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 Title: Extraction of Papain from Papaya Leaves and Estimation of Its Activity in Terms of Ammonia

Aim: To extract papain from papaya leaves and estimate its activity by measuring the amount of ammonia released.

Introduction: 

Papain is a cysteine protease enzyme found in papaya (Carica papaya) leaves and latex. It hydrolyzes proteins into peptides and amino acids and has applications in food, pharmaceutical, and textile industries. The enzyme activity can be estimated by quantifying the ammonia released during protein hydrolysis using Nessler’s reagent.

Theory: 

Papain hydrolyzes proteins by cleaving peptide bonds, releasing amino acids and ammonia as byproducts. The ammonia produced can be detected and quantified using Nessler’s reagent, which forms a yellow to brown color complex proportional to the ammonia concentration.

Principle: 

The extracted papain hydrolyzes casein or another protein substrate, releasing ammonia. Nessler’s reagent reacts with ammonia to produce a yellow-brown complex, which is measured spectrophotometrically at 405 nm to determine enzyme activity.

Requirements:

• Fresh papaya leaves

• Sodium phosphate buffer (pH 7.0, 50 mM)

• Sodium chloride (NaCl)

• EDTA (Ethylenediaminetetraacetic acid)

• Cysteine hydrochloride

• Acetone

• Casein solution (1% w/v)

• Nessler’s reagent

• Trichloroacetic acid (TCA) (10% w/v)

• Water bath (37°C)

• Centrifuge

• Spectrophotometer

• Test tubes and pipettes

Procedure:

1. Extraction of Papain: 

• Wash fresh papaya leaves and homogenize them in 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl, 5 mM EDTA, and 5 mM cysteine hydrochloride. 
• Filter the homogenate through muslin cloth and centrifuge at 10,000 rpm for 15 minutes at 4°C. 
• Collect the supernatant as the crude papain extract.

2. Enzyme Assay: 

• Prepare the reaction mixture with 1 mL of 1% casein solution and 1 mL of sodium phosphate buffer (pH 7.0). 
• Add 0.5 mL of the papain extract and incubate at 37°C for 10 minutes. 
• Stop the reaction by adding 2 mL of 10% TCA and centrifuge the mixture at 5,000 rpm for 10 minutes. 
• Take 1 mL of the supernatant and add 1 mL of Nessler’s reagent. 
• Incubate at room temperature for 5 minutes and measure absorbance at 405 nm using a spectrophotometer.

3. Standard Curve Preparation: 

• Prepare ammonia standard solutions (0.1–1.0 mM) and react with Nessler’s reagent. 
• Measure absorbance and plot a standard curve.

4. Calculation of Enzyme Activity:

• One unit of papain activity is defined as the amount of enzyme releasing 1 µmol of ammonia per minute under standard conditions.

• Enzyme activity (U/mL) is calculated using:

Observation:

• Formation of a yellow to brown color indicates the presence of ammonia.

• The intensity of the color is proportional to the amount of ammonia released.

Result: The activity of papain was determined by measuring the ammonia released from protein hydrolysis using Nessler’s reagent. The enzyme unit was calculated accordingly.

Conclusion: Papain was successfully extracted from papaya leaves, and its activity was estimated based on ammonia release. This method provides an efficient way to evaluate the proteolytic activity of papain for industrial and research applications.