protease production and assay

PROTEASE ENZYME PRODUCTION AND ASSAY

AIM: To isolate the protease enzyme producing organism from the environment and assay the enzyme production

PRINCIPLE: Proteolytic enzymes are ubiquitous in occurrence found in all living organisms and are essential for cell growth and differentiation. These enzymes not only play an important role in the cellular metabolic processes also gained considerable attention in the industrial community. Commercial proteases are mostly produced from various bacteria. Culture conditions play significant role on growth and production of protease by bacteria. Based on their acid-base behaviour, proteases are classified into three groups i.e. acid, neutral and alkaline proteases. The majority of commercial alkaline proteases are produced by bacteria, especially Bacillus sp. Several Bacillus species involved in protease production are B. cereus, B. sterothermophilus, B. mojavensis, B. megaterium and B. subtilis. Identification and characterization of microbial proteases are pre-requisite for understanding their role in pathogenesis. Proteases are also useful and important components in biopharmaceutical products. Currently, Protease performs numerous varieties of activities in detergent, food, pharmaceutical, leather, laundry, food processing etc.

MATERIALS REQUIRED:

Media and reagents: Bacterial or Fungal culture, Skim milk agar, Casein agar, SD Agar, Potato Dextrose Agar, Lacto phenol cotton blue &Gram’s staining kit, Folin’s reagent, IMViC Media, TSI Agar, phosphate buffer, 1% casein solution, 20% TCA (trichloroacetic acid).

Glassware: Petri plates, Conical flasks, Pipettes, Glass slides

Instruments: Spectrophotometer, Microscopes, Centrifuge, Colony counter, Waterbath.

PROCEDURE: (For bacteria)

1.      Sampling: The test soil samples were collected from the different sites of DRNGPSC Campus garden soil with aid of sterile spatula from 4-5 cm depth in to sterile plastic bags.

2.      Soil samples were air dried at room temperature.

 Isolation and purification:

3.       Isolation of bacteria from soil carried out by serial dilution method and isolated bacterial colonies were purified by sub culturing and stored as slants at 4±2⁰ C.

4.      Characterization of bacterial culture Various biochemical tests were performed for the identification and characterization of isolated bacteria viz Gram staining, Catalase test, Casein hydrolysis, IMViC test, Urease test, Nitrate test.

5.      Morphological, cultural, physiological and biochemical properties of the isolated strains were studied according to the methods given in Bergey’s manual of systemic bacteriology.

 Enzymatic study:

6.      Qualitative screening of bacteria (for protease)

The bacteria were streaked on casein hydrolysed medium and plates were incubated at 35±2⁰ C for 24 hours. A clear zone around the growth indicates proteolytic activity of the strains and visible difference in the extent of zone of clearing was recorded for proteolytic activity

7. Standard curve Prepared different concentration of protein (10¹ - 10¹⁰) in different test tubes by taking 0.1ml of protein and 0.9ml of distilled water and so on.

8. Added 1mlof protein and 1 ml of 1N NaOH in test tube and kept in water bath at 100^o C for 10 min. Tubes were cooled at room temperature and after 10 min add 1ml Folin’s reagent and leave it for 30 min and absorbance was measured at 750 nm.

     9. Quantitative determination (Proteolytic activity): Casein-yeast extract-dextrose broth were used for the enzymatic activity.

    10. Protease Assay The Protease was assayed by 3ml of bacterial supernatant (centrifuged 24hr old culture at 5000 rpm for 20 min) take in sterile test tube add 3ml phosphate buffer and 3ml 1% casein solution and place it on water bath at 35^o C. now add 5ml 20% TCA (trichloroacetic acid) added in reaction.

     11. Content was immediately mixed after adding Folin Ciocalteu Reagent in the mixture after 30 min 6 ml distilled water was added. Now absorbance was measured at 650 nm in every 24 hr till 72 hrs.

PROCEDURE: (For fungi)

1.      Organism and inoculums preparation fungal strains were isolated from soil of DRNGPASC garden soil, Coimbatore, by serial dilution plate method.

2.      Fungus were isolated from 10^-3 - 10^-4 dilutions by plating into Potato Dextrose Agar (PDA) medium. Isolated fungal cultures were screened for protease enzyme production.

3.      The organisms were identified using lacto phenol cotton blue mounting method.

4.      The isolated culture (Aspergillus flavus) was purified by routine sub-culturing and stored at 4^oC for further use.

5.      Enzyme assay by plate zone method:  The activity of alkaline protease was also measured by casein and gelatin plate technique. One unit of enzyme activity was defined as the amount of enzyme in 25µl of enzyme solution that produced a clear zone of 1mm² at pH 8 and 30°C for 18 hours. 25µl - 1mm² 1U 50µl - 20 mm² 10U 100µl - 30 mm² 15U The 10 U for 50µl and 15U of activity for 100µl were achieved by alkaline protease.

Figure: Casein and gelatin was hydrolyzed by alkaline protease showed a clear zone.