COMPLETE PROCESS OF ISOLATION OF L-ASPARAGINASE

1. 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:

2. Hydrolases:In biochemistry, a hydrolase is an enzyme
that catalyzes the hydrolysis of a chemical bond
SOME COMMERCIALLY IMPORTANT HYDROLASES:
1. LIPASE
2. PROTEASE
3. ASPERGINASE
4. TANNASE
SOURCES OF L-ASPERGINSE: 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.

3. Mechanism of Action as Food Processing Aid:
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 L-
asparagine 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 L-
asparagine for acrylamide formation. The enzyme will be
inactivated by denaturing during the heating step.

4. MATERIAL AND METHODs:-
Media used:-
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

5. 4.Screening Medium( for L- asparaginase)
Na₂HPO₄ 6 g/L
KH₂PO₄ 3 g/L
NaCl 0.75 g/L
L- asparagine 10 g/L
PROCEDURE:
Collection of soil sample
The soil sample was collected from the garden area of Delhi University South 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.

6. Simpler methods for isolation of a pure culture
include:
(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.

7. PLATING
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.
Then , colony were developed on the agar surface and we characterize the
colony according to their morphology.

8. 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.

9.  Fig: slant
 Gram Staining
Gram positive bacteria have a thick mesh-like cell wall made of
peptidoglycan (50-90% of cell wall), which are stained purple by crystal violet,
whereas Gram-negative bacteria have a thinner layer (10% of cell wall), which
are stained pink by the counter-stain. Alcohol does not readily penetrate to
decolorize the cell wall of the previously applied crystal violet stain. Gram-
negative cells have a thinner cell wall through which the alcohol readily
penetrates. The crystal violet is removed from these cell walls that are then
stained with the safranin counterstain.

10. There are four basic steps of the Gram stain:-
(1) Applying a primary stain (crystal violet) to a heat-fixed (death by heat)
smear of a bacterial culture.
(2) The addition of a trapping agent (Gram's iodine)
(3) Rapid decolorization with alcohol or acetone, and
(4) Counterstaining with safranin.
(5) Observe the slide under the microscope

11. Negative Staining
Negative staining is an established method for contrasting a thin specimen
with an optically opaque fluid. In this technique, the background is
stained, leaving the actual specimen untouched, and thus visible. This
contrasts with 'positive staining', in which the actual specimen is stained.
To conduct a proper negative stain the following procedure should be
followed:
1. Place a very small drop (more than a loop full--less than a free falling drop
from the dropper) of nigrosin near one end of a well-cleaned and flamed
slide.
2. Remove a small amount of the culture from the slant with an inoculating
loop and disperse it in the drop of stain without spreading the drop.
3. Use another clean slide to spread the drop of stain containing the organism
using the following technique.

12. 4. Rest one end of the clean slide on the center of the slide with the stain. Tilt the clean slide
toward the drop forming an acute angle and draw that slide toward the drop until it
touches the drop and causes it to spread along the edge of the spreader
slide. Maintaining a small acute angle between the slides, push the spreader slide
toward the clean end of the slide being stained dragging the drop behind the spreader
slide and producing a broad, even, thin smear.
5. Allow the smear to dry without heating.
6. Focus a thin area under oil immersion and observe the unstained cells surrounded by the
gray stain

13. Screening Media
After preparing plates of screening media, point inoculation was performed.
The plate was divided into 8 parts and a point was marked in each of the eight
sections.
The slant of the all the colonies were taken one by one.
And with the help of loop a small amount of colony was picked from slant.
Then the loop was touched at the point marked in the plate.
These steps were repeated until all the colonies were inoculated.
The plates were then incubated at 37 ͦC for 12-24 hours.
Growth Curve
Growth is an orderly increase in the quantity of cellular constituents. The growth of
microorganisms reproducing by binary fission can be prepared by plotting as the
logarithm of the number of viable cells versus the incubation time.
Lag Phase:
Bacteria are becoming "acclimated" to the new environmental conditions to which
they have been introduced (pH, temperature, nutrients, etc.). There is no
significant increase in numbers with time.

15. Exponential Growth Phase:
The living bacteria population increases rapidly with time at an exponential growth in
umbers, and the growth rate increasing with time. Conditions are optimal for growth.
Stationary Phase:
With the exhaustion of nutrients and build-up of waste and secondary metabolic
products, the growth rate has slowed to the point where the growth rate equals the
death rate. Effectively, there is no net growth in the bacterial population.
Death phase:
The depletion of nutrients and the subsequent accumulation of metabolic waste
products and other toxic materials in the media will facilitates the bacterium to move
on to the Death phase. During this, the bacterium completely loses its ability to
reproduce. Individual bacteria begin to die due to the unfavourable conditions and
the death is rapid and at uniform rate. The number of dead cells exceeds the number
of live cells. Some organisms which can resist this condition can survive in the
environment by producing endospores .

16.  Enzyme Activity
Effect of temperature
The temperature has a vital role to play in activity of any enzyme. The readings of
the enzyme activity were calculated for temperatures ranging from 20 ͦC to 70 ͦC.
In which the highest activity was noticed at 60 ͦC. The following readings were
observed and subsequent graphs were plotted.
Temperature( ͦC) Enzyme
Activity(IU/mL)
20 203.829
30 274.283
40 351.664
50 450.456
60 555.818
70 168.63

17. 203.829
274.283
351.664
450.456
555.818
168.63
0
50
100
150
200
250
300
350
400
450
500
550
600
0 10 20 30 40 50 60 70 80
EnzymeActivity(IU/mL)
Temperature( ͦC)
Enzyme Activity
Enzyme Activity

18. Effect of pH
The pH has a vital role to play in activity of enzyme. The readings of the Enzyme
activity were measured from pH ranging from 2 to 12. In which the highest
activity was noticed at pH 9. The following readings were observed and
subsequent graphs were plotted.
pH Enzyme Activity(IU/mL)
4 6.715
5 24.31
6 70.073
7 91.7896
8 129.912
9 150.456
10 91.7896
11 62.389
12 50.6963

19. 6.715
24.31
70.073
91.7896
129.912
150.456
91.7896
62.389
50.6963
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5 6 7 8 9 10 11 12 13
EnzymeActivity
pH
Enzyme Activity
Enzyme Activity