PROTEASE ENZYME PRODUCTION FROM FOOD WASTE

 

PROTEASE ENZYME PRODUCTION FROM FOOD WASTE

 

AIM: To isolate protease producing organism and assay the enzyme protease from food waste.

 

MATERIALS REQUIRED: Skim milk agar, Baal media for enzyme production, Centrifuge, azocasein, Tris HCl, Trichloroacetic acid, NaOH, UV-visible spectrophotometer, Bovine serum albumin

 

PROCEDURE:

I. Isolation of protease producing bacterial isolates:

 

1.      Food waste samples were randomly collected from near the house.

2.      One gram of the soil was suspended in 9 mL of sterile distilled water.

3.      After a serial dilution (10¹ to 10⁶) of this suspension with sterile distilled water, 100 mL from each diluted suspension was spread on skim milk agar (SMA) plates (2.0% skim milk, 1.0% glucose, 1.5% agar and pH 9.0) and incubated at 37⁰C for 24-48 h.

4.      Bacteria showing clear zones due to hydrolysis of casein in milk were selected as protease producers.

5.      Based on the ratio of clear zone, screening of protease producer was carried out and then pure cultures were obtained on SMA media.

 

II. Production of crude protease in the shake flask:

1.      A basal media (l.0% glucose, 0.5% peptone, 0.5% yeast extracts, 0.1% K₂HPO₄, 0.01% MgSO₄ and 0.5% Na2CO₃; pH 9.0) was used for alkaline protease production unless noted.

2.      For seed culture, 5 ml of sterile basal media was inoculated with an isolated colony and incubated at 37⁰C and 120 rpm for 20 h.

3.      The seed culture was then transferred to 45 ml production media in a 250 ml conical flask and incubated under the conditions as indicated.

4.      The cell free supernatant was obtained by centrifugation at 8000 rpm for 20 min at 4⁰C and used for determining the protease activity or further study.

 

III. Enzyme assay and protein estimation:

 

1.      The reaction mixture was (500 ml of 1.0% azocasein in 50 mM Tris HCI, pH 9.0 and 1 ml of enzyme solution) incubated for 30 min at 40⁰C.

2.      The reaction was stopped by adding 2.8 ml of 5.0% (w/v) trichloroacetic acid (TCA), and was kept at 4⁰C for 15 min.

3.      The precipitate was removed by centrifugation at 8,000 rpm for 20 min.

4.      A 2 ml of the supernatant was added to 2 ml of 1.0M NaOH and the absorbance was measured at 440 nm using a UV-visible spectrophotometer.

5.      One unit of protease activity was defined as the amount of enzyme that produced an increase in absorbance of 0.01 under the above assay conditions.

6.      For negative control, 5.0% of the TCA solution was added before addition of enzyme preparation. Total protein concentration was determined with Bradford protein assay kit using bovine serum albumin as a standard protein.

ACKNOWLEDGE: We are thankful the authors who have been published this article in open source. Al Hakim, Farhana Rumzum Bhuiyan,Asif Iqbal,Tanvir Hossain Emon,Jahed Ahmed,Abul Kalam Azad. Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes. Heliyon 4 (2018) e00646. doi: 10.1016/j.heliyon.2018. e00646