ASPARAGINASE

BY:
T.CH.ROHITHA
K.SREEJA

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 ABSTRACT

L-asparaginase is an important enzyme as therapeutic agents used in combination with other

drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes
strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it
was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the
GenBank database under accession number KJ467538. L-asparaginase was purified from the
crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange
chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified
enzyme were carried out. The optimum pH, temperature and incubation time for maximum
L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum
substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were
0.01007 M and 95.08 Uml(-1)min(-1), respectively. The half-life time (T1/2) was 184.91 min
at 50 °С, while being 179.53 min at 60 °С. The molecular weight of the subunits of L-
asparaginase was found to be approximately 53 kDa by SDS-PAGE analysis. The purified L-
asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-
fold. The present work for the first time reported more information in the production,
purification and characterization of L-asparaginase produced by newly isolated
actinomycetes Streptomyces fradiae NEAE-82.

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 Index

1.Introduction
a) History
b) Uses
i. Medical
ii. Food manufacturing
c) Side effects
d) Mechanism of action
i. As a food processing aid
ii. AS a drug

e) Regulations Amending the Food and Drug Regulations

f) Executive summary

2. Review of literature
3. Materials & Methods
a) Sample collection and isolation
b) Screening for enzyme product
c) Pure culture preparation
d) Production Media preparation and Inoculation
e) Gram’s Staining
f) Identification of Bacteria
g) Downstream processing
i. Centrifugation
ii. Salt Precipitation
iii. Dialysis
iv. Qualitative Study
v. Quantitative study
4. Results and Discussion
5. Conclusion
6. Reference

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 1.Introduction
Asparaginase is an enzyme that is used as a medication and in food manufacturing. As a
medication, L-asparaginase is used to treat acute lymphoblastic leukemia (ALL), acute
myeloid leukemia (AML), and non-Hodgkin's lymphoma. It is given by injection into a
vein, muscle, or under the skin. A pegylated version is also available. In food manufacturing
it is used to decrease acrylamide.
Common side effects when used by injection include allergic reactions, pancreatitis, blood
clotting problems, high blood sugar, kidney problems, and liver dysfunction. Use
in pregnancy may harm the baby. As a food it is generally recognized as safe. Asparaginase
works by breaking down the amino acid known as asparagine without which the cancer cells
cannot make protein.
Asparaginase was approved for medical use in the United States in 1978. It is on the World
Health Organization's List of Essential Medicines, the most effective and safe medicines
needed in a health system. The wholesale cost in the developing world is about US$42.00 per
10,000 IU vial. This amount in the United Kingdom costs the NHS 613.00 pounds. It is often
made from Escherichia coli or Erwinia chrysanthemi.

Asparaginase - Clinical data

a) History
The discovery and development of asparaginase as an anti-cancer drug began in 1953, when
scientists first observed that lymphomas in rat and mice regressed after treatment with guinea
pig serum. Later it was found out that it is not the serum itself which provoke the tumour
regression, but rather the enzyme asparaginase.
After researchers comparing different kinds of asparaginases, the one derived
from Escherichia coli and Erwinia chrysanthemi turned out to have the best anti-cancer

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ability. E. coli has thereby become the main source of asparaginase due to the factor that it is
also easy to produce in large amount.

b) Uses

i. Medical
E. coli strains are the main source of medical asparaginase. Branded formulations (with
different chemical and pharmacological properties) available in 1998 include Asparaginase
Medac, Ciderolase, and Oncaspar 5 (Crasnitin has been discontinued.) Spectrila is a new
recombinant E. coli asparaginase.
Asparaginase produced by Dickeya dadantii (formerly called Erwinia chrysanthemi) instead
is known as crisantaspase (BAN), and is available in the United Kingdom under the trade
name Erwinase.
One of the E. coli asparaginases marketed under the brand name Elspar for the treatment
of acute lymphoblastic leukemia (ALL) is also used in some mast cell tumorprotocols.
Unlike most of other chemotherapy agents, asparaginase can be given as an intramuscular,
subcutaneous, or intravenous injection without fear of tissue irritation.

ii. Food manufacturing

The most common use of asparaginases is as a processing aid in the manufacture of food.
Marketed under the brand names Acrylaway and PreventASe, asparaginases are used as a
food processing aid to reduce the formation of acrylamide, a suspected carcinogen, in starchy
food products such as snacks and biscuits.

c) Side effects
The main side effect is an allergic or hypersensitivity reaction; anaphylaxis is a
possibility. Additionally, it can also be associated with a coagulopathy as it decreases protein
synthesis, including synthesis of coagulation factors (e.g. progressive isolated decrease
of fibrinogen) and anticoagulant factor (generally antithrombin III; sometimes protein
C & S as well), leading to bleeding or thrombotic events such as stroke. Bone marrow
suppression is common but only mild to moderate, rarely reaches clinical significance and
therapeutic consequences are rarely required. Other common side effects include pancreatitis.
These side effects mainly attributes to the dual activity of L. Asparaginase as it can also
hydrolysis L. Glutamine to Glutamic acid and ammonia.

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 d) Mechanism of action

i. As a food processing aid

Acrylamide is often formed in the cooking of starchy foods. During heating the amino
acid asparagine, naturally present in starchy foods, undergoes a process called the Maillard
reaction, which is responsible for giving baked or fried foods their brown color, crust, and
toasted flavor. Suspected carcinogens such as acrylamide and some heterocyclic amines are
also generated in the Maillard reaction. By adding asparaginase before baking or frying the
food, asparagine is converted into another common amino acid, aspartic acid,
and ammonium. As a result, asparagine cannot take part in the Maillard reaction, and
therefore the formation of acrylamide is significantly reduced. Complete acrylamide removal
is probably not possible due to other, minor asparagine-independent formation pathways. As
a food processing aid, asparaginases can effectively reduce the level of acrylamide up to 90%
in a range of starchy foods without changing the taste and appearance of the end-product.

ii. As a drug
The rationale behind asparaginase is that it takes advantage of the fact that acute
lymphoblastic leukemia cells and some other suspected tumor cells are unable to synthesize
the non-essential amino acid asparagine, whereas normal cells are able to make their own
asparagine; thus leukemic cells require high amount of asparagine.[16] These leukemic cells
depend on circulating asparagine. Asparaginase, however, catalyzes the conversion of L-
asparagine to aspartic acid and ammonia. This deprives the leukemic cell of circulating
asparagine, which leads to cell death.

e) Regulations Amending the Food and Drug Regulations

Column I Column II Column III Column IV

Item
No. Maximum Level of
Permitted in or
Use
Additive Permitted Source Upon
(1) Good
(1) Bread; Flour; Whole
Manufacturing
wheat flour
Practice
Aspergillus niger ASP72; Aspergillus (2) Good
A.3 Asparaginase
oryzae(pCaHj621/BECh2#10) Manufacturing
Practice
(2) Unstandardized foods

f) Executive summary
Issue: The Food and Drug Regulations (“the Regulations”) control the sale and use of food
additives in Canada, listing the permitted food additives and how they may be used. Health

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Canada has received submissions from industry requesting that the Regulations be amended
to permit the use of the enzyme asparaginase from the following new genetically modified
sources of micro-organisms: Aspergillus oryzae(pCaHj621/BECh2#10) and Aspergillus
niger ASP72. The use of the enzyme asparaginase would be permitted in the production of
bread, flour, whole wheat flour and unstandardized foods, all at a maximum level of use
consistent with good manufacturing practice. Asparaginase is used in certain foods in order
to reduce the amount of the amino acid asparagine that is already present in foods. Under
specific cooking conditions, asparagine can react with certain carbohydrates in the food to
form acrylamide, a potential human carcinogen. Reducing the amount of asparagine in the
food prior to cooking or processing would reduce the amount of acrylamide that can be
formed. Enabling the use of asparaginase in food processing is part of the risk management
approach adopted by Health Canada to reduce consumers’ exposure to acrylamide from food
sources.

Description: The amendments enable the use of the enzyme asparaginase produced from the
genetically modified sources of micro-organisms as described above. The amendments
permit the use of this food additive in the manufacturing of unstandardized foods, and in the
production of bread, flour and whole wheat flour for which there are standards set out in
Division 13, Grain and Bakery Products, of the Regulations.

Business and consumer impacts: The amendments benefit consumers by reducing the
formation of acrylamide in some processed foods, thereby helping to protect their health and
safety. These amendments also benefit some food manufacturers by providing them with a
safe option to reduce acrylamide formation in certain foods without compromising the
quality, flavour or other characteristics of the food.

Domestic and international coordination and cooperation: The purpose for using
asparaginase in the manufacturing of food is to reduce the amount of asparagine in food,
thereby reducing the formation of acrylamide, dietary exposures to which have been
identified as a potential human health concern by the Joint Food and Agricultural
Organization (FAO) / World Health Organization (WHO) Expert Committee on Food
Additives (JECFA). (see footnote 2) JECFA has called for strategies to reduce exposure to
acrylamide. Health Canada’s scientists participated in the JECFA review of acrylamide.
Health Canada agrees with JECFA’s call for strategies to reduce exposure to acrylamide.
Health Canada’s risk management approach to reduce exposure to acrylamide involves
encouraging industry to develop reduction strategies. The use of asparaginase as described
above is consistent with Health Canada’s risk management approach by providing food
manufacturers with a safe option to reduce acrylamide formation in certain processed foods.
Other countries have already approved the use of the enzyme asparaginase. Asparaginase
from Aspergillus oryzae (pCaHj621/BECh2#10) is permitted for use in the United States,
Australia, New Zealand and Denmark, and complies with international specifications as it
received a positive evaluation at the 68th meeting of JECFA in June 2007. Asparaginase
from Aspergillus oryzae has been assigned by JECFA an acceptable daily intake (ADI) of
“not specified,” confirming that asparaginase does not pose a hazard to the health of the
consumer for its intended purpose in manufacturing certain foods. Such non-numerical ADIs
are assigned to food additives for which no toxicological concerns are identified through

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their intended use. Similarly, asparaginase from Aspergillus niger ASP72 also received a
positive evaluation at the 69th meeting of JECFA in June 2008, where it has been assigned
an ADI of “not specified.” Asparaginase from Aspergillus niger ASP72 is permitted for use
in Australia, China, Denmark, Mexico, the Netherlands, New Zealand, Russia, Singapore,
Switzerland and the United States.

Issue
Health Canada has received submissions from industry requesting that Table V to section
B.16.100 of the Food and Drug Regulations (“the Regulations”) be amended to permit the
use of the enzyme asparaginase from the following new genetically modified sources of
micro-organisms: Aspergillus oryzae(pCaHj621/BECh2#10) and Aspergillus niger ASP72.
These amendments permit the use of asparaginase in the production of bread, flour, whole
wheat flour and unstandardized foods, all at a maximum level of use consistent with good
manufacturing practice (GMP).
Asparaginase is an enzyme that hydrolyzes an amino acid, asparagine, to aspartic acid. The
purpose for using asparaginase in food manufacture is to reduce the amount of asparagine in
food, thereby reducing the formation of acrylamide in baked or fried food products.
Acrylamide is an industrial chemical and can also naturally form in certain foods, particularly
plant-based foods that are rich in carbohydrates and low in protein, during processing or
cooking at high temperatures. The greatest source of exposure of the general population to
acrylamide is the diet. The highest concentrations of acrylamide have been detected in potato
chips and French fries, although it has been found in other foods as well. Acrylamide is the
product of a chemical reaction between asparagine and reducing sugars that takes place when
certain foods are baked or fried at temperatures exceeding 120°C. Both asparagine and
reducing sugars are commonly found in many raw food materials.
Dietary exposure to acrylamide has been identified as a potential human health concern by
JECFA. (see footnote 3) Therefore, acrylamide was considered a high priority for assessment
of risk to human health by the Government of Canada due to its high potential for exposure
to consumers. Acrylamide was included in one of the priority groups of chemical substances
under the Government of Canada Chemicals Management Plan. A science-based screening
assessment of acrylamide conducted by the Government of Canada concluded that
acrylamide is potentially harmful to human health. Additional research is being undertaken in
order to fully understand the risks acrylamide poses to human health.
The enzyme asparaginase meets the definition of a food additive under the Regulations.
Prescriptive regulations set out in Division 16 of Part B of the Regulations control the sale
and use of food additives in Canada. The Tables to section B.16.100 list each permitted food
additive according to their purpose of use and specify in which foods and at what levels they
may be incorporated. Table V to section B.16.100 lists food enzymes that may be used as
food additives. Asparaginase is not currently listed in Table V. Therefore, amendments to
Part B of the Regulations are required to enable the use of asparaginase as a food additive.

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Objectives
A screening assessment of acrylamide (see footnote 4) was completed in 2009 as part of the
federal government’s Chemicals Management Plan, in which detailed information about uses
of acrylamide, its environmental impacts, and its potential risks to ecological and human
health was presented. The release of the Government of Canada’s screening assessment to
minimize consumers’ exposure to acrylamide from food sources reaffirmed Health Canada’s
approach: (1) to press the food industry towards the development and implementation of
acrylamide reduction strategies by food processors and the food service industry; (2) to
update and reissue its consumption advice to consumers on how to limit their exposure to
acrylamide from food sources, based on new scientific findings; and (3) to coordinate its risk
communication efforts with key international food regulatory partners. On August 22, 2009,
the Minister of Health announced that acrylamide may pose a risk to human health and
recommended that acrylamide be added to Schedule 1 (List of Toxic Substances) of
the Canadian Environmental Protection Act, 1999. The proposed Order to add acrylamide to
Schedule 1 was published in Part Ⅰ of the Canada Gazette (see footnote 5) on October 3,
2009. The regulatory amendments permitting the use of the enzyme asparaginase address the
risk management approach adopted by Health Canada to reduce consumers’ exposure to
acrylamide from food sources.
Health Canada has conducted a safety and efficacy assessment of asparaginase for the uses
described above. The evaluation considered the toxicological aspects of the use of the food
additive, as well as relevant microbiological and nutritional factors. Asparaginase has been
determined to be effective for its intended purpose, that is in reducing the amount of
asparagine in foods such as bread, crackers and cookies, and cut potato products, sliced
potato products and potato chips, thereby reducing the formation of acrylamide in these
products. When used as described above, asparaginase does not pose a hazard to the health of
the consumer. Further, Health Canada has concluded that the sources of micro-organisms
mentioned above have a history of safe use and have been shown not to present toxicological
concerns.
The enzyme asparaginase from the sources of micro-organisms mentioned above complies
with international specifications established at the 68th and 69th JECFA meetings, (see
footnote 6),(see footnote 7) confirming that asparaginase is a substance of very low toxicity.
The enzyme asparaginase from the sources of micro-organisms noted above also meets the
general and additional requirements for enzymes as outlined in the Fifth Edition of the Food
Chemicals Codex (see footnote 8) and the First Supplement to the Fifth Edition. Based on its
safety and efficacy assessment and in accordance with section B.16.003, the Minister
recommends that amendments be made to the Regulations to enable the uses of asparaginase
as described above.

Description
The amendments enable the use of the enzyme asparaginase produced from the genetically
modified sources of micro-organisms as described above. Table V to section B.16.100 of the
Regulations is amended to permit the use of asparaginase in the production of bread, flour,
whole wheat flour and unstandardized foods, all at a maximum level of use consistent with

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GMP. The standards for flour, whole wheat flour and bread in Division 13, Grain and Bakery
Products, are also amended to permit the use of the enzyme asparaginase in the foods
mentioned above.
ASPG (Uniprot code Q86U10), also named 60 kDa Lysophospholipase, is a protein that
should have asparaginase, lysophospholipase, transacylase and PAF (platelet-activating
factor) acetylhydrolase activities.
The catalytic domain of ASPG is located in its N-terminal part that contains also an ankyrin
repeat, whereas the C-terminal region of the protein includes four ankyrin repeats. The rat
form of ASPG was characterized and asparaginase, lysophospholipase, and PAF
acetylhydrolase activities were demonstrated . The asparaginase activity of the N-terminal
region of human ASPG was described in detail .
We previously reported that ASPG is a new molecular partner of the serine-threonine kinase
SGK1 . The main effect of ASPG that we observed in eukaryotic cells was the down-
regulation of the epithelial sodium channel (ENaC) activity, which is a feature associated
with the decrease of cell malignancy . We also suggested that ASPG, through its
lysophospholipase activity, can play a critical role in the control of cell proliferation
mediating the conversion of lysophosphatidylinositol into glycerophosphoinositol, which is
an important intracellular messenger derived from RAS pathway . These findings and the
enzymatic activities of ASPG suggest that it represents a key element in the inhibition of
tumor cell growth.
The therapeutic role of L-asparaginases is based on their ability to hydrolyze L-asparagine
into L-aspartate and ammonia, depriving tumor cells of a critical metabolite. More
specifically, leukemia cells require large amounts of L-asparagine in order to sustain the
rapid malignant growth. In contrast, the supply of L-asparagine is dispensable for healthy
cells that can synthesize the amino acid in sufficient amounts by their L-asparagine
synthetase (ASNS). Clinical trials demonstrated the effectiveness of L-asparaginases in the
treatment of pediatric and adult acute lymphoblastic leukemia (ALL) patients and the use of
L-asparaginases appears to be promising in the therapy of other hematologic malignancies
and solid tumors . All commercial L-asparaginases are bacterial-derived enzymes that cause
immunological reactions neutralizing the therapeutic effects and inducing adverse reactions
in more than 50% of cancer cases . Thus, the adoption of a human asparaginase enzyme
could overcome the side effects associated with the administration of bacterial proteins.
PAF-AH (PAF acetylhydrolase) catalyzes the biochemical conversion of PAF into the
biologically inactive lyso-PAF by removing the acetyl group at the sn-2 position. It also
metabolizes glycerophospholipids containing short, oxidized, and/or fragmented sn-2 acyl
group that are typically generated during inflammation and oxidant stress. The potential role
of PAF-AHs as anticancer enzymes is still controversial since their ability to act both as
oncoproteins and tumor suppressor proteins, depending on the metabolized substrate and the
targeted cell cycle phase . Anyway, there are several pieces of evidence showing that PAF
and related phospholipids can act as tumorigenic agents stimulating proliferation, increasing
the expression of anti-apoptotic genes and inducing cell migration . Therefore, PAF-AHs,

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converting platelet-activating factor in a biologically inactive form may decrease PAF levels
contrasting the tumorigenesis . There is also evidence suggesting that PAF-AHs can limit
multiple key steps involved in the dissemination of tumor cells .
In the present study, we characterize the asparaginase and PAF-acetylhydrolase activities of purified
human ASPG exploring its effect on the viability of leukemia cells.

Expression and purification of GST, GST-ASPG and GST-ASPG

The vectors pGEX-4T3, pGEX-4T3-ASPG and pGEX-4T3-ASPG (T19A) were used to
transform E.coli BL21 cells. Bacteria were exponentially grown at 37°C in LB medium
supplemented with 100 μg/mL ampicillin and subsequently induced to express fusion
proteins by 0.5 mM isopropyl-thiogalactopiranoside (IPTG) for 3h at 37°C. Cells were then
centrifuged at 4.000 x g for 15 min, washed with 1X cold phosphate-buffered saline (PBS),
resuspended with lysis buffer containing 1% triton X-100, 1% glycerol, 10% sarkosyl, 4
mg/mL lysozyme, 20 mM CHAPS, 10 mM NaF, 2 mM orthovanadate in cold PBS 1X
supplemented with protease inhibitors (Roche Molecular Biochemicals) and finally sonicated
three times for 30 seconds using a Hielscher UP50H sonicator (0.5 cycles, amplitude 100).
Fusion proteins were purified from crude E.coli extract by single-step affinity
chromatography using Glutathione Sepharose® 4B-beads (GE Healthcare) according to the
manufacturer's instructions and finally dialyzed at 4°C with 1 mM EDTA, 50 mM NaCl, 50
mM Tris/HCl pH 8 buffer. The concentration of each purified GST-fusion protein was
estimated by Coomassie Brilliant Blue R-250 staining using a standard curve of BSA.

Asparaginase and glutaminase assays

Nesslerization procedure was applied for the determination of ammonia liberated upon
deamination of L-asparagine. Different dilutions of recombinant GST-protein were incubated
with 25 mM of Tris/HCl pH 8.6 and L-asparagine (concentration depending on the assay) at
37°C for 30 min. The reaction was stopped adding trichloroacetic acid. Ammonia
concentrations was evaluated by reading the absorbance at 436 nm after 5 min of Nessler's
reagent addition in a microplate reader (Thermo Fisher). The ammonia produced in the
reaction was determined by comparing with a standard curve obtained with ammonium
sulfate. One international unit (IU) of the enzyme was defined as the amount of the enzyme
that liberates one μmole of ammonia per min under the assay conditions. Glutaminase
activity was determined following the procedure described above using 15 mM of L-
glutamine instead 15 mM of L-asparagine. All experiments were performed in triplicate and
repeated more times with different preparations of the recombinant proteins.

PAF acetylhydrolase assay

The PAF-AH activity of recombinant GST-fusion proteins was detected using PAF
acetylhydrolase Assay Kit (Abcam) that can detect free thiol groups that are released upon

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hydrolysis of the acetyl thioester bond at the sn-2 position of 2-thio PAF, the modified
substrate used in the assay. The reaction was started adding a range of 10–60 nM of purified
GST-fusion proteins [GST, GST-ASPG and GST-ASPG (T19A)] to the sample mixture
containing 200 μM 2-thio-PAF, 0.1 mM of DTNB and 100 mM Tris/HCl pH 7.2. The
product of the reaction catalyzed by PAH-AH reacts with DNTB forming 5-thio-2-
nitrobenzoic acid, a compound that has a maximum absorbance peak at 412 nm. The
absorbance, evaluated by a microplate reader (Thermo Fischer) of 5-thio-2-nitrobenzoic acid
was used to calculate the International Unit of PAF-AH activity of our recombinant proteins.
All experiments were performed in triplicate and repeated more times with different
preparations of the recombinant proteins.

Cell culture
K562 (Human immortalized myelogenous leukemia), NALM-6 (Human B cell precursor
leukemia) and MOLT-4 (human T lymphoblast; acute lymphoblastic leukemia) cell lines
were cultured in RPMI-1640. PBMCs were isolated from blood by density gradient
centrifugation using Biocol Separating Solution (Biochrom GmbH), and cultured in RPMI
supplemented with 4% of phytohemagglutinin (Life Technology). Human Dermal
Fibroblasts-adult (HDFa) cells were cultured in DMEM. All the media were supplemented
with 10% of Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (from stock solution
of 10.000 units penicillin and 10 mg streptomycin per mL). The cells were grown at 37°C in
a 5% CO2 atmosphere incubator.

Cytotoxicity assay
Leukemia cell lines and PBMCs were seeded in 96-well plates at a density of 1x104
cells/well, HDFa cells were seeded at a density of 5x103cells/well. All the cells were
incubated with different concentrations of purified recombinant proteins for 24h. Microscopy
was performed on a Leica DMI400B automated Inverted Microscope, images were captured
using a Leica DFC 350 FX camera (10X Magnification) and acquired by Leica Application
Suite Software
Cells were then treated with the CCK8 reagent (Sigma), following manufacturer's
instructions. Cell viability was evaluated by determining the concentration of WST-8-
formazan, a compound with a typical absorbance at 450 nm, which formation is directly
proportional to the number of living cells. Absorbance was read by using a microplate reader
(Thermo Fisher). The viability of cells was also evaluated by the Trypan Blue exclusion
assay. Experiment of comparison of cytotoxic activity between GST-ASPG and bacterial
Asparaginase was performed by using E. Coli asparaginase (Sigma). Cytotoxicity of GST-
ASPG on leukemia cells was also evaluated in the presence of D-asparagine (200 mM), L-
asparagine (50 mM) and PAF (10μM), using the CCK8 assay.

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Caspase assay
To allow direct determination of the percent of live, apoptotic, dead and necrotic populations,
distinguished by the presence or absence of activated caspases and/or an intact plasma
membrane [14], 2x104 K562 cells/mL, treated with 700 ng of GST, GST-ASPG and GST-
ASPG (T19A) for 6, 12 and 24h, were used for Guava Caspase Assay according with the
manufacturer’s instructions (Millipore, 4500–0500). Four populations of cells were thus
distinguished in this assay: Lower-left quadrant: viable cells (negative for both Caspase and
7-AADreagents); Lower-right quadrant: cells in the middle stages of apoptosis (positive for
Caspase Reagent and negative for 7-AAD); Upper-right quadrant: cells in the late stages of
apoptotic or dead (Positive for both Caspase and 7-AAD reagents); Upper-left quadrant:
necrotic cells (negative for Caspase Reagent and positive for 7-AAD reagent).

Statistical analysis
GraphPad Software Prism 5 was used for regression and graphical analysis. To determine the
statistical significance of each effect student’s T-test or the one-way analysis of variance
(ANOVA) test followed by Bonferroni post-test for multiple comparisons was used.
Statistical significance was set at p < 0.05.

Regulatory and non-regulatory options considered

Under the Regulations, the use of food additives in foods sold in Canada is controlled
through prescriptive regulations. Specifically, Division 16 of Part B of the Regulations sets
out specific requirements for food additive submissions and requires that the Minister notify
the person filing the submission whether or not she intends to recommend to the Governor in
Council that the Regulations be amended. Evaluation of available data supports the safety
and effectiveness of the enzyme asparaginase produced from the genetically modified
sources of micro-organisms Aspergillus oryzae (pCaHj621/BECh2#10) and Aspergillus
nigerASP72, in the above specified uses.
The only regulatory options available to address these submissions are for the Minister to
recommend or not to the Governor in Council that the Regulations be amended to permit the
uses as described above for this food additive. Based on its safety and efficacy assessment,
the Minister recommends that the uses for this food additive be enabled.

Rationale
There is no anticipated increase in cost to government from the administration of these
amendments to the Regulations. Furthermore, compliance costs incurred by the
manufacturers are not considered to be a factor because the use of food additives is optional.

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These amendments benefit consumers by reducing the formation of acrylamide in some
processed foods, thereby reducing the levels of acrylamide in the food supply and helping to
protect the health and safety of consumers. In addition, these amendments provide some food
manufacturers with a safe option to reduce acrylamide formation in certain foods without
compromising the quality, flavour or other characteristics of the food.

Consultation
The amendments permit the use of this food additive in certain foods for which there are
standards set out in Division 13, Grain and Bakery Products, of the Regulations. The Baking
Association of Canada and the Canadian Food Inspection Agency (CFIA) were consulted
during the development of this regulatory project and neither expressed any objection to the
use of this food additive as described above.
Two public consultations on the proposal to amend the Regulations to permit the use of
asparaginase in certain food products were conducted through postings on Health Canada’s
Web site in 2009. The final consultation closed on February 21, 2010, although comments
received after this date were accepted.
Health Canada received more than 600 comments from consumers, health professionals,
health organizations, food and agricultural organizations, and from a parliamentarian. The
majority of the comments did not support Health Canada’s proposal. However many
comments were based on misconceptions and inaccuracies about how asparaginase would be
used as a food additive. In response to the large volume of questions that were received,
Health Canada has prepared a document entitled Questions and Answers Regarding Health
Canada’s Proposal to Amend the Food and Drug Regulations to Permit the Use of the
Enzyme Asparaginase in Certain Food Products. This document provides responses to the
most common questions that were posed during the consultation periods and also attempts to
address the misconceptions and correct the inaccuracies about how asparaginase would be
used as a food additive

Clarification of information
A number of comments raised concerns to the effect that Health Canada was proposing to
add a chemotherapeutic prescription drug to junk food as a strategy for cancer prevention.
Some comments asked for clarification about possible side effects on human health,
including allergic reactions, of the use of asparaginase in foods, while others questioned the
use of genetically modified organisms to our foods or the use of enzymes in food processing.
Enzymes have been used in food production for centuries. Most of the enzymes that are used
today by the food industry are isolated from sources of micro-organisms, some of which are
genetically modified to optimize the production of the desired enzyme. The enzyme
asparaginase itself does not contain any genetically modified material; however, the micro-
organism that produces it has been genetically modified.
Any confusion about the use of asparaginase may have resulted from the fact that it is also
used as an injectable prescription drug to treat some forms of cancer. However, it should be
emphasized that it is not possible for asparaginase, when used in food, to have a therapeutic

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effect on the human body. In order to act as a chemotherapeutic agent, asparaginase must be
in its active form to be effective and it must be injected into muscle or veins and not given
orally. When used in manufacturing food, asparaginase exerts its effect before the food is
cooked. The heat from cooking inactivates the enzyme asparaginase that is present. Inactive
asparaginase, consumed as part of a food, would be digested in the stomach like other
proteins (including other enzymes) in food and would not have any therapeutic effects on the
body. When used in food processing, asparaginase can only act on the food to which it has
been added. Asparaginase would be used in certain foods in order to reduce the amount of
the amino acid asparagine that is already present in foods. Under specific cooking conditions,
asparagine can react with certain carbohydrates in the food to form acrylamide, a potential
human carcinogen. Reducing the amount of asparagine in the food prior to cooking or
processing would reduce the amount of acrylamide that can be formed.
It is very unlikely that the consumption of residual asparaginase in food would cause an
allergic reaction or hypersensitivities. Based on an internationally accepted approach to
determine the potential allergenicity of proteins (including enzymes), there was no evidence
of any similarity between the asparaginase protein sequence with known toxins or food
allergens. Asparaginase has been used in other jurisdictions (European Union, United States,
Australia and New Zealand) with a history of safe use.
Asparaginase and human health
Comments were received which questioned Health Canada’s role in conducting toxicity
studies for asparaginase. Health Canada does not conduct toxicity studies for food additive
submissions from industry. Pursuant to the Regulations, when industry makes a request that a
food additive be added to, or a change made in, the Tables to section B.16.100 of the
Regulations, the request shall be accompanied by a submission in which data and detailed
reports are submitted for review by Health Canada’s scientists. Health Canada’s scientists
review all safety studies provided as part of the submission, as well as any available studies
published in the scientific literature. If insufficient safety data or other deficiencies in the
submitted data are identified, additional information or studies are requested by Health
Canada. Health Canada will consider making a regulatory amendment to permit the use of a
food additive, under specified conditions, if Health Canada’s scientists are satisfied that the
food additive is safe and does not pose a hazard to the health of the consumer and is effective
for the intended purpose of use.
Health Canada received comments expressing concern about the effects of asparaginase or its
breakdown products on human health. Various toxicity studies were evaluated. There are no
known health risks associated with the breakdown products of asparagine from the enzymatic
reaction that takes place during food processing. The enzyme asparaginase is added to foods
prior to the frying and baking process in order to reduce acrylamide formation during
processing and cooking at high temperatures. As mentioned above, the enzyme asparaginase
is inactivated when the food to which it has been added is cooked; therefore, no active
enzyme remains in food products when they are consumed. Further, the residual inactive
asparaginase, as for any other protein, would be broken down by acid and digestive enzymes
in the human gastrointestinal tract and eliminated through the digestive system.
Health Canada’s approach to mitigating the risks posed by acrylamide in food

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Other comments questioned the real benefits to human health to using asparaginase to reduce
acrylamide levels in foods. JECFA has determined that the estimated dietary exposure of
acrylamide from certain foods may be a human health concern and has called for strategies to
reduce exposure to acrylamide. (see footnote 9) Health Canada’s scientists concur with
JECFA’s recommendations. Health Canada is proactively responding to JECFA’s
recommendation to reduce exposure to foodborne acrylamide and has strongly encouraged
the food industry to develop and implement acrylamide reduction strategies. Some food
processors, in Canada and internationally, have adjusted their cooking instructions to ensure
that acrylamide levels are reduced in their food products, whether prepared at home or in a
restaurant. The use of asparaginase in food processing is another option for reducing
acrylamide formation in food that the food industry has proposed.
It is difficult to determine if past exposure to acrylamide in food in Canada has had a
detrimental effect on human health. However, it is well known that the diet has changed over
the years to include more carbohydrate-rich foods that are baked or fried, thus exposure to
acrylamide has likely increased over time. Since acrylamide is a known carcinogen in
laboratory animals, Health Canada is working proactively to reduce human exposure to
acrylamide from food. Allowing for the use of asparaginase in certain foods is considered
one of several options available to reduce dietary exposure to acrylamide, including the
continued promotion of a healthy diet in accordance with Health Canada’s Eating Well with
Canada’s Food Guide (www.hc-sc.gc.ca/fn-an/food-guide-aliment/index-eng.php).
Food additive regulation and use in Canada
A number of comments requested information on how Health Canada would regulate the
level of use of asparaginase in foods and how it would ensure that this enzyme would not be
used in additional foods. If the food industry wishes to use asparaginase in additional foods
in the future, a new submission would have to be sent and a new safety and efficacy
assessment would have to be undertaken before an amendment could be made to the food
additive tables in the Regulations. All enzymes that are used as food additives in Canada,
including the new enzyme asparaginase, have a permitted maximum level of use that is
consistent with GMP. The Regulations define “Good Manufacturing Practice” as the amount
of a food additive that does not exceed the amount that is required to accomplish the purpose
for which that food additive is permitted. Since the use of enzymes or other food additives
add to the food manufacturer’s costs, there is no motivation on the part of the food
manufacturer to add a greater concentration of a food additive than is necessary to achieve
the desired technical effect.
Informing the public of the use of asparaginase in foods
Other comments requested clarification on whether asparaginase would be labelled on
prepackaged food products so that consumers can avoid the products containing asparaginase
if they choose. Pursuant to the Regulations, most prepackaged products containing more than
one ingredient must carry a list of all ingredients, including components, if any, subject to the
exceptions set out in Regulations. A consumer may therefore have to inquire with retailers to
determine if their products contain asparaginase.

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L-asparaginase
Generic name: Asparaginase
Trade names: Elspar®, Kidrolase®
Other names: Erwinia L-asparaginase
Chemocare.com uses generic names in all descriptions of drugs. Kidrolase and Elspar are
trade names for asparaginase. L-asparaginase or erwinia l-asparaginase are other names for
asparaginase. In some cases, health care professionals may use the trade names elspar or
kidrolase or other names l-asparaginase or erwinia l-asparaginase when referring to the
generic drug name asparaginase.
Drug type: L-asparaginase is an anti-cancer ("antineoplastic" or "cytotoxic") chemotherapy
drug. This medication is classified as an "enzyme." (For more detail, see "How this drug
works" section below).
What This Drug Is Used For:

 Acute lymphocytic leukemia (ALL)

Note: If a drug has been approved for one use, physicians may elect to use this same drug for
other problems if they believe it may be helpful.

How This Drug Is Given:

 Asparaginase is given as an injection into a large muscle (intramuscular or

IM). Depending on your dose, the medication may need to be divided into two
injections.
 Also may be given into the vein as an infusion (intravenous or IV). This method has
higher risk of allergic reaction so often a test dose is given first.
 There is no pill form of aspariginase.
 The amount of aspariginase that you will receive, and how it is given, depends on
many factors, including your height and weight, your general health or other health
problems, and the type of cancer or condition being treated. Your doctor will
determine your dose and schedule.

Side Effects:
Important things to remember about the side effects of asparaginase:

 Most people do not experience all of the side effects listed.

 Side effects are often predictable in terms of their onset and duration.
 Side effects are almost always reversible and will go away after treatment is
complete.
 There are many options to help minimize or prevent side effects.
 There is no relationship between the presence or severity of side effects and the
effectiveness of the medication.

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The following side effects are common (occurring in greater than 30%) for patients taking
asparaginase:

 Fever, chills (see flu like symptoms)

 Nausea and vomiting
 Allergic reaction, (sudden onset of wheezing, itching, rash, face swelling, agitation,
low blood pressure). You will be monitored closely for this reaction.
 Poor appetite
 Stomach cramping
 Central neurotoxicity: excessive sleepiness, depression, hallucinations, agitation,
disorientation or seizure. Less commonly seen stupor, confusion and/or coma.

These side effects are less common (occurring in about 10-29%) of patients receiving
asparaginase:

 Mouth sores
 Pancreatitis (inflammation of the pancreas) in up to 10% of patients. Mainly noted in
blood tests that return to normal after therapy is discontinued. Rarely may be severe
causing symptoms. Symptoms of acute pancreatitis include: (pain in the upper
abdomen that worsens with eating, swollen and tender abdomen, nausea, vomiting,
fever, and rapid pulse).
 Blood test abnormalities (Increased blood glucose level - some refer to this as
"sugar").
 Increases in blood tests measuring liver function. These return to normal once
treatment is discontinued (see liver problems).
 Blood clotting disorders, increases risk of both bleeding and clotting.

Not all side effects are listed above. Some that are rare (occurring in less than 10% of
patients) are not listed here. However, you should always inform your health care provider if
you experience any unusual symptoms.

When to contact your doctor or health care provider:

Contact your health care provider immediately, day or night, if you should experience any of
the following symptoms:

 Fever of 100.4° F (38° C) or higher, chills (possible signs of infection).

The following symptoms require medical attention, but are not an emergency. Contact your
health care provider within 24 hours of noticing any of the following:

 Nausea (interferes with ability to eat and unrelieved with prescribed medication)
 Vomiting (vomiting more than 4-5 times in a 24 hour period)
 Persistant upper abdominal pain or pain that worsens with eating
 Abdominal swelling
 Diarrhea (4-6 episodes in a 24-hour period)
 Unusual bleeding or bruising

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  Swelling, redness and/or pain in one leg or arm and not the other
 Extreme fatigue (unable to carry on self-care activities)
 Yellowing of the skin or eyes
 Unusual thirst, need to urinate frequently
 Confusion, excessive sleepiness, hallucinations (seeing, hearing or feeling things that
are not there), agitation, or disorientation (not able to recognize familiar
surroundings)

Always inform your health care provider if you experience any unusual symptoms.
Precautions:

 Before starting asparaginase treatment, make sure you tell your doctor about any
other medications you are taking (including prescription, over-the-counter, vitamins,
herbal remedies, etc.). Do not take aspirin, products containing aspirin unless your
doctor specifically permits this.
 Do not receive any kind of immunization or vaccination without your doctor's
approval while taking asparaginase.
 Asparaginase may be inadvisable if you have had a hypersensitivity (allergic)
reaction to asparaginase. If you have had a reaction to Elspar®, Erwinia L-
asparaginase may be used with caution.
 Inform your health care professional if you are pregnant or may be pregnant prior to
starting this treatment. Pregnancy category C (use in pregnancy only when benefit to
the mother outweighs risk to the fetus).
 For both men and women: Do not conceive a child (get pregnant) while taking
asparaginase. Barrier methods of contraception, such as condoms, are recommended.
Discuss with your doctor when you may safely become pregnant or conceive a child
after therapy.
 Do not breast feed while taking this medication.
 Those who have a history of pancreatitis (inflammation of the pancreas) should not be
treated with asparaginase.

Self-Care Tips:

 Drink at least two to three quarts of fluid every 24 hours, unless you are instructed
otherwise.
 For flu-like symptoms, keep warm with blankets and drink plenty of liquids. There
are medications that can help reduce the discomfort caused by chills.
 You may be at risk of infection so try to avoid crowds or people with colds, and
report fever or any other signs of infection immediately to your health care provider.
 Wash your hands often.
 To help treat/prevent mouth sores, use a soft toothbrush, and rinse three times a day
with 1/2 to 1 teaspoon of baking soda and/or 1/2 to 1 teaspoon of salt mixed with 8
ounces of water.
 Use an electric razor and a soft toothbrush to minimize bleeding.
 Avoid contact sports or activities that could cause injury.

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  To reduce nausea, take anti-nausea medications as prescribed by your doctor, and eat
small, frequent meals.
 Avoid driving and tasks that require being alert until your response to this drug is
well understood.
 Avoid sun exposure. Wear SPF 15 (or higher) sunblock and protective clothing.
 In general, drinking alcoholic beverages should be kept to a minimum or avoided
completely. You should discuss this with your doctor.
 Get plenty of rest.
 Maintain good nutrition.

If you experience symptoms or side effects, be sure to discuss them with your health care
team. They can prescribe medications and/or offer other suggestions that are effective in
managing such problems.
Monitoring and Testing:
You will be checked regularly by your doctor while you are taking asparaginase, to monitor
side effects and check your response to therapy. Periodic blood work to monitor your
complete blood count (CBC), blood clotting factors, pancreatic enzymes, blood sugar as well
as the function of other organs (such as your kidneys and liver) will also be ordered by your
doctor.

How This Drug Works:

All cells need a chemical called asparagine to stay alive. Normal cells can make this
chemical for themselves, while cancer cells cannot. Asparaginase breaks down asparagine in
the body. Since the cancer cells cannot make more asparagine, they die.
When asparaginase breaks down asparagine it is broken down into 2 chemicals, aspartic acid
and ammonia. The neurologic side effects seen with asparaginase (such as, confusion,
excessive sleepiness, agitation, disorientation, or coma) are related to increased levels of
these chemicals circulating in the body.
Antineoplastic Drugs
Asparaginase is an enzyme that catalyzes the hydrolysis of l-asparagine to l-aspartic acid
and ammonia. Asparaginase products are isolated from different types of bacteria,
namely Escherichia coliand Erwinia chrysanthemi. They inhibit protein synthesis in tumor
cells by depriving them of the amino acid asparagine. This drug is phase specific, with the
greatest activity in the G1 phase of the cell cycle. Clinical use is confined presently to acute
lymphocytic leukemia. Asparaginase products may produce acute hypersensitivityreactions
with hypotension, sweating, bronchospasm, and urticaria. Other effects in patients taking l-
asparaginase include the possibility of hepatitis, pancreatitis, and altered production
of coagulation factors resulting in either increased bleeding or increased clotting risk. A
second formulation of asparaginase is pegaspargase (PEG-l-asparaginase), which

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has polyethylene glycol covalently linked to the asparaginase structure to
decrease immunogenicity and to prolong its half-life.

Respiratory Toxicology
l-Asparaginase, a polypeptide of bacterial origin, is important in the treatment of
childhood acute lymphoblastic leukemia. Pneumonitisattributable to l-asparaginase has not
been reported. However, the rates of allergic reactions,
including anaphylaxis and bronchospasm, are high for l-asparaginase compared to other
chemotherapeutic agents. Depending on the specific agent used, the route of administration,
and concomitant therapies, the risk is up to 8% per administered dose, 33% by the fourth
dose , and 2–75% overall . Pegylated asparaginase was approved for use in acute
lymphoblastic leukemia in patients who developed hypersensitivity to the native form of
asparaginase and has been demonstrated to be less immunogenic

REVIEW OF LITERATURE

Article-1

TOPIC: PRODUCTION OF ASPARAGINASE

Authors

Bruno , Da Silva FVS1, Costa-Silva TA1, Apolinario AC1, Santos JHPM1,2, Keynesian’s
EK1, Monteiro G1, Rangel-Yagi CO1, Bunyanian B3, Junior AP1.

This article was published in the year 2018

ABSTRACT
L-Asparaginase (ASNase) is a vital component of the first line treatment of acute
lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over
53,000 people worldwide by 2020. More recently, ASNase has also been shown to have
potential for preventing metastasis from solid tumors. The ASNase treatment is, however,
characterized by a plethora of potential side effects, ranging from immune reactions to severe
toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious
new products tailored to minimize adverse reactions while increasing patient survival have
been devised. In the following pages, the reader is invited for a brief discussion on the most
recent developments in this field. Firstly, the review presents an outline of the recent

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improvements on the manufacturing and formulation processes, which can severely influence
important aspects of the product quality profile, such as contamination, aggregation and
enzymatic activity. Following, the most recent advances in protein engineering applied to the
development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis
resistant and less immunogenic) using techniques such as site-directed mutagenesis,
molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards,
the attention is shifted toward nanomedicine including technologies such as encapsulation
and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing
the results of the most innovative and representative academic research, the review provides
an overview of the products already available on the market or in the latest stages of
development. With this, the review is intended to provide a solid background for the current
product development and underpin the discussions on the target quality profile of future
ASNase-based pharmaceuticals.

CONCLUSION

Since its initial discovery as a drug in the early 1950' by J. G. Kidd and collaborators,
ASNase has become one of the cornerstones of chemotherapeutic treatments, especially of
acute lymphoblastic leukemia (ALL) (Kidd, 1953). Recent studies also point toward its
potential for the treatment of solid tumors (Knott et al., 2018). Despite of the groundbreaking
medical innovation of the first ASNase therapy and its success in extending the lives of
millions of people over the last decades, most of the products currently in the market lack
desirable pharmaceutical characteristics. Those include, but are not limited to, an extended
blood serum half-life as well as reduced immunogenicity and toxicity.
The development of improved ASNases is not an easy task, given the microbiological source
of the enzyme, which can result in immunogenicity. In addition to that, serum proteases may
degrade the enzyme causing further loss of activity and increased immunogenicity due to
additional exposition of epitopes after cleavage. To face those challenges, researchers have
nowadays a wide array of biomolecular and biochemical tools at their disposal to aid in the
improvement of ASNase, tailoring the enzyme for its application.
The development of biobetter ASNases starts at the process development. This is in line with
the classical QbD maxim “to begin process development with the end (the product) in mind”
(Juran, 1992). Following this idea, several research groups have looked into new
microbiological sources of ASNases, with special focus on the eukaryotic ones such as
Saccharomyces cerevisiae, Aspergillus sp. and Proteus vulgaris, which might deliver
ASNases with reduced immunogenic potential. In accordance to the same guiding principle,
genetically engineered and/or chemically modified ASNases have also been investigated,

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with special attention to site-directed mutation, PEGylation and nanoencapsulation. As J. M.
Juran so wisely put it: “the product is the process” and some of the product's immunogenicity
results from the presence of contaminating biomolecules and protein aggregates, both of
which should be removed in the purification steps. Therefore, novel purification strategies
are also worth investigating.
Thus, as demonstrated in this review, drawing inspiration from QbD principles, new
technologies have been applied to improve the ASNase quality profile. However, there is a
need for a deeper understanding of the mechanisms involved in the treatment with ASNase in
order to create new and effective strategies to improve this biopharmaceutical.

Article-2

TOPIC: L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia

asparaginase

AUTHORS
Rob Pieters, MD, PhD,1 Stephen P Hunger, MD,2 Joachim Boos, MD,3 Carmelo Rizzari,
MD,4 Lewis Silverman,

Abstract
Asparaginases are a cornerstone of treatment protocols for acute lymphoblastic leukemia
(ALL) and are used for remission induction and intensification treatment in all pediatric
regimens and in the majority of adult protocols. Extensive clinical data have shown that
intensive asparaginase treatment improves clinical outcomes in childhood ALL. Three
asparaginase preparations are available; the native asparaginase derived from Escherichia
coli (E. coli-asparaginase), a pegylated form of this enzyme (PEG-asparaginase) and a
product isolated from Erwinia chrysanthemi, i.e. Erwinia asparaginase. Clinical
hypersensitivity reactions and silent inactivation due to antibodies against E.coli-
asparaginase, lead to inactivation of E-Coli asparaginase in up to 60% of cases. Current
treatment protocols include E. coli-asparaginase or PEG-asparaginase for first-line treatment
of ALL. Typically, patients exhibiting sensitivity to one formulation of asparaginase are
switched to another product to ensure they receive the most efficacious treatment regimen
possible. Erwinia asparaginase is used as a second- or third-line treatment in European and
US protocols. Despite the universal inclusion of asparaginase in such treatment protocols,

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there is much debate regarding the optimal formulation and dosage of these agents. This
manuscript provides an overview of available evidence to make recommendations for
optimal use of Erwinia asparaginase in the treatment of ALL.

Conclusion
Advances in therapies for ALL have led to improved long-term survival rates for pediatric
and adult patients. Asparaginases form a cornerstone of ALL treatment protocols with three
main preparations for use in treatment protocols: the native E. coli-asparaginase, a pegylated
form (PEG-asparaginase) and an alternative enzyme isolated from Erwinia chrysanthemi,
referred to as Erwinia asparaginase. Despite the availability of these agents, much debate
remains regarding the optimal formulation and dose for the treatment of pediatric and adult
ALL patients. This manuscript aims to provide recommendations, based on data available in
the literature, to ensure optimal use of Erwinia asparaginase. Patients who receive an
asparaginase as first-line treatment for ALL and develop anti-asparaginase antibodies should
be switched to another asparaginase preparation to ensure maximum survival benefit.
Monitoring of asparaginase levels is preferable to assess the extent of serum asparagine
depletion and to identify cases of silent inactivation. Erwinia asparaginase is a valid second-
or third-line therapy, depending upon protocols, regulatory factors and availability. Evidence
from published studies suggests that Erwinia asparaginase should be administered at a dose
of at least 20 000 IU/m2 three-times weekly, via either the IV or IM route. Further clinical
and pharmacokinetic studies of Erwinia asparaginase will help to optimize the use of this
agent.

Article-3

AUTHORS:
Vassilios; Aramis and Prakash Nidhi Tiwari

Abstract:

The discovery of the tumor-inhibitory properties of asparaginase (ASNase) began in the early
1950s with the observation that guinea pig serum-treated lymphoma-bearing mice underwent
rapid and often complete regression. About 4000 cases of acute lymphoblastic leukemia
(ALL) are diagnosed very year in the US and many more through out the world. The
majority of these cases are in children and young adults, making ALL the most common
form of malignancy in these age groups. The treatment protocols of ALL are complex and
use 6–12 drugs. Consequently, the improvement in the protocol design has improved
significantly the success rate for long-term event-free survival in the past 20–30 years, which
is now approximately 75% for patients afflicted with the higher risk ALL features and just
above this percentage for patients with standard or good features. Despite this success,
approximately 15% of patients die from ALL, making leukemic relapse the most common
cause of treatment failure in pediatric oncology. ASNases have been the cornerstone of ALL

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therapies since the late 1970s. Native or pegylated L-asparaginase (ASNase or PEG-ASNase)
are highly specific for the deamination of L-asparagine (Asn) to aspartic acid and ammonia.
Depletion of Asn leads to a nutritional deprivation and inhibition of protein biosynthesis,
resulting in apoptosis in T-lymphoblastic leukemias, which require Asn from external
sources. The reactions of the host exposed to repeated ASNase treatments as well as the up-
regulation of the mammalian enzymes to overcome the ASN-depletion toxic condition are of
significant importance and may make us relearn the lessons on this important antileukemic
drug.

CONCLUSION:

Anise is a tetramer protein that deaminates Asn and Gln. ASNase inhibits protein synthesis in
T-cells. The average IC50 concentration of PEG-ASNase is 0.4 IU/mL. Gln deamination is
necessary for optimal Asn deamination and, therefore, leukemia blast kill. PK–PD analyses
show that PEG-ASNase provides a better day 7/14 bone marrow response. ASNase activity
of 0.4–0.7 IU/mL provides greater than 90% depletion in vivo (with hepatic asparagine
biosynthesis) (CCG-1961 and CCG-1962).

Better Asn and Gln deamination are associated with improved EFS. High titer anti-ASNase
antibody is found in some children with no clinical allergy (silent hypersensitivity). IgG
antibody neutralizes ASNase activity. Anti-ASNase antibody production leads to inferior
treatment outcome in patients with ALL. The purpose of intensive therapy in ALL is also the
prevention of immunization to ASNase. The longer activity and less immunogencity with
PEG-ASNase may increase EFS and the quality of life. Neutralizing antibody development
against native ASNase has cross-reactivity with PEG-ASNase but not with Erwinia ASNase.
The dosing of all formulations should be monitored for activity. The early use of PEG-
ASNase may require lower dosing and may be more cost effective. A more intensive ASNase
dosing may be required to provide high trough levels of 0.4–0.7 IU/mL in relapsed ALL
patients.
ASNase therapy for the treatment of newly diagnosed and recurrent-refractory leukemias is
an important milestone in the efforts to achieve high EFS, overall survival, with little
toxicities and events in patients with these diseases. Maintaining optimal ASNase enzymatic
activity at 0.4–0.7 IU/mL, low anti-ASNase antibodies in these patients, and optimal
depletion of Asn and Gln play a vital role in achieving these goals. Hence, appropriate
monitoring of these parameters during therapy is of the utmost importance. Prevention of
relapses in leukemias could be further ensured with intensive ASNase dosing (weekly or
biweekly) and appropriate monitoring of these parameters during treatment of newly
diagnosed leukemias. Future studies should examine additional aspects that would promote
effective ASNase therapy, like the role of asparagine synthetase, gene expression profiles,
and micro-array technology in order to categorize leukemia patients and search for inherent
resistance or refractoriness to ASNase.

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 Article-4

Topic: L-ASPARAGINASE: A REVIEW ON CURRENT SCENARIO AND FUTURE

PROSPECTS
MICROBIAL

Authors:

S. Kumar Jha*, Divya Pasrija, Rati Kumari Sinha, Hare Ram Singh, Vinod Kumar Nigam
and Ambrish Sharan Vidyarthi
This was published on 01 september 2012

ABSTRACT
L-Asparaginase , also known as L-asparagine amidohydrolase is the enzyme with anti-tumor
activity and is well accepted as a chemotherapeutic agent against the acute lymphoblastic
leukemia and lymphosarcoma. This article has briefly touched nearly all the industrial and
clinical aspects of L-Asparaginase and provides the recent update of the topic. The article
includes a brief introduction to the topics, mechanism of action, a little information about the
structure, sources of enzyme, purification, optimum conditions for the enzyme production,
recombinant strains for higher productivity and formulation of the enzyme.

CONCLUSION:
The discovery of the fact that L-Asparaginase is responsible for the action of the guinea-pig
serum against the acute lymphoblastic leukemia has set a milestone in the field of medicine.
After this discovery detailed information about the enzyme has been dug out. It has been
proved that L-Asparaginase from Escherichia coli and Erwinia carotovora has anti-
neoplastic activity against cancer and is being used as anti-cancer drug. But, it is observed
that the action of enzyme is coupled with some side effects. Moreover, the yield of enzyme
was not enough to fulfil the demand of the drug. Solid state fermentation is being adopted all
over the world for the production of the enzyme as it has many advantages over submerged
fermentation. So, it created the need to discover new sources and techniques toenhance the
yield and decrease the side effects of the enzyme. Enzyme isolated from different sources has
different optimized conditions for production and activity. The structure of enzyme predicted
from E. coli has four identical units. Recombinant work and formulation of enzyme is also in
progress, yet there is still a long way to go. Moreover, L-Asparaginase has spread its arms in
food industry also, as an ingredient which reduces the toxicity of baked food by significantly
reducing the amount of acrylamide in food items. It is a need of the time, medicine and food
industry to dig a lot more to satisfy the thirst of the drug

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
Article - 5
TOPIC : Evaluation of recombinant human growth hormone secretion in E. coli. using the L-
asparaginase II signal peptide.

Authors: Divya Pasrija, Rati Kumari Sinha, Hare Ram Singh,

This was published in 2006

ABSTRACT

Human Growth Hormone (hGH) or somatotropin, a single chain polypeptide contains 191
amino acid residues with a molecular mass of 22 kDa, is synthetized and secreted by the
anterior pituitary gland . The mature form of the hGH is created from its 217 amino acid
precursor after removal of the signal peptide (26 amino terminal residues). Owing to a
substantial role of the hGH in various biological functions, it has a wide range of therapeutic
applications like hormone therapy of hypopituitary dwarfism, skin burns, bone fractures,
bleeding ulcers, HIV wasting syndrome and genetic disorders such as Turner's and Down's
syndromes . Since this protein does not need post-translational modifications like
glycosylation, Escherichia coli (E. coli) host is considered a prokaryotic powerhouse for
industrial production of recombinant hGH . Notwithstanding different advantages of E. coli,
including appropriate growth on low-cost media, fast biomass accumulation and extensive
knowledge of its genetics and physiology , there are some obstacles in acquiring considerable
yields of correctly folded recombinant proteins. Failure of protein to rapidly reach a correct
folding and disulfide bond formation leads to protein degradation by proteases and
accumulation as inactive aggregates in the cytoplasm called inclusion bodies . Transferring
the protein into the bacterial periplasmic region of E. coli or directly into the extracellular
medium, using an appropriate signal peptide at the N-terminal of the protein, is an applicable
approach to solve these problems . Efficient secretion of the recombinant protein is achieved
using an optimal signal sequence, which is compatible with the secretory protein.
Unfortunately, there is no general rule for selecting a suitable signal peptide to guarantee
successful protein secretion. Therefore, trial-and-error experimental approach is universally
applied to evaluate different signal peptides . Uptill now, various signal peptides have been
evaluated for secretory production of hGH in E. coli, like OmpA , PhoA , pelB , LTB , npr ,
StII , DsbA , penicillinase and natural hGH signal peptide

27 | P a g e
conclusion

High-level cytoplasmic expression of recombinant hGH in E. coli frequently leads to

aggregation of mis-folded protein . To overcome this obstacle, an alternative expression
approach can be applied to secrete the protein into the periplasmic space of the E. coli by
inserting a signal sequence to the N-terminal of the hGH gene. It has been demonstrated that
naturally secreted proteins like hGH, are universally considered to be appropriate candidates
for the secretory production in heterologous systems . An optimal balance between all stages
of the secretory cascade is required for a prosperous secretion process. One of the most
important factors that has a considerable effect on all steps of secretion pathway and finally,
on the yield of the recombinant secreted protein is the signal peptide . This short amino-
terminal extension is then proteolytically removed by specific enzymes called signal
peptidases during translocation to yield the correctly folded proteins with the formation of
proper disulfide bonds Due to the lack of universal rule for selecting an appropriate signal
peptide, extensive trial-and-error research has been undertaken to express periplasmic
recombinant proteins using different signal peptides . E. coli L-asparaginase II, a periplasmic
enzyme with high affinity for L-asparagine, has been applied clinically for treatment of acute
lymphoblastic leukemia The ansB gene encoding this enzyme naturally comprises a
secretory signal peptide of 22 amino terminal residues . A previous report indicated efficient
secretory expression of recombinant hirudin III in E. coli exploiting L-asparaginase II signal
sequence . Another study represents the improvement of recombinant cyclodextrin
glucanotransferase secretion in the presence of mutant L-asparaginase II signal peptide .
However, to our knowledge, there is no experimental study to evaluate this signal peptide in
connection with hGH and its probable effect on appropriate secretion of this protein.
Therefore, this objective was pursued in the present study.

Article-6
Topic: Screening, Production and Optimization of L-Asparaginase From Soil Bacteria
Isolated in Ibadan, South-western Nigeria.

Authors: Keynesian’s EK1, Monteiro G1, Rangel-Yagi CO

Abstract

L-Asparaginase producing bacteria were isolated from soil samples under optimum
conditions using submerged fermentation. Their abilities to produce asparaginase at

28 | P a g e
different pH, temperature, incubation temperature, utilization of different carbon and
nitrogen sources as well as their specific conditions for optimal activity were also
investigated. Streptococcus spp. D1, Bacillus polymyxa and Streptococcus spp. D2
showed optimum asparaginase production at pH 8 with activities of 11.6 U/ml, 8.8 U/ml
and 7.9 U/ml respectively. The pH 7 was observed as optimum pH for Bacillus firmus
(8.8 U/ml) while optimum pH 6 was observed for Bacillus circulans and Paenibacillus
validus.

Maximum L-asparaginase productivity was attained at a temperature of 45C by

Bacillus firmus, Streptococcus sp. D1 and Bacillus circulans with activity of 4.6 U/ml,
5.6 U/ml and 3.8 U/ml respectively while 35C incubation temperature was optimum for
Paenibacillus validus and Bacillus polymyxa with enzyme activity of 6.2 U/ml and 6.1
U/ml respectively. Mannitol supported the maximum asparaginase production of
Bacillus circulans, Streptococcus sp. D2 and Bacillus polymyxa while maltose was
observed as the best carbon source for Streptococcus sp. D1 and Bacillus firmus; and
sucrose for Paenibacillus validus. The optimum nitrogen source was casein for Bacillus
circulans (7.57 U/ml) and Streptococcus spp. D1 (6.19 U/ml), Yeast extract for Bacillus
polymyxa (7.037 U/ml) and Bacillus firmus (5.368 U/ml) while NaNO3 supported
optimum L-asparaginase production for Streptococcus sp. D2 (6.006 U/ml) and
Paenibacillus validus (4.754 U/ml).

conclusion

Sixty-one asparaginase producing bacteria were isolated from five different soil
samples. This may be attributed to the fact that soil is a rich source for potential
enzyme producing organisms.

In this study, the result of screening test revealed that asparaginase producing bacteria were
able to hydrolyse L-asparagine since they utilized L- asparagine as their substrate and
breakdown asparagine to L-aspartate and ammonia which further reacts with water to
produce NH4OH, hence the pH of the medium is basic which subsequently changes the
medium from yellow to pink, hence the pinkish zone observed around the colony of
asparaginase producing bacteria. This is in accordance with the work of Gulati et al. [15]
who proved that colour transformation was due to L-asparaginase production. The bacterial
isolates with highest asparaginase activity were identified as Bacillus spp., Streptococcus
spp. and Paenibacillus spp. This is also similar to the report of Kamble and Khade [16] who
reported Bacillus spp. and Paenibacillus spp. to be a good asparaginase producing bacteria.

The optimum incubation temperature at which Bacillus firmus, Streptococcus spp. D1 and
Bacillus circulans showed highest asparaginase yield was 45C; this was in contrast with the
report of Narayana [17] on the optimum incubation temperature for Streptomyces
albidoflavus. Bacillus polymyxa and Paenibacillus validus had their highest production at
incubation temperature of 35C, which was similar to the work of Borkotaky and Bezbaruah

29 | P a g e
[18] that reported optimum incubation temperature for L-asparaginase activity by Erwinia sp.

Among the physical parameters, pH of the growth medium plays an important role by
inducing morphological changes in the microorganisms and in enzyme secretion. The result
of the effect of pH showed that maximum asparaginase yield by Streptococcus spp. D1,
Streptococcus spp. D2 and Bacillus polymyxa was at pH8. This observation agrees with the
work of Dhevagi and Poorani [19] who reported maximum L-asparaginase by Streptomyces
sp. PDK7 at pH 8.0 to 8.5.

The effect of various concentration of mannitol (0.1 % to 0.5 %) on L-asparaginase

production showed that Bacillus firmus and Streptococcus sp. D1 had their optimum
asparaginase production at 0.1 % while the least asparaginase production by all the bacterial
isolates was observed at 0.5 % mannitol concentration except Bacillus circulans and
Paenibacillus validus. This agrees with the work of Thae and Ellaiah [20] who stated that
increase in mannitol concentration (from 0.1 %- 0.2 %) resulted in decreased asparaginase
production and that increase in mannitol concentration above 0.1% resulted in the
accumulation of mannitol in the cultivation medium.

The best sucrose concentration for maximum L- asparaginase production by all the bacterial
isolates was 0.3 % except Streptococcus sp. D1 which was 0.1 % sucrose concentration. In
contrast, Praveen et al. [21] observed higher titres of L-asparaginase by Serratia marcescens
when medium was supplemented with 1.5 % sucrose concentration. Susmika and Mandal
[22] have reported sucrose as the best carbon source for L-asparaginase production. This may
be due to the inductive effect of sucrose and its remarkable efficiency in asparaginase
production, being an inexhaustible source of carbon compared to other carbon sources and it
also helps in stabilizing the enzyme [23].

The optimum maltose concentration at which Bacillus firmus, Streptococcus sp. D2,
Paenibacillus validus showed optimum asparaginase production was 0.4 % maltose
concentration. This agrees with the work of Amena et al. [24] who reported 0.5 % maltose
concentration as the best concentration for optimum L- asparaginase production for
Streptomyces gulbargensis. In contrast, Deokar et al. [25] observed maximum L-
asparaginase production by Erwinia carotovora at 1.13 % maltose concentration.

Reports have shown that lactose was the best carbon source for L-asparaginase
production [26, 27]. The best lactose concentration for maximum asparaginase
production by most bacteria was 0.5%. In contrast, Liu and Zajic [28] observed
enhanced L- asparaginase production with 1 % lactose concentration.

The best nitrogen source for maximum asparaginase production by Bacillus firmus and
Bacillus polymyxa was yeast extract. This agrees with the work of Verma [29] who
reported yeast extract to be important for cell growth and L-asparaginase synthesis.

30 | P a g e
The best KNO3 concentration for extracellular asparaginase production was 0.4 %
KNO3 concentration for Bacillus firmus and Streptococcus sp. D2. Least L-
asparaginase production was observed at 1 % KNO3 concentration for Bacillus
polymyxa, Bacillus circulans and Paenibacillus validus. This agrees with the work of
Singh and Srivastava [30] who reported reduction in asparaginase production at 1 %
KNO3 concentration.

Maximum L-asparaginase production for most of the isolates was recorded at 0.2 %
NaNO3 concentration, an observation similar to that of Makky et al. [31] who observed
NaNO3 to be optimum for L-asparaginase activity of Bacillus spp. KK2S4.

Bacillus polymyxa had its highest L-asparaginase activity at 0.6 % yeast extract
concentration while the least asparaginase activity by all isolates was 0.8 %
concentration except Streptococcus sp. D2 and Bacillus circulans. This agrees with the
work of Hosamani and Kaliwal [9] that showed that 0.5 % yeast extract gave optimum
L-asparaginase production. This result was similar to the work of Verma [29] who
reported the importance of yeast extract at low concentration for cell growth and L-
asparaginase synthesis. In contrast, Kavitha and Vijayalakshimi [32] showed that 1.5 %
yeast extract concentration was optimum for L-asparaginase production. Also, Deokar
et al. [25] showed that 1.74 % yeast extract concentration was optimum for L-
asparaginase synthesis by Erwinia carotovora.

Increase in casein concentration resulted in decreased asparaginase, an observation

which agrees the work of Thaer and Ellaiah [20] that showed gradual decline in L-
asparaginase production with increased casein concentration. Report have also shown
that casein is essential for cell growth and L-asparaginase synthesis, but in high
concentration, the production of L-asparaginase is inhibited which might be due to the
presence of high substrate concentration and induction of proteolytic enzyme

ARTICLE –7
AUTHORS:
Dharmaraj et al , Kamble eT
This was published on 1999
ABSTRACT

L-asparaginase is an anti-neoplastic agent used in the

chemotherapy of lymphoblastic leukaemia. The present work deals with production of
extra-cellular Lasparaginase from marine actinomycetes, using submerged fermentation.
Marine actinomycetes Streptomyces associated with marine sponge Callyspongia diffusa
was isolated using specific ISP medium. Sponge-associated Streptomyces was
characterized by conventional methods, and identified as Streptomyces noursei MTCC
10469. Production of Lasparaginase by submerged fermentation was carried out using
medium Tryptone Glucose Yeast extract (TGY) broth. The enzyme was purified to near
homogeneity by ammonium sulphate precipitation, dialysis, gel filtration on Sephadex G

31 | P a g e
100 column, CM Sephadex C-50 and SDS-PAGE. The enzyme was purified at 98.23
folds, and showed a final specific activity of 78.88 IU/mg, with 2.14% yield. SDS-PAGE
of the purified enzyme revealed an apparent molecular weight of 102 kDa for it. The
optimum pH, temperature and incubation time of L-asparaginase was found to be 8, 50ºC
and 35 min, respectively. The study suggests that marine actinomycetes, particularly
Streptomyces, may be used as a potential source of L-asparaginase.

Conclusion

Due to the hazards of chemotherapeutic drugs and its painful

effects, L-asparaginase are emerging as safer source of anticancer enzymes. Genetic
studies and enzyme purification studies has always been simpler with regards to bacteria
hence bacteria are also preferred source for L-asparaginase production. Study so far
reports some of the bacterial L-asparaginase is allergic and therefore there is a
considerable need to find alternative bacterial L-asparaginase. We have employed a
media containing L-asparagine as principle carbon source for screening of l-asparaginase
producing bacteria. Twenty isolates were obtained after screening various soil samples. A
qualitative assay was performed in order to select the efficient bacteria. Out of these
isolates twelve efficient bacteria are further selected for physiological studies and carbon
sources utilization. Optimization of pH and temperature is also studied. We have also
performed a comparative assay of these isolates for Lasparaginase production. The
bacterial genera belong to Paenibacillus species, B. subtilis, Aneurinibacillus species,
Staphylococcus species, S. saprophyticus, Brevibacillus species and Micrococcus species


MATERIALS AND METHODS:

32 | P a g e
SAMPLE COLLECTION

The Bacterial strain of Pseudomonas aeruginosa was collected from MTCC Chandigarh.

SCREENING FOR ENZYME PRODUCT

The strain was screened for L-asparaginase production using a method in which modified M9
medium (composition for 1 l: 6.0 g Na2HPO4·2H2O; 3.0 g KH2PO4; 0.5 g NaCl2; 5.0 g
Lasparagine; 1mole/litre of 2.0 ml MgSO4.7H2O; 0.1mole/litre of 1.0ml CaCl2.2H2O; 10 ml
of 2.0% w/v glucose; and 20.0 g agar) incorporated with a pH indicator (phenol red) was
used (Gulati et al., 1997). L-asparaginase activity was identified by formation of pink zone
around colonies.

Media: Modified M9 Media

Composition of the Modified Media

Media pH -7.0

Preparation of various concentration of dye

33 | P a g e
  2.5% stock solution of phenol red dye was prepared in ethanol and the pH was
adjusted to 7.0 using 1mole/litre of sodium hydroxide.
 The stock solution of the dye, ranging from 0.04ml to 0.3ml, was added to
1000ml of modified M9 medium, given final dye concentrations of 0.001% to
0.009% respectively.

Procedure

 20ml of 0.005% of the above dye containing media was poured into a Petri plates.
 Two control plates were also prepared by using modified M9 medium .Among them
one was without dye and the other one was without asparagine (instead containing
NaNO3 as nitrogen source).
 The strain was inoculated in Petri plates.
 The plates were incubated for 18hrs at 37oC.
 The zone diameter where measured after 18hrs of incubation

Pure culture preparation

 Take 1.25 grams of asparaginase base, 0.5 agar agar and mix in 50ml of distilled
water.
 Clean the sterilize laminar air flow with ethanol.
 Take two petri dishes and clean them with ethanol in the presence of laminar airflow
and sterilize them.
 On the uv light for 5 mins to kill the other bacterial organisms present.
 After 5 mins, put off the uv and don’t go near the uv light as it causes mutations.
 Take asparaginase media and mix it thoroughly in 50ml of distilled water.
 Put the solution micro oven and give 3 heat boil.
 Take that off after 3 heat boils and cover it with a foil.
 Leave until it cools.
 After cooling pour that asparaginase media into petri dishes in the presence of
laminar air flow.
 Leave the dishes until it solidifies.
 After the solidification put the asparaginase media in incubator for 2 days to increase
the bacterial growth.

Production media preparation and inoculation

 Clean the sterilize laminar air flow with ethanol in presence of flow.
 Take two petri dishes and clean with ethanol in the presence of laminar airflow and
sterilize them.
 Put On the uv light for 5 mins to kill the other bacterial organisms present.

34 | P a g e
  After 5 mins put off the uv and don’t go near to the uv light as it causes mutations.
 Take asparaginase media and mix it thoroughly in 50ml of distilled water
 Put the solution in micro oven and give 3 heat boil
 Take the solution out after 3 heat boils and cover it with a foil.
 Leave until it cools.
 After cooling pour that asparaginase media into petri dishes in the presence of
laminar air flow.
 Leave the dishes until it solidifies.
 After the solidification put that asparaginase media in incubator for 2 days to increase
the bacterial growth.

Preparation of nutrient agar media:

 Take 50ml conical flask and pour 25ml of distilled water in it.
 In that add 0.125grams of peptone, 0.075gms of beef extract ,0.125gms of
Nacl and 0.125gms of agar agar.
 Mix it thoroughly and heat it in micro oven up to three heat boils
 Then cover it with foil.
 Leave it undisturbedly until it cools
 Clean the sterilize laminar air flow with ethanol in presence of flow.
 Take two petri dishes and clean with ethanol in the presence of laminar
airflow and sterilize them.
 Put On the uv light for 5 mins to kill the other bacterial organisms present.
 After 5 mins off the uv and don’t go near the uv light as it causes mutations.
 Take asparaginase media and mix it thoroughly in 50ml of distilled water
 Put the solution in micro oven and give 3 heat boil
 Take the solution off after the 3 heat boils and cover it with a foil.
 Leave until it cools.
 After cooling pour that nutrient agar media into petri dishes in the presence of
laminar air flow.
 Leave the dishes until it solidifies.

Serial dilution

 Collect some soil from the plant at the underneath level of the plant.
 Take 10 test tubes and wash it thoroughly.
 Add 10ml of distilled water in each test tube.
 Add a pinch of soil in first test tube.
 Take a micro pippete of range 200-1000micro liters. With the help of micro pippete
suck 10ml of the solution from the first test tube and pour it in second test tube.
 Repeat the process above up to tenth test tube.

Spreading

35 | P a g e
  Clean the laminar air flow with ethanol.
 Light the Bunsen burner.
 Take a petri dish with full of ethanol.
 Take out the spreader and put the spreader in ethanol.
 Take the spreader near to the Bunsen burner and sterilize it.
 Take out the nutrient agar media petri dish and split out the soil solution with the help
of spreader.
 Repeat the process for the other petri dish.
 Keep the petri dishes in incubator for 1 day.

Inoculation

streaking
 Bring the asparaginase media and nutrient agar media to the laminar airflow.
 Lighten up the Bunsen burner
 Take a loop and put it in Bunsen burner flame up until the loop appears red
hot colour.
 Take loop full of asparagine bacteria from the asparaginase media plate
 Streak it in nutrient agar media plate.
 Repeat the same process for the other plate
 Put the media in incubator for 1 day.

Gram staining
 Take a grease free glass slide
 keep a drop of sterilized distilled water
 Take a loop full of test bacteria(asparganase) and mix it with a drop of
distilled water on a slide to prepare a thin smear.
 Heat fix the smear.

Gram staining protocol

 Add 2-3 drops of crystal violet on to the smear and keep it for 40-60 seconds.
 Wash gently under the tap water
 Add 2-3 drops of grams iodine and retain for 20 seconds.
 Wash the slide under the tap water.
 Add 70% ethyl alcohol (decolorizing agent) drop by drop until the excess
colour goes away.
 Add 2-3 drops of saffron (counter strain)
 Retain for 60 seconds and wash under running tap water.
 Air dry the smear
 Absorb it under 100x oil immersion object.

Qualitative assay of asparaginase

Downstream process
centrifugation

36 | P a g e
  Take 10ml of distilled water
 Take a loop full of test bacteria and mix it in distilled water.
Centrifugation is a process of mixing the 44% ammonium sulphate, in 10ml
of chilled supernatant at 6000rpm for 10mins .44% of ammonium sulphate is
equal to 4.44 grams of ammonium sulphate.
By certifuging the chilled supernatant the high molecular weight molecules
goes down and the lower molecular weight molecules will float on the surface
.

Salt precipitation

 Take 10ml of supernatant and put it on the icepack.

 Add pinch by pinch of ammonium sulphate to mix thoroughly.
 Pour the solution in 2 test tubes and put the test tubes for overnight incubation
at 4degree Celsius.
 Centrifuge at 10000rpm for 10 minutes.

Dialysis

Introduction

Dialysis is a separation technique that facilitates the removal of small, unwanted compounds
from macromolecules in solution by selective and passive diffusion through a semi-
permeable membrane. A sample and a buffer solution (called the dialysate, usually 200 to
500 times the volume of the sample) are placed on opposite sides of the membrane. Sample
molecules that are larger than the membrane-pores are retained on the sample side of the
membrane, but small molecules and buffer salts pass freely through the membrane, reducing
the concentration of those molecules in the sample. Changing the dialysate buffer removes
the small molecules that are no longer in the sample and allows more contaminants to diffuse
into the dialysate. In this way, the concentration of small contaminants within the sample can
be decreased to acceptable or negligible levels.

37 | P a g e
 Qualitative study

Total Protein Estimation by Lowry’s Method

Objective

To determine the concentration of proteins by Lowry’s method.

Reagents Required

1. BSA stock solution (1mg/ml),

2. Analytical reagents:

(a) 50 ml of 2% sodium carbonate mixed with 50 ml of 0.1 N NaOH solution

(0.4 gm in 100 ml distilled water.)

(b) 10 ml of 1.56% copper sulphate solution mixed with 10 ml of 2.37%

sodium potassium tartarate solution. Prepare analytical reagents by mixing 2
ml of (b) with 100 ml of (a)

3. Folin - Ciocalteau reagent solution (1N) Dilute commercial reagent (2N)

with an equal volume of water on the day of use (2 ml of commercial reagent
+ 2 ml distilled water)

Principle

The phenolic group of tyrosine and trytophan residues ( amino acid) in a

protein will produce a blue purple color complex , with maximum absorption
in the region of 660 nm wavelength, with Folin- Ciocalteau reagent which
consists of sodium tungstate molybdate and phosphate. Thus the intensity of
color depends on the amount of these aromatic amino acids present and will
thus vary for different proteins. Most proteins estimation techniques use

38 | P a g e
 Bovin Serum Albumin (BSA) universally as a standard protein, because of its
low cost, high purity and ready availability. The method is sensitive down to
about 10 μg/ml and is probably the most widely used protein assay despite its
being only a relative method , subject to interference from Tris buffer, EDTA,
nonionic and cationic detergents, carbohydrate, lipids and some salts. The
incubation time is very critical for a reproducible assay. The reaction is also
dependent on pH and a working range of pH 9 to 10.5 is essential.

Procedure

1. Different dilutions of BSA solutions are prepared by mixing stock BSA

solution (1 mg/ ml) and water in the test tube as given in the table. The final
volume in each of the test tubes is 5 ml. The BSA range is 0.05 to 1 mg/ ml.

2. From these different dilutions, pipette out 0.2 ml protein solution to

different test tubes and add 2 ml of alkaline copper sulphate reagent
(analytical reagent). Mix the solutions well.

3. This solution is incubated at room temperature for 10 mins.

4. Then add 0.2 ml of reagent Folin Ciocalteau solution (reagent solutions) to

each tube and incubate for 30 min. Zero the colorimeter with blank and take
the optical density (measure the absorbance) at 660 nm.

5. Plot the absorbance against protein concentration to get a standard

calibration curve.

6. Check the absorbance of unknown sample and determine the concentration

of the unknown sample using the standard curve plotted above. BSA Water
Sample conc. Sample vol Alk. CuSO4 Lowry reagent O.D.

TUB PROTEIN(mg/ml Water(ml Alk Folins OD

E ) ) CuSO4(ml reagent(ml
) ) Commented [yb1]:
Blank 0 2 0.2 0.2 0
S1 0.2 0.8 0.2 0.2 0.02
1
S2 0.4 0.6 0.2 Leav 0.2 Leav 0.04
e e 2
S3 0.6 0.4 0.2 Upto 0.2 Upto
0.06
3
S4 0.8 0.2 0.2 10 0.2 30 0.08
4

39 | P a g e
 S5 1.0 0 0.2 min 0.2 Mins 0.09
6
T1 0.6 1.4 0.2 0.2 0.27
8
T2 1.0 1 0.2 0.2 0.09
0

Write short notes on the following points in the report:

1. Beer-Lamberts law

2. Compare Folin-Lowry method with other methods of protein estimation.

Phosphate buffer preparation:

Prepare 800ml of distilled water in a suitable container.

 Add 20.209 grams of sodium phosphate dibasic hepta hydrate to the solution.
 Add 3.394 grams of sodium phosphate mono basic mono hydrate t the
solution.
 Adjust the solution to the final desired PH using HCL or NAOH.
 Add distilled water until volume is 1 liter.

Collect the pellat of protein sulphate and dissolve it in phosphate buffer.

COMPONENTS MASS(gms) Molarity(M)

Sodium phosphate 20.209 0.0754
dibasic heptahydrate
Sodium phosphate 3.394 0.0246
monobasic monohydrate

Result and discussion

 In pure culture preparation we used to produce bacteria in certain condition of
incubator. At that particular temperature and pressure the production bacteria
will be speed up. As a result, after two days the bacteria will be appears as
clumsy and bubbled structure.

 In production media preparation and inoculation, the asparaginase bacteria

inoculated in nutrient agar media. After 1 day a streak of colonial bacteria will
appear.it is used for the further test bacterial result.
 In gram strain process we will identify the bacteria under the microscope.as a
result gram positive bacillai no spore had appeared for asparaginase enzyme
production bacteria.
 In countrification process higher molecular weight substances like protein
settle down in the pellet and lower molecular weight substances like

40 | P a g e
 phosphate will float in the pellat. This separation will occur a 6000rpm for
10mins.for further removal of sulphate we use to centrifuge that crude subtract
in 10000rpm for 10 mins.
 In dialysis process certain amount of protein will be present on the membrane
and the other waste materials will be passed out by semi permeable
membrane.
 In qualitative study certification, salt precipitation and dialysis methods play
crucial role.
 In quantitative study the certain concentration of protein will be calculated.

Conclusion
The L asparaginase from gram positive basicilli no spour was purified in glutaminase free
form which can reduce the possibility of side effects during the course of anti-cancer therapy.
The enzyme showed good stability over a wide range of physiological conditions as PH and
temperature.in the next step of our Project L asparaginase 1 will be used as potential
candidate for treatment of acute lymphoblastic Lukemia.

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