L-Asparaginase, an enzyme which has great biological importance. It is used as a drug against acute lymphoblastic leukaemia. Not merely the ALL it is besides to bring around many other types of malignant neoplastic diseases . -Asparaginase is besides used as an immunosuppressive agent in some immunological responses. . Kidd foremost reported the antitumor activity of L-Asparaginase in 1953 which was subsequently proved by Broom . The enzyme L-Asparaginase in known by Kidrolase, Elspar, Colaspase . The action of L-Asparaginase is associated by doing the indispensable aminoacid Aspragine scarce to the cancerous cells. Because the cancerous cells are in demand of big sums of L-Asparaginase for their endurance, and therefore they die. The L-Asparaginase enzyme is non merely used as a drug but is besides used as a nutrient processing agent by forestalling the formation of acrylamide, a powerful carcinogen formed upon endorsing the starchy nutrient points above 120oC by agencies of Millards reaction .
The drug Acts of the Apostless by catalysing the amino acerb Asparaginase into aspartic acid and ammonium hydroxide. The aspartic acid therefore formed is converted into fumaric acid and oxaloacetic acid, an intermediate by-product of the TCA rhythm. Asparaginase, hence is really utile in keeping the degrees of amino acid and N balance within the cells .
The purpose of the present probe is to place the fungous Microorganism Aspergillus niger as it was observed that the eucaryotic fungal micro-organisms are possible for L-Asparaginase production, and to heighten the enzyme production by utilizing the inexpensive natural stuff for Solid State Frementation. Very few surveies are reported for the fungous manufacturers.
Mechanism of action
The fungous micro-organism for the present probe were taken from the old survey . It was observed that the F5 being showed high zone of clearance and was maximum at 0.1 % phenol concentration.
The settlement morphology showed a black to brown colored settlement which indicated the presence of Aspergillus Niger. The microscopic observation was performed to corroborate the being with Lactophenol cotton blue discoloration and viewed under 45X.
The different substrates used for the survey were the Orange peel+ Corn flour ( OCF ) and the Corn Flour + Green peas Husk ( CGH ) . The substrates were prepared by rinsing several times with H2O followed thrice with 95 % ethyl alcohol. Guaranting proper drying, they should be made into all right pieces and assorted exhaustively.
The solid province 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 unfertile 250ml Erlenmeyer flask and to each of these flasks 20g of the substrate ( OCF ) and ( CGH ) are added. To each of these flasks, 1 milliliter of the inoculant is added and incubated for 8 yearss at 30 OC and the wet content is adjusted to 45 % .
The enzyme L-Asparaginase is extracted by adding 30ml of 0.01M phosphate buffer at pH 6.2 and whirling for 45min. This is followed by 8000rpm for 25min at 4oC. The enzyme check is done utilizing the Nesselers Reagent and the enzyme is used for farther probe.
One unit of L-Asparaginase activity is defined as that sum of enzyme which catalyses the formation of 1AµMol of ammonium hydroxide per minute and is expressed as IU.
The media optimisation is performed by analyzing the specific activity of the enzyme at different pH, Temperature, Carbon Source and Nitrogen Source.
The consequence 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 severally and incubated for 5 yearss at 30oC.
The consequence of temperature was studied by taking 20,25,30,35 and 40 oC at pH 7.4 incubated for 5 yearss and the specific activity was determined.
The consequence of Carbon beginning was studied by taking glucose, maltose, amylum and Xylose incubated at pH 7.4 for 5 yearss.
The consequence of Nitrogen beginning was studied by taking the followers, Peptone, Urea, Yeast infusion, and NaNO3, incubated for 5 yearss at pH 7.4 and the enzyme activity was determined.
The settlement morphology and the microscopic staining technique by Lactophenol bluish discoloration confirmed the being to be Aspergillus Niger.
The media used for the solid province agitation is as follows:
Name of Chemical
FeSO4. 7 H2O
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 yearss of incubation.
Orange Peel+Corn Flour ( OCF )
Corn Flour +Green peas Husk ( CGH )
The substrate Corn Flour +Green peas Husk ( CGH ) showed the maximal activity of the enzyme with 12.9 IU/ml.
The consequence of pH on the enzyme production for both the media was studied and the specific activity was determined.
SPECFIC ACTIVITY IU/ml
The maximal 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 severally.
The present probe proceeded by taking different temperatures 20,25,30,35, & A ; 40. The specific activity is determined after incubating for 5 yearss at pH 7.4 for both the substrates.
Temperature ( oC )
SPECFIC ACTIVITY ( IU/ml )
The maximal 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 severally.
The consequence of C beginning on the production of enzyme was studied.
SPECFIC ACTIVITY ( IU/ml )
The maximal activity for OCF was found to be with the C beginning Xylose with 15.4 IU/ml and CGH was found to be with Carbon beginning 14.6 IU/ml severally.
The consequence of Nitrogen beginning on the production of enzyme was studied by taking Peptone, Urea, Yeast Extract and NaNO3.
SPECFIC ACTIVITY ( IU/ml )
L-Asparaginase is an enzyme with great curative value. Much of the survey is done with the bacterial isolates and less studies were recorded with fungous micro-organisms. In the present survey we have used two different natural stuffs as substrates which are really easy available. The consequences showed really 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.