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

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Purification and viability determination of polyphenol oxidase

Purpose requirement

1. Learning to extract and purify polyphenol oxidase

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

Experimental principle

Many plants are gradually browned in the air when they are mechanically damaged. This is the result of contact reaction between polyphenol oxidase and polyphenols which are separated from each other when the plant cells are broken.

Polyphenol oxidase is a copper enzyme that catalyzes the conversion of phenolic compounds into hydrazine. In recent decades, studies on browning of plant tissues at home and abroad have shown that browning of tissues is mainly caused by PPO acting on phenolic substances in natural substrates.

Reagents and equipment

1, reagent

0.25 mol/L potassium phosphate buffer (ph7.2); 0.15 mol/L catechol solution; 10 mmol/L Tris-HCI 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 pulp tissue, add 200mL, ice acetone (about 20 °C), homogenize with high-speed tissue masher for 5min, filter the mixture with medium-speed filter paper on the funnel rack, and wash the residue with ice acetone repeatedly, filter until it becomes White powder, this powder is acetone powder.

(2) Preparation of enzyme solution: Weigh 1g of acetone powder, add 20mL, 0.025mol/L phosphate buffer (pH7.2), homogenize with magnetic stirrer for 30min at 0°C, centrifuge at 12000r/min for 30min, take The supernatant was obtained as a crude enzyme extract.

2. Purification of polyphenol oxidase

Add NH4SO4 to the above enzyme solution to 80% saturated solution, stir for 30 min, overnight, centrifuge at 12000 g for 30 min, and dissolve the precipitate in 0.025 mol/L phosphate buffer (pH 7.2) for 10 mmol/L, Tris-HCI ( pH 8.3) dialysis for 18 h, 3 times during the period, which is the purification of polyphenol oxidase.

3. Determination of the activity of polyphenol oxidase

Using catechol as a substrate, add 2.75 mL, 0.25 mol/L phosphate buffer (pH 7.2), 0.1 mL, 0.15 mol/L catechol solution in a 1 cm cuvette, and mix at room temperature. After 3 minutes of storage, 0.1 mL of enzyme solution was added, and after 5 s, the change of A420 value was started within 1 min, and the amount of enzyme required for the enzyme activity to change 0.001 per minute of light absorption was 1 vitality unit.

Precautions

The polyphenol oxidase is easily inactivated, and the enzyme is preferably extracted at a low temperature.