Polyphenol oxidase purification and viability determination - Database & Sql Blog Articles

Purification and Viability Determination of Polyphenol Oxidase

Purpose Requirement

1. Learn how to extract and purify polyphenol oxidase.

2. Master the principle and method for measuring the activity of polyphenol oxidase.

Experimental Principle

Many plants gradually turn brown when exposed to air after mechanical damage. This browning is caused by the reaction between polyphenol oxidase (PPO) and phenolic compounds that are released when plant cells are broken.

Polyphenol oxidase is a copper-containing enzyme that catalyzes the oxidation of phenolic compounds into quinones. In recent years, research on plant tissue browning has shown that this process is mainly due to PPO acting on phenolic substances in natural substrates.

Reagents and Equipment

1. Reagents:

- 0.25 mol/L potassium phosphate buffer (pH 7.2)

- 0.15 mol/L catechol solution

- 10 mmol/L Tris-HCl solution (pH 8.3)

- Ammonium sulfate

2. Materials:

- Pear

3. Equipment:

- Tissue homogenizer

- Funnel

- Filter paper

- Spectrophotometer

Method of Operation

1. Extraction of Polyphenol Oxidase (PPO):

(1) Acetone powder preparation: Take 100g of pear pulp, add 200mL of ice-cold acetone (about 20°C), and homogenize using a high-speed tissue masher for 5 minutes. Filter the mixture with medium-speed filter paper on a funnel rack, and wash the residue with ice-cold acetone repeatedly until it turns into white powder. This is the acetone powder.

(2) Preparation of enzyme solution: Weigh 1g of acetone powder and add 20mL of 0.025mol/L phosphate buffer (pH 7.2). Homogenize with a magnetic stirrer at 0°C for 30 minutes. Centrifuge at 12,000 r/min for 30 minutes. The supernatant obtained is the crude enzyme extract.

2. Purification of Polyphenol Oxidase:

Add ammonium sulfate to the enzyme solution to reach 80% saturation. Stir for 30 minutes, then let it stand overnight. Centrifuge at 12,000 g for 30 minutes. Dissolve the precipitate in 0.025 mol/L phosphate buffer (pH 7.2) and dialyze in 10 mmol/L Tris-HCl (pH 8.3) for 18 hours, performing dialysis three times during this period. This is the purified polyphenol oxidase.

3. Determination of Enzyme Activity:

Use catechol as the substrate. In a 1 cm cuvette, add 2.75 mL of 0.25 mol/L phosphate buffer (pH 7.2) and 0.1 mL of 0.15 mol/L catechol solution. Mix at room temperature. After 3 minutes, add 0.1 mL of enzyme solution and start recording the change in A420 value within 1 minute. One unit of enzyme activity is defined as the amount of enzyme required to cause an absorbance change of 0.001 per minute.

Precautions

Polyphenol oxidase is highly sensitive and easily inactivated. It is recommended to extract the enzyme at low temperatures to maintain its activity.