Protease Enzyme Assay

Aim:

To determine the protease activity of given bacterial culture.

Introduction:

            Protease enzyme is naturally present in all organisms and it corresponds to 1-5% of total protein content Protease is the third largest group of industrial enzymes and has a worldwide sale of 60%. Proteases can hydrolyse peptide bonds in proteins and they are also called peptidase or proteinase or proteolytic enzymes . Proteases are classified into three groups based on their acid base behavior that is, acid, neutral, and alkaline proteases . Acid proteases have a pH range of about 2.0-5.0 and they are produced only by fungi. Neutral pH of protease ranges from 7.0-8.0 and they are mainly of plant origin and finally proteases with pH above 8 are said to be alkaline proteases. Proteolytic enzymes are ubiquitous in nature and they are found in all living organisms such as plants, animals and microbes.

            The protease producing bacterial strains are as Bacillus subtilis, Bacillus licheniformis, and Bacillus thuringiensis.

Materials required:

Casein 

Carbonate buffer

10 % TCA (Trichloro acetic acid solution)

Bacterial broth culture

Folin ciocalteau reagent

Sodium carbonate

Preparation of 0.44M Na2CO3

0.2 M solutions each of sodium carbonate anhydrous (21.2 g/L) and sodium hydrogen carbonate (16.8 g/L) were prepared. 50 ml of 0.2 M sodium carbonate solution was pipette into a 100 ml volumetric flask and made upto the mark with 0.2 M sodium hydrogen carbonates.

Tyrosine Stock solution:

200 mg of Tyrosine was dissolved in distilled water to make 100 ml in a volumetric flask, which resulted in 200 mg  µg/ml.

Estimation of Protease :

Casinase activity determination by using 2% casein  in 0.2 M carbonate buffer  (pH 10) as substrate. Casein solution (0.5 ml) with an equal volume of suitably diluted enzyme solution was incubated at 55°C. 

After 10 min, the reaction was terminated by the addition of 1 ml of 10% trichloroacetic acid. 

The mixture was centrifuged.

The supernatant was added 5 ml of 0.44 M Na2CO3 and 1ml of two-fold diluted Folin Ciocalteau reagent. 

After 30 min, the colour developed was read at 660 nm against a reagent blank prepared in the same manner.

Tyrosine served as the reference standard. The optical density of these solutions was measured in a Shimadzu (Japan) spectrophotometer.

One unit of enzyme activity was defined as the amount of enzyme that released one ug of tyrosine per ml per min.

Tyrosine Standard:

Into a series of 10 ml volumetric flasks 1,2, 3,4, and 5 ml of standard stock solution of tyrosine was taken and distilled water was added to make upto 10 ml mark in each volumetric flask. 

Mixed well and the optical density was measured at 660 nm after developing the colour as described above, against a reagent blank prepared in same manner.

A standard curve was constructed taking concentration of Tyrosine µg/ml on X-axis and corresponding optical density on Y-axis.

Result: