PRODUCTION OF PROTEASE ENZYME BY ASPERGILLUS NIGER THROUGH SSF USING RICE BRAN AS SUBSTRATE

ABHISHEK PRAKASH (U07BT001)

ANAND KUMAR RAWANI (U07BT008)

ANIL KUMAR (U07BT009)

MANISH KUMAR DASH (U07BT036)

A.STANLEY MATHEW (U07BT066)

DEPARTMENT OF BIOTECHNOLOGY

AIM

To isolate Aspergillus niger from onion for the extraction of protease enzyme and estimation of protease activity.

OBJECTIVE To isolate and identify the micro organisms

Aspergillus niger from natural sources (onion).

Aspergillus niger was isolated in PDA. Screening of Aspergillus niger was done using

lactophenol cotton blue staining technique. SSF was done to extract protease enzyme

using Aspergillus niger. The extracted crude enzyme was put in the

casein plate and the zone of clearance was observed.

To find out the concentration of protein , Lowry’s method was used.

Protease enzyme assay was done using spectrophotometric studies.

METHOD 1. ISOLATION OF Aspergillus niger FROM ONION

Aspergillus niger spores from onion is inoculated in potato dextrose agar and incubated at room temperature for 3-5 days.

PROCEDURE 100 ml of the potato dextrose agar media was prepared as per the

composition and autoclave at 120°C 151 lb pressures for 15 min. 10 test tubes were taken and then 9 ml of distilled water was poured in

each of the test tube , plugged and autoclave at 120°C 151lb pressure for 15 min.

PDA media was poured into 5 petridishes and allowed to solidify. The onion was serially diluted and subjected to planting.

1ml of the diluted suspension was added to the petridishes from various dilutions.

Incubated at 25°C for 2-3 days.

2. IDENTIFICATION OF FUNGI THROUGH STAINING TECHNIQUE Lacto phenol cotton blue was used to stain the fungus. In a clean glass slide, 2 to 3 drops of lacto phenol cotton blue was

transferred to the slide in a sterilized condition. Using the flame sterilized forceps , a little amount of fungi was transferred

from the colony to the staining solution. The fungal mass was smeared into small pieces properly without losing

the integrity using needles. Slide is covered with the cover sip and observed under microscope.

FIG. - MICROSCOPIC VIEW OF Aspergillus niger

3. INOCULUM PREPARATION PD broth media was prepared as per the composition and dissolve it in

150ml of distilled water. The media was autoclaved at 121°C at 151lb pressure for 15min. After autoclaving, cool the media and the media was poured into

petriplates under sterile condition . A small amount of fungal growth was transferred from the masterplate to

the broth in the conical flask to the test tubes. The conical flask and test tubes were incubated at 25°C.

4. MASS PRODUCTION OF ASPERGILLUS NIGER USING SOLID STATE FERMENTATION

500g of rice bran was mixed with 500ml of distilled water , 5g of casein was added in the mixture. It was autoclaved and allowed to cool. The cultivation was carried out in the Czapek Dox medium as the following composition (g/l):

K2HPO4 - 1 ; MgSO4.7H2O - 0.5; KCL - 0.5; FeSO4.7H2O – 0.01; SUCROSE – 30g

The culture broth of A.niger was added to the medium flask and shaken well;

This should be done in sterile condition. The solid state fermentation was followed using rice barn as substrate

culture and incubated at 25°C for 7-8 days.

5. EXTRACTION OF CRUDE ENZYME SOLUTION The rice bran culture was mixed with 5 litres of water. The mixing should be done under cold conditions. Then the solution was filtered through a filtrate cloth. The solution was [filtrate] was subjected to centrifugation. The extracted solution was transferred to the centrifuge tubes. The rpm was set to 10,000 for 10 minutes. The supernatant was collected and pellet was discarded.

6. SCREENING FOR PROTEASE ENZYME USING PLATE ASSAY METHOD

Agar was distilled in 75ml of distilled water , autoclaved at 121°C for 15 min at 15lb pressure.

Milk powder was dissolved in 25ml of water by heating to 50°C. The milk solution was sterilized at 113°C T, 8lb pressure for 20min.

Allowed to the cool at 57°C and combine in one flask. It was poured into a petri plate and allowed to solidify. The fungal smear was taken and added to the centre of the media and

incubated for 2-3 days at 25°C. Zone of clearance was observed in the plate which shows the presence of

protease in crude extract.

7. PROTEIN ESTIMATION BY LOWRY’S METHOD

Protein solution are treated with alkaline copper sulphate solution and foline phenol the blue color complex which is due to the reduction of phospho tungstate and phospho molybdate in folic reagent by tyrosin and cryptophan residue of protein. The intensity of blue colour development at 640 nm calorimetrically.

  SUMMARY AND CONCLUSION

Aspergillus niger was isolated from onion and it was identified by Lactophenol cotton blue.

Fungal colony was subcultered and screened for protease activity. Bulk quantity of enzyme was produced by solid state fermentation and it was

subjected to Extraction and Downstream Processing. Total protein content was estimated by using Lowry method and its activity was

measured.

From the data presented in this report, it can be concluded that the current protease activity of this strain may be quiet different from the other Aspergillus species in its properties. The protease concentration was highest on seventh day which shows that with optimum conditions , the maximum yield can be enhanced.

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