapy. Soil

microbial isolates were screened for potential producers of L-Asparginase using phenol red
indicator growth medium and the microbes producing the largest hydrolysis zone was selected. It
can isolate and characterised by biochemical tests and it was belonged to Bacillus sp. The present
study reports production and characterization of purified L-Asparginase from actinomycetes
isolate. L-Asparginase is an enzyme which is chemotherapeutic agent used in cancer treatment,
in food industry the enzyme asparginase is used to diminish the quantity of acrylamide that is
present in fried and baked starchy foods and it is carcinogenic in nature.

Keywords - L-asparaginase, Bacillus, Actinomycetes, Purification, Anticancer

INTRODUCTION

L-Asparginase is the enzyme which converts L-Asparginine to aspartic acid and ammonia, has
been used as a chemotherapeutic agent (Fisher and Wray,2002). It has increased attention in
recent years for it’s anti carcinogenic potential (Manna et al,1995). Clinical action of this enzyme
is attributed to the reduction of L-Aspargine; tumour cells unable to synthesis this amino acid are
selectively killed by L-Aspargine deprivation. Though several L-asparaginases of bacterial origin
have been developed and their potential usage in clinical trials have been studied to prevent the
progress of L-asparagine -dependent tumors, mainly, the success hitherto has been rather limited,
and most other treatments must be interrupted due to severe side effects and immunological
reactions in the patients.
In most of the microorganisms, L-Asparginase accumulates as an intracellular product. Enzyme
localiza-tion in bacteria has been carriedout using various methods (Marr, 1960). In L-
asparaginase producing strains, the existence of both periplasmand cytoplasm enzyme have been
reported (Schwartz etal., 1966). The study on the localization of any enzyme
plays a vital role in the development of bioprocess.
L-Asparaginase (E.C. 3.5.1.1) is an amidohydrolase that is found in a wide range of organisms
i.e. plants, microbes and animals that catalyzes the hydrolysis aspargine into aspartic acid and
ammonia. It is difficult to extract it from plants and animals therefore microorganisms are
assessed as potential sources for the production L-Asparaginase enzyme (Arima et al 1972) .
This enzyme has significant applications in the therapeutics and food industry. In therapeutics L
asparaginase act as anti-tumor agent and show tumor inhibitory properties. Acute lymphoblastic
leukemia (ALL) is mainly treated by Asparaginsae. Normal body cells can produce L-asparagine
by using asparagine synthetase enzyme, while certain malignant cells are not able to synthesize
aspargine and require an external source of L-asparagine for their growth.
In All treatment with L-Asparaginase, in the body of the patient all the circulating asparagine is
hydrolyzed into ammonia and aspartic acid as a result of which absorption of asparagine by
tumor cells is prevented and hence divesting the tumor cells of their extracellular L-asparagine
source.

The cancerous cells cannot carry out the de novo asparagine synthesis (Killander et al., 1976).
Anaphylaxis, thrombosis, pancreatitis, coagulation abnormality, liver dysfunction,
hyperglycemia, and cerebral dysfunction are side effects that are associated with L-Asparaginase
therapy. L asapaginase can evoke immune response in which antiasparaginase antibodies are
formed in the body. This enzyme also plays role in food industry to stop the production of a
carcinogen named as acrylamide at high temperatures such as frying or baking of starchy food items.
L- asparagine is an essential amino acid required for the growth of normal cells, L- asparagine
can be obtained by either diet or by the synthesis via asparagine synthetase located on the
chromosome number 7 in humans. L-Asparaginase is also use glutamine as substrate but it have
less affinity for glutamine than L-Asparagine.Glutamine donates an amino group to L-asparagine
synthetase for de novo pathway for asparagine biosynthesis, therefore Asparaginase decreases
the level of glutamine and also help in supporting the reduced level of asparagine and so
contributes the chemotherapic effect of L-Asparginase (Zeidan et al., 2009).
L-Asparginase can also be absorbed from external sources.Cancerous cell lack the enzyme
aspergine synthtase and hence are unable to synthesize their own aspargine. Hence the cancerous
cells are dependent on L-asparagine present in the blood of the patient. Tumor cells require an
enormous quantity of L-asparagine for their rapid growth. L-asparagine is limited in the
circulating pool when the enzyme L-asparaginase is present resulting in the death of tumor cells.
This approach helps in the use of L-asparaginase as an anti-cancerous agent.
In addition to clinical applications of L-asparaginase have potential application in food industry.
It can be used in eliminating acrylamide formation during frying of starchy foods. Amino acids
and reducing sugars present in starchy food, when heated, give a characteristic aroma and flavor.
When L-asparagine and reducing sugars react, it leads to the formation of acrylamide. Addition
of L-asparaginase enzyme before baking or frying the food leads to conversion of L-asparagine
to aspartic acid. Thus, the enzyme reduces the risk of acrylamide formation in food. (Amena,
2002)
APPLICATIONS

Role of L-Asparaginase in Amino Acid Metabolism

For the production of different amino acids like lysine, threonine, and methionine are
biosynthesized by L-Asparaginase. These amino acids are known as aspartic family amino acids.
In addition to Kreb’s cycle aspartic acid is synthesized by L-asparaginase. This aspartic acid is
further used as a direct precursor of threonine and lysine.

Role of L-Asparaginase in Food industry

In food processing L-asparaginase is also extensively used enzyme. Current advances in food
technology revealed that a crystalline solid that is colorless and odorless in nature is produced by
Millard reaction, is known as acrylamide (IUPAC name is 2-propenamide). Millard reaction
occurs when starchy foods are fried or baked at 120 °C. Acrylamide is act as neurotoxin and
considered as carcinogenic agent and therefore it is harmful for human.
Asparaginase can be used in food industry to reduce acrylamide formation in starchy foods. To
avoid acrylamide production pre-treatment of starchy foods such as potato and bread dough is
done with L-Asparaginase. Recombinant asparaginase from Aspergillus oryzae was tested on
various food items like ginger biscuits, semisweet biscuits, sliced potato chips, the results
showed 34–92% reduction in acrylamide content. In past, some other researches have dealt with
this application of L-Asparaginase that can decrease the side effects of acrylamide having foods
without damage their features.
Anticancer drug

L-Asparaginase plays a vital role in treatment of Acute Lymphoblastic Leukemia with vincristine
and glucocorticoid. L-asparaginase is a famous chemotherapeutic agent which is used in
combination with other drugs to treat certain malignancies such as ALL (mostly in childs), acute
myelocytic leukemia. L-asparagine, act as an important amino acid required for synthesis of
protein, for various tumor cells and cell growth, whereas L-asparaginase converts L-asparagine
to aspartate that is why presence of L-Asparaginase destruct the growth factors of malignant cells
as a result of which depletion of asparagine occur and malignant and ultimately tumor cells die.

Role of L-Asparaginase in Biosensor

To study the levels of asparagine in leukemic patients and food industry biosensors of L-
Asparaginase are developed. A number of spectroscopy techniques like XPS, XRD, TEM, and
SEM are recently used for the analysis of L-asparagine, but the main disadvantage is their high
cost. As compare to these expensive techniques, biosensor technology can be a reliable, cheap,
and user-friendly approach. The mechanism of action of the biosensor is based on asparaginase
activity, ammonium ions produced from the hydrolysis of asparagine cause a change in pH
resulting in the change of color and absorption. (Basha N.S. ,2009)
REVIEW OF LITRATURE

In recent times marine microbes are consider as to use for the production of bioactive
compounds. A potential source of actinomycetes is marine environment from where these
microorganisms can be isolated and used for antibiotics and bioactive compounds production.
Actinomycetes are synthsize L-Asparaginase that kill malignant cells and show cytotoxic effects
on myelogenous leukemia (Dhevagi & Poorani, 2006). Six strains of actinomycetes streptomyces
aureofasciculus, Streptomyces chattanoogenesis, Streptomyces hawaiiensis, Streptomyces
orientalis, Streptomyces canus, and Streptomyces olivoviridis were screened in marine fish (sahu
et al., 2007).
In 2011 Dharmaraj reported L-Asparaginase of marine actinomycetes where purified L-
Asparaginase exhibited 78.88 IU/mg specific activity at pH 8. L-Asparaginase formation from
various actinomycetes like Streptomyces ABR2 and Streptomyces albidoflavus have been
explored (Sudhir et al., 2012).
L-asparagine, act as an important amino acid required for synthesis of protein, for various tumor
cells and cell growth, whereas L-asparaginase converts L-asparagine to aspartate that is why
presence of L-Asparaginase destruct the growth factors of malignant cells as a result of which
depletion of asparagine occur and malignant and ultimately tumor cells die (Salzer et al., 2014).
L-asparaginase is present in plants, animals, and microbes, but only selected L-asparaginase
from E. chrysanthemi and E. coli was permitted for chemotherapy for ALL treatment (Verma et
al., 2012). However latest research shows that autophagy is associated with L-asparaginase
treatment, so it is recommended to use anti-autophagy drug in combination with L-asparaginase
for the treatment of ALL. Inhibition of autophagy enhances L-asparaginase-induced cytotoxicity
and overcomes the acquired resistance to L-asparaginase in ALL cells (Takahashi et al., 2017).
Study of morphology of actinomycetes
Studies have been made to understand each aspect of actinomycetes growth and survival under
various condition. In the past, Shirling and Gottlieb (1966) examined isolates for pigmentation,
colour of aerial mycelium and related morphological features. Abbas using the same method, did
grow cultures for 4 weeks and observation were made at weekly intervals for morphological
properties of colony, cells and spores (Abbas 2006).
Even variation of the media for growth was done for morphological study, like Sahin et al.,
(2002) chose oatmeal agar as the medium of growth and visual examination on the basis of aerial
spore mass colour, substrate mycelia pigmentation and colouration of media by diffusible
pigments was done, and at the same time peptone-yeast extract in iron agar plates were used to
observe production of any dark colored melanin pigments Also there is confinement of study and
investigation of fine structure of germinating spores to Streptomyces genera as put forth by
Kalakoutswl and Agre (1973). Hence, there has been suggestions for need of modification of
colour grouping method, and objective color determination method (Pridham 1965).

Isolations within India

Screening of actinomycetes have been undertaken for many attributes like four different strains
from laterite soil in Guntur region of Andhra in India (Kavitha et al., 2010).15 strains of
actinomycetes were isolated from Lucknow in Uttar Pradesh (Pandey et al., 2011) Five
actinomycetes strains of Isoptericola variabilis was isolated from 25 samples of Cauvery river
basin. (Muthu et al., 2013). six strains (in total) of actinobacteria were isolated from the soil
samples collected from various arid and semi regions around Jaipur, Jhunjhunu, Sikar of
Rajasthan (Masand et al., 2015).10 isolates with distinct respective morphology were isolated
and purified on starch casein agar from forest soils of Mahabubnagar district, Andhra Pradesh by
Balakrishna et al., (2012)

Throughout the world

Similarly, other investigations carried through-out the world shows evidence of the ability of
actinomycetes to inhabit many parts of the world. Heng et al., (2015) have isolated 110
Streptomyces isolates from samples of peat soil of Malaysia. A thermophilic actinomycetes
Thermasporomyces composti gen. nov., sp. nov. was isolated from compost (Yabe et al., 2011).
31 strains of potential antibiotic producing actinomycetes from sediments as well as water of
Tana Lake, Ethiopia were isolated by Gebreyohannes et al., (2013). 60 actinomycetes isolates
were isolated from soil samples that were collected from different selected locations of Saudi
Arabia. (Ababutain et al., 2012). A total of foryt four strains of actinomycetes were isolated from
Caspian Sea sediments at a depth of 5-10 m (Mohseni et.al. 2013).
AIM AND OBJECTIVES

AIM – Isolation, characterization and production of L-Asparaginase from Actinomycetes.

OBJECTIVES –

1-Collection of soil sample

2-Isolation of Actinomycetes
3-Screening of Actinomycetes for production of L- asparginase
4-Characterization of Actinomycetes
5-Production of L- asparginase
MATERIALS & METHODOLOGY

1.Sample -
Soil sample (1gram)
Actinomycetes Antibiotic
2.Glasswares-
Conical Flask
Beaker
Petriplates
Test tubes
3.Instruments –
Autoclave
Cyclomixture
Micropipette
Incubator
4.Chemicals –
Ethanol
Sodium hydroxide
Indole
Methyl red
Voges Proskauer
Citrate
5.Media -
Nutrient agar media
Modified M9 medium (KH2PO4 , L-aspargine , MgSO4 , CaCl2 , Glucose , agar )

6.PH Indicators –
Phenol Red Indicator
 1. Isolation of microorganism

Soil sample was collected from root nodules. The collected soil sample were dried at room
temperature and made into fine powder with the help of mortor and pestle under the sterile
condicition. Then tenfold serial dilution was prepared with one gram of soil sample using
distilled water. Diluted sample were spread on modified M9 Medium (KH2PO4 0.3% L-
aspargine 6.0% , MgSO4 0.15% , CaCl2 1% , Glucose 5% , Agar 2.5%)
To isolate the Actinobacteria the serially diluted sample was spresd on M9 medium plate.The
medium was supplemented with streptomycin as a antibacterial , antifungal agents to avoide
unwanted bacterial colonies. After inoculation the petri plates were stored in incubator at 37˚C
for 10 days. After incubation discrete colonies were observed.

2. Identification of Actinobacteria species

Morphological & biochemical characterization was performed for identification of isolated
actinobacterial strains. Biochemical test such as a IMViC test (indole test, methyl red test, voges
proskaur test and citrate utilization test were performed. Microscopic identification was done by
gram’s staining method.

3. Screening of Actinobacteria for L-Asparginase activity

The isolates were screened for L-Asparginase production. Modified M9 medium (Na2HPO4 0.3
% , KH2PO4 0.3% , L-aspargine 6.0% , MgSO4 0.15% , CaCl2 1% , Glucose 5% , Agar 2.5%)
was used for plate assay method for L-Asparginase production . A 2.5% of stock solution of
phenol red pH indicator was added to M9 medium and maintain Ph 6.8 Medium L-Aspargine act
as a source of carbon nitrogen and also as substrate. The media was incubated at 30˚C for 5 days
.The active strains cleave L-Aspargine to aspartic acid and ammonia leading to alkaline pH
which show changes in color of medium from yellow to dark pink or red. Colonies with pink
zone were consider as a positive strains for L-Asparginase and selected for further studies.
4. Production of L-Asparginase and quantitative assay

L-Asparginase production by the isolate was carried out by submerged fermentation. The
sterilized production media (Na2HPO4 0.3 %, KH2PO4 0.3% , L-aspargine 6.0% , MgSO4
0.15%, CaCl2 1% , Glucose 5%, Agar 2.5%) was inoculated with a loop-full of bacterial culture
and was incubated in a rotary incubator at 30˚C for 48hrs.

5. Purification of L-asparaginase

At a fixed interval, known volume of samples was withdrawn and centrifuged in a cooling
centrifuge (Eppendorf) at 8000 × g for 15 minutes at 4°C. The supernatant served as the crude
enzyme preparation. Enzyme was assayed using the to 0.5 ml of crude enzyme, 0.5 ml of 1 mM
L-asparagine and 1 ml of 0.5 M Tris-HCl buffer (pH 6) and incubated at 37°C for 10 min. The
reaction was stopped by adding 1 ml of phenol reagent, 1 ml of 1N sodium hydroxide. The
mixture was kept for 15 min. Absorbance was recorded at 600 nm using ultraviolet-visible
spectrophotometer using appropriate blank. (El-Bessoumy AA , 2004)
Media preparation –Nutrient Agar Modified M9 medium

Serial Dilution of soil sample Isolated colonies from soil sample

M9 Medium plates
Identify L-Asparginase by using phenol red indiactor

Biochemical test
RESULTS AND DISCUSSION

Isolation of actinobacteria

Actinomycetes were isolated from different soil sample, 5 to 6 colonies were isolated & screened
for L-Asparginase production using modified M9 medium with phenol red pH indicator. Due to
the production of ammonia the pH of the medium increased. Hence the addition of pH indicator
changed its color and this forms the basis of assay. Phenol red was used as pH indicator in the
medium. Plates containing phenol red were yellow at lower pH value and changed to dark pink
at higher pH value. Hence, a dense dark pink color is produced if microorganism produces L-
asparaginase.

Characterization of actinomycetes

5 to 6 colonies were isolated, produced i.e white, wrinkled colonies without pigmentation.
Growth was observed after incubation. The strains were identified as actinobacterial species by
morphological characterization, biochemical characterization and enzymatic properties. (Ravi Av
2016)
Table No.1
Morphological characterization
Characterization Result

Size 1.2 cm

Shape Round

Surface Rough

Color Chalk white

Opacity Opaque

Gram character Gram Positive

Margin Regular

Table No.2
Biochemical characterization
Characteristics Result
Gram’s nature Positive
Indole Negative
Methyl Red Negative
Voges-Proskauer Positive
Citrate Positive
Carbon utilization Result
Glucose Positive
Fructose Positive
Maltose Positive
Lactose Positive
Sucrose Positive
Screening of L-asparginase activity

Actinobacterial isolates were screened for ability to produce L-Asparginase enzyme. The isolates
show s positive result exhibiting pink zone in rapid plate assay. All the positive strains were
considered as active strains. It produce high enzymatic activity in rapid plate assay method based
on the zone.

Production and Purification of L-Asparaginase

L-Asparginase production by the isolate was carried out by submerged fermentation. The
isolated Streptomyces sp. was inoculated in production media and incubated for 120 hours. It was
observed that Streptomyces sp. had a highest enzyme activity. (El-Bessoumy AA , 2004)
CONCLUSION

The present study that the isolates of actinomycetes were screened for L-Asparginase production.
L-Asparginase with maximum enzymatic activity at optimized conditions under selected
parameters. In conclusion, L-Asparginase from different microbial sources shows such properties
which makes it an important enzyme in both pharmaceuticals and food industries. Further studies
should be conducted for reducing cost of the enzyme production. It should be done by increasing the
yield of L-asparaginase through optimization of the production process and by improvement of
strains. Although bacterial L-Asparaginase is clinically used for all treatment and it also induces
certain adverse reactions. Novel producers of L-asparaginases should be explored which may
have less side effects while clinical use.
REFERENCES

1. El-Bessoumy, A.M. Sarhan and J.Mansour, 2004. Production, isolation, and purification of L-
Asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. IJBMB.,
37(4): 387–393.

2. Amena, S., N. Vishalakshi,M.Prabhaka, A. Dayanand and K.Lingappa.2010. Production

purification and characterization of L-asparaginase from Streptomyces
gulbargensis.BJM., 41: 173–178.
Abdel-Fattah YR, Olama ZA (2002). L-asparaginase production by Pseudomonas aeruginosa in
solid-state culture: evaluation andoptimization of cultureconditions using factorial designs.
ProcessBiochem., 38: 115-122.

3. Amena S, Vishalakshi N, Prabhakar M, Dayanand A, Lingappa K(2010). Production,

purification and characterization of Lasparaginasefrom Streptomyces gulbargensis.
Brazil J. Microbiol.,41: 173-178.

4. Asselin BL, Lorenson MY, Whitin JC (1993). Comparativepharmacokinetic studies of three

asparaginase preparations. J. Clin.Oncol., 11: 1780-1786.

5. Cornea CP, Lupescu I, Vatafu I, Savoiu VG, Campeanu GH (2000).

Isolation and characterization of some L-asparaginase producingrecombinant Escherichia coli
strains. Roum. Biotechnol. Lett., 5: 471-478.

6. Dhevagi P, Poorani E (2006). Isolation and characterization of Lasparaginasefrom marine

actinomycetes. Ind. J. Biotechnol., 5: 514-520.

7. Distasio JA, Salazar AM, Nadji M, Durden DL (1982). Glutaminase-freeasparaginase from

Vibrio succinogenes: an antilymphoma enzymelacking hepatotoxicity. Int. J. Cancer., 30: 343-
347.
8. Eden OB, Shaw MP, Lilleyman JS, Richards S (1990). Non-randomizedstudy comparing
toxicity of Escherichia Coli and Erwiniaasparaginase in children with leukaemia. Med. Pediat.
Oncol., 18:497-502.

9. Egorova OV, Nikolayeva VM, Suzina NE, Donova MV (2004). Localization of 17_
hydroxysteroid dehydrogenase in Mycobacteriumsp. VKM Ac-1815D mutant strain. J. Steroid.
Biochem. Mol. Biol., 94:519-525.

10. El-Bessoumy AA, Sarhan M, Mansour J (2004). Production, isolation, and purification of L-
asparaginase from Pseudomonas Aeruginosa50071 using solid-state fermentation. J. Biochem.
Mol. Biol., 37: 387-393.

11. Fisher SH, Wray Jr LV (2002). Bacillus subtilis 168 contains two differentially regulated
genes encoding L-asparaginase. J. Bacteriol.,184: 2148-2154.

12. Ho PPK, Milikin EB, Bobbitt JL, Grinnan EL, Burck PJ, Frank BH, BoeckLD, Squires RW
(1970). Crystalline L-asparaginase from Escherichiacoli B. J. Biol. Chem., 245: 3708-3715.
Khamna S, Yokota A, Lumyong L (2009). L-asparaginase production byactinomycetes isolated
from some Thai medicinal plant rhizospheresoils. Int. J. Integrative Biol., 6: 22-26.

13. Laemmli UK (1970). Cleavage of structural proteins during theassembly of the head of
bacteriophage T4. Nature., 227: 680-685.

14. Ravi Av, Kanapala S, Sai Babu VN, Bodaiah B, Poda S. Partial purification, characterization
and optimization of anti-leukemic enzyme L-Asparaginase from mangrove soil actinobacteria. J
Pharm Res 2016;10:502-11.
15. Sahu MK, Sivakumar K, Poorani E, Thangaradjou T, Kannan L. Studie on asparaginase
enzyme of actinomycetes isolated from estuarine fishes. J Environ Biol 2007;28:465-74.
16. Kavitha A, Vijayalakshmi M. A study on L-asparaginase of Nocardia levis MK-VL_113.
ScientificWorldJournal 2012;2012:160434.
17. Basha NS, Rekha R, Komala M, Ruby S. Production of extracellular anti-leukaemic enzyme
L-Asparaginase from marine actinomycetes by solid state and submerged fermentation:
Purification and characterization. Trop J Pharm Res 2009;8:353-60.
18. Kiranmayi MU, Poda S, Vijayalakshmi M. Production and optimization of L-asparaginase by
an actinobacterium isolated from nizampatnam mangrove ecosystem. J Environ Biol
2014;35:799-805.
19. Mohamed SA, Elshal MF, Kumosani TA, Aldahlawi AM. Purification and characterization
of asparaginase from Phaseolus vulgaris seeds. Evid Based Complement Alternat Med
2015;2015:309214.
20. Bhagat J, Kaur A, Chadha BS. Single step purification of Asparaginase from endophytic
bacteria Pseudomonas oryzihabitans exhibiting high potential to reduce acrylamide in processed
potato chips. Food Bioprod Process 2016;99:222-30.