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

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Purification and Viability Determination of Polyphenol Oxidase

Purpose Requirement

1. Learn how to extract and purify polyphenol oxidase (PPO).

2. Master the principle and method for measuring PPO activity.

Experimental Principle

When plant tissues are damaged, they gradually turn brown in the air. This browning is due to the reaction between polyphenol oxidase and phenolic compounds that are released when the cells break open.

Polyphenol oxidase is a copper-containing enzyme that catalyzes the oxidation of phenolic compounds into quinones. Recent studies have shown that tissue browning is mainly caused by 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 (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), grind using a high-speed tissue masher for 5 minutes. Filter the mixture with medium-speed filter paper, wash the residue with ice-cold acetone repeatedly until it turns white, resulting in acetone powder.

(2) Enzyme solution preparation: Weigh 1g of acetone powder, add 20mL of 0.025 mol/L phosphate buffer (pH 7.2), stir at 0°C for 30 minutes using a magnetic stirrer. Centrifuge at 12,000 rpm for 30 minutes, and collect the supernatant as the crude enzyme extract.

2. Purification of Polyphenol Oxidase

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

3. Determination of Polyphenol Oxidase Activity

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

Precautions

Polyphenol oxidase is highly sensitive and easily inactivated. It is best to extract the enzyme at low temperatures to maintain its activity. All steps should be performed quickly and under controlled conditions to prevent degradation.