2. Enzyme : All living organisms from bacteria
to man are built and maintained by biological
catalysts called enzymes. These enzymes are
made from proteins which have each been
evolved over millions of years to perform very
specific biochemical tasks.
Enzyme classification:
3. SOME COMMERCIALLY IMPORTANT HYDROLASES:
1. LIPASE
2. PROTEASE
3. ASPERGINASE
4. TANNASE
SOURCES OF L-ASPERGINASE: This enzyme is widely
distributed, being found in animal, microbial and plant
sources. it's presence in guinea pig serum was first
reported by Clementi (Clement,l922). The enzyme is
present in liver and kidney of certain birds, rats and
chicken(Krebs, 1950). Large number of microorganisms
that include Erwinia caratovora, Pseudomonas stutzeri,
Pseudomonas aerugenosa and E.coli. It has been observed
that eukaryotic microorganisms like yeast and fungi have a
potential for asparaginase production.
4. Asparaginase is intended for use as a processing aid during food
manufacture to reduce the level of L-asparagine by its hydrolysis
to L-aspartic acid and ammonia. Free L-asparagine present in
food is the main precursor of acrylamide, which is considered to
be a probable human carcinogen. Acrylamide is formed from
Lasparagine and reducing sugars primarily in starchy foods that
are baked or fried at temperatures above l20 ͦ C.
Asparginase will be used during preparation of carbohydrate-
rich foods that are major sources of dietary acrylamide, such as
bread and other cereal-based products, baked and fried potato-
based products, and reaction flavours (also known as “thermal
process flavours"). The levels of L-asparagine would be reduced
in these foods prior to heating, thereby reducing the availability
of Lasparagine for acrylamide formation. The enzyme will be
inactivated by denaturing during the heating step.
5. 1.Enrichment media for isolation of microorganism from soil
Nutrient Broth 13 g/L
Glucose 20 g/L
Peptone 20 g/L
KH₂PO₄ 1 g/L
K₂HPO₄ 0.5 g/L
Distilled Water to make 1000 mL
Maintain pH at 7.0 at 37 ͦC
2.Nutrient Agar medium
Nutrient Agar 28 g/L
Agar Powder 4 g/L
3. Nutrient Broth Medium
Nutient Broth 13 g/L
Distilled Water to make 1000 ml
6. Na2HPO4.2H2O 6g/L
Glucose Stock solution 10ml
L-asparaginase 5g/L
KH₂PO₄ 3 g/L
MgSO4.7H20 2g/L
CaCl2.2H2O 1g/L
NaCl 0.5 g/L
Agar 2g/L
Distilled Water to make 1000 mL
Maintain pH at 7.0 at 37 ͦC
7. Collection of soil sample
The soil sample was collected from the garden area of
Jadavpur University Campus.
Culture Enrichment
The soil samples collected were then added to the
enriched media prepared earlier. Soil sample was added
4g/L of enriched media for microbes present in the soil to
grow. For that they were incubated overnight in shaking
incubator at 37°C.
Isolation of pure cultures
In natural habitats microorganisms usually grow in
complex mixed populations containing several species.
This presents a problem for the microbiologist because a
single type of microorganism cannot be studied
adequately in a mixed culture. One needs a pure culture, a
population of cells arising from a single cell, to
characterize an individual species.
8. (i) Spread plating on solid agar medium with a glass spreader and
(ii) Streak plating with a loop. The purpose of spread plating and streak
plating is to isolate.
SPREAD PLATE TECHNIQUE:
In this technique, the number of bacteria per unit volume of sample was
reduced by serial dilution before the sample was spread on agar plate.
Serial Dilution
●12 test tubes were taken and 9 mL of distilled water in each test tube was
added and autoclaved.
●These test tubes were then marked from 10-1 to 10-12 serially.
●1 mL of medium (containing the soil sample) was added in test tube
marked as 10-1.
●Now take 1 mL of sample from the test tube marked 10-1 and add it to
the tube marked 10-2 and repeat the sample process till 10-12.
Nutrient Agar plates were made and marked and these plates were then
marked from 10-1 to 10-12 serially.
9. A spreader was taken and heated to make it sterile. Now from
each test tube 50 μL of sample was taken and added to the
corresponding agar plate. With the help of spreader, spreading
was done till the surface of agar becomes rough.
10. Slant Preparation
For slant preparation, nutrient agar was prepared in a
flask (250 mL).
The flask was then placed in the heating mantle and
was continuously shaken for even heating. The flask
was heated till it boils.
The flask was then removed from the heating mantle.
Test tubes were placed in the stand and media was
poured in the test tube
Test tubes were sealed by the cotton plugs and then
autoclaved.
After autoclaving they were kept at an angle to
prepare the slants.
11. The L-Asparaginase activity was determined by detection
of ammonia or L-aspartic acid.
The assay procedure is based on direct Nesslerization of
ammonia. Ammonia is estimated by detecting the optical
density at 425nm.
The activity was determined in 0.05 M-borate buffer (pH
8.5) at 37ºC in the presence of 10mM-substrate
(asparagines).
Nesslerization method was used and the optical density
was measured at 425nm.
The other method is an automated technicon method. Both
took advantage of the observation that the incidence of
turbidity which is often encountered in the Nessler’s
reaction is lower in the presence of EDTA and when alkali
is added immediately prior to Nesslerization. The 0.05M
borate buffer (pH 8.5) was used.
12. Standard graph was prepared by treating 1ml
of 0.25, 0.5, 0.75 and 1mM ammonium
sulphate with trichloroacetic acid, NaOH and
Nessler’s reagent. It was prepared by
dissolving 132 mg of ammonium sulphate in
100 ml distilled water in a volumetric flask.
Working solution of 1mM: It was prepared by
taking 5 ml of stock solution and made upto
50 ml with distilled water. From this 0.25 and
0.5 mM were prepared.
13. Into a series of test tubes, 2 ml of (0.05M) borate buffer was
taken. To this 1 ml of each 1 mM, 0.75 mM, 0.5 mM and 0.25
mM of working solutions were added.
From this 1 ml sample was withdrawn and delivered into 2.5 ml
of 0.1N trichloroacetic acid. To this 1 ml of 1N NaOH was added.
0.2 ml of EDTA (0.1M) was added to each sample to overcome
the encountered turbidity.
After 2 min., 0.5 ml of Nessler reagent was added. The OD was
measured after 5 min. at 425 nm.
Blank was prepared by adding 1 ml of water instead of
ammonium sulphate solutions. The data is shown in .
A standard curve was constructed by taking ammonium sulphate
(μM/ml) on X-axis and corresponding optical density on Y-axis
and the data is shown in Figure 1.
Unit: . One LA unit (IU) was defined as the amount of enzyme,
which liberates 1μ mole of ammonia per min under the optimal
assay conditions.
14. Optimization of pH
The selected strain of the organism was grown at
different pH i.e. 5,6,7,8 at 37 C for 24 hr. After
24 h the fermented broth was centrifuged and
the clear supernatant was used for assay of the
enzyme at 396nm.
Optimization of Temperature
The selected strain of the organism was grown at
different temperature i.e.27,32,37,42C for 24
hr. After 24 h the fermented broth was
centrifuged and the clear supernatant was used
for assay of the enzyme at 396nm.
19. Asparaginase is used with or without other
cancer drugs to treat acute lymphocyctic
leukemia(ALL). It works by starving tumor
cells of needed nutrients and slowing tumor
cell growth.
As a food processing aid. Acrylamide is often
formed in the cooking of starchy foods, by
adding asparaginase before baking or frying
the food, asparagineis converted into another
common acid, aspartic acid, and ammonium.
20. Asparginase is an interesting enzyme with
important application in pharmaceutical and food
industry.
Discoveries are being followed by an intensive
work aiming to increase the process productivity
to enable and extend the use of the enzyme
mainly in food industries.
In the present study 13 microbial sample were
isolated from soil sample capable of producing
asparaginase enzyme out of which the strain
marked MR7 was selected as the most potent
one. The strain was capable to produce
maximum of the desired enzyme at pH 5 and
temperature at 27ºC.
