L-Asparaginase, an enzyme which has great biological importance. It is used as a drug against acute lymphoblastic leukemia. Not only the ALL it is also to cure many other types of cancers [2]. -Asparaginase is also used as an immunosuppressive agent in some immunological responses.[3]. Kidd first reported the antitumor activity of L-Asparaginase [4] in 1953 which was later proved by Broom [5]. The enzyme L-Asparaginase in known by Kidrolase, Elspar, Colaspase [6]. The action of L-Asparaginase is associated by making the essential aminoacid Aspragine scarce to the cancerous cells. Because the cancerous cells are in requirement of large amounts of L-Asparaginase for their survival, and hence they die. The L-Asparaginase enzyme is not only used as a drug but is also used as a food processing agent [7] by preventing the formation of acrylamide, a potent carcinogen formed upon backing the starchy food items above 120oC by means of Millards reaction [8].
The drug acts by catalyzing the amino acid Asparaginase into aspartic acid and ammonia. The aspartic acid thus formed is converted into fumaric acid and oxaloacetic acid, an intermediate byproduct of the TCA cycle. Asparaginase, hence is very useful in maintaining the levels of amino acid and nitrogen balance within the cells [9].
The aim of the present investigation is to identify the fungal Microorganism Aspergillus niger as it was observed that the eukaryotic fungal microorganisms are potential for L-Asparaginase production, and to enhance the enzyme production by using the cheap raw material for Solid State Frementation. Very few studies are reported for the fungal producers.
Mechanism of action [10]
MATERIALS AND METHODS:
MICROORGANISM SCREENING:
The fungal microorganism for the present investigation were taken from the previous study [11]. It was observed that the F5 organism showed high zone of clearance and was maximum at 0.1% phenol concentration.
IDENTIFICATION OF THE FUNGUS:
The colony morphology showed a black to brown coloured colony which indicated the presence of Aspergillus niger. The microscopic observation was performed to confirm the organism with Lactophenol cotton blue stain and viewed under 45X.
ENHANCING THE PRODUCTION OF THE ENZYME USING SOLID STATE FERMENTATION:
SELECTION OF THE SUBSTRATE:
The different substrates used for the study were the Orange peel+ Corn flour (OCF) and the Corn Flour + Green peas Husk (CGH). The substrates were prepared by washing several times with water followed thrice with 95% ethanol. Ensuring proper drying, they should be made into fine pieces and mixed thoroughly.
FERMENTATION PRODUCTION MEDIA:
The solid state fermentative media is prepared by adding peptone-1g, L-asparagine-15g, K2HPO4 -2.5g, glycerol-3g, MgSO4.7H2O-2g, FeSO4.7H2O-1g, Kcl-2g , Distlled Water-1000ml.
100ml of this media is transferred into 2 sterile 250ml Erlenmeyer flask and to each of these flasks 20g of the substrate (OCF) and (CGH) are added. To each of these flasks, 1 ml of the inoculum is added and incubated for 8 days at 30 OC and the moisture content is adjusted to 45%.
ENZYME EXTRACTION:
The enzyme L-Asparaginase is extracted by adding 30ml of 0.01M phosphate buffer at pH 6.2 and spinning for 45min. This is followed by 8000rpm for 25min at 4oC. The enzyme assay is done using the Nesselers Reagent [12] and the enzyme is used for further investigation.
ENZYME ACTIVITY:
One unit of L-Asparaginase activity is defined as that amount of enzyme which catalyses the formation of 1µMol of ammonia per minute and is expressed as IU.
The media optimization is performed by studying the specific activity of the enzyme at different pH, Temperature, Carbon Source and Nitrogen Source.
EFFECT OF pH ON THE ENZYME PRODUCTION:
The effect of pH on the production of the enzyme is studied for both the substrates by taking pH 2,3,4,5,6,7,8, and9 respectively and incubated for 5 days at 30oC .
EFFECT OF TEMPERATURE ON THE ENZYME PRODUCTION:
The effect of temperature was studied by taking 20,25,30,35 and 40 oC at pH 7.4 incubated for 5 days and the specific activity was determined.
EFFECT OF CARBON ON THE ENZYME PRODUCTION:
The effect of Carbon source was studied by taking glucose, maltose, starch and Xylose incubated at pH 7.4 for 5 days.
EFFECT OF NITROGEN ON THE ENZYME PRODUCTION:
The effect of Nitrogen source was studied by taking the following , Peptone, Urea, Yeast extract, and NaNO3, incubated for 5 days at pH 7.4 and the enzyme activity was determined.
RESULTS AND DISCUSSIONS:
IDENTIFICATION OF THE FUNGUS:
The colony morphology and the microscopic staining technique by Lactophenol blue stain confirmed the organism to be Aspergillus niger.
FERMENTATION MEDIA COMPOSITION:
The media used for the solid state fermentation is as follows:
S.No.
Name of Chemical
g/l
1.
L-Asparaginase
15.0
2.
K2HPO4
2.5
3.
Glycerol
3.0
4.
MgSO4. 7H2O
2.0
5.
FeSO4. 7 H2O
1.0
6.
KCl
2.0
7.
Dist. Water
1000
100ml of this media is taken in 250ml of an Erlenmeyer flask. To this 20g of the substrate is added. The specific activity is determined after 8 days of incubation.
S.No.
Substrate Name
IU/ml
1.
Orange Peel+Corn Flour (OCF)
8.6
2.
Corn Flour +Green peas Husk (CGH)
12.9
The substrate Corn Flour +Green peas Husk (CGH) showed the maximum activity of the enzyme with 12.9 IU/ml.
EFFECT OF pH:
The effect of pH on the enzyme production for both the media was studied and the specific activity was determined.
S.No
pH
SPECFIC ACTIVITY IU/ml
OCF
CGH
1.
2
2.8
2.5
2.
3
4.1
3.9
3.
4
4.5
4.6
4.
5
5.1
6.5
5.
6
8.9
9.2
6.
7
15.4
13.7
7.
8
12.5
16.5
8.
9
11.6
15.3
The maximum activity for OCF was found to be at pH 7 with 15.4 IU/ml and CGH was found to be 16.5 IU/ml respectively.
Effect of Temperature:
The present investigation proceeded by taking different temperatures 20,25,30,35, & 40. The specific activity is determined after incubating for 5 days at pH 7.4 for both the substrates.
S.No.
Temperature( oC)
SPECFIC ACTIVITY (IU/ml)
OCF
CGH
1.
20
8.2
9.4
2.
25
13.3
14.7
3.
30
13.9
15.4
4.
35
14.8
16.6
5.
40
14.2
15.9
The maximum activity is found at the temperature 35 OC for both the substrates with the specific activity of 14.8 IU/ml and 16.6 IU/ml respectively.
EFFECT OF CARBON SOURCE:
The effect of carbon source on the production of enzyme was studied.
S.No.
CARBON SOURCE
SPECFIC ACTIVITY (IU/ml)
OCF
CGH
1.
Glucose
11.0
10.0
2.
Maltose
12.5
13.3
3.
Starch
13.5
14.6
4.
Xylose
14.2
12.1
The maximum activity for OCF was found to be with the carbon source Xylose with 15.4 IU/ml and CGH was found to be with Carbon source 14.6 IU/ml respectively.
EFFECT OF NITROGEN SOURCE:
The effect of Nitrogen source on the production of enzyme was studied by taking Peptone, Urea, Yeast Extract and NaNO3.
S.No.
NITROGEN SOURCE
SPECFIC ACTIVITY (IU/ml)
OCF
CGH
1.
Peptone
12.3
19.7
2.
Urea
10.3
16.3
3.
Yeast Extract
15.2
17.9
4.
NaNO3
11.9
14.2
CONCLUSION:
L-Asparaginase is an enzyme with great therapeutic value. Much of the study is done with the bacterial isolates and less reports were recorded with fungal microorganisms. In the present study we have used two different raw materials as substrates which are very easily available. The results showed very clearly that between the two substrates used i.e. the Orange +Corn flour (OCF) and Corn flour + Green pea Husks (CGH), the CGH has showed high activity which proves to be the best substrate.
Cantarelli MA, Pellerano RG, Marchevsky EJ, Camina JM. (Title of article). Anal Sci, 2011; 27 (1): 73-8.
