Detection method of pectinase

Detection of pectinase activity

[Purpose]

This test method is used for the catalytic activity of pectinase. The method is suitable for various solid and liquid pectinase preparations.

[description]

This method is suitable for quality analysis and quality control of pectinase. But it is not a prediction of the absolute activity of products of our company and other companies. The final enzyme activity of various enzyme preparations can still play a good catalytic activity under good experimental operation.

[principle]

Pectin mainly exists between plant primary wall and cells, and pectin is the matrix polysaccharide of cell wall. Pectin includes two acidic polysaccharides (polygalacturonic acid and rhamnolic acid) and three neutral polysaccharides (arabinan, galactan and arabinogalactan). Pectinase is essentially a polygalacturonase hydrolase. Pectinase hydrolyzes pectin and mainly produces β -galacturonic acid. Sodium iodate method can be used to quantify galacturonic acid, thus determining the activity of pectinase.

[Definition of pectinase active unit]

1g (or 1ml liquid enzyme) enzyme powder, under the condition of 50.0℃ and pH3.5, the amount of enzyme catalyzing pectin hydrolysis to produce 1μ g galacturonic acid per minute is one active unit.

1. reagents and instruments

* All reagents used in this standard are analytically pure without any explanation.

1. 1 acetic acid

1.2 iodine

1.3 potassium iodide

1.4 concentrated sulfuric acid

1.5 Pectin (Sigma Company)

1.6 sodium thiosulfate

1.7 sodium carbonate

1.8 soluble starch

1.9 water bath pot

1. 10 iodine volumetric flask

2. Preparation of reagents

2.1acidic water with pH value of 3.5

Adjust distilled water to 3.5 with acetic acid.

2.2 1% pectin solution:

Accurately weigh 1g analytically pure pectin, dissolve in acid water, boil, cool, and filter to 100ml.

2.2 0. 1N iodine solution:

Accurately weigh 5g of potassium iodide, dissolve it in distilled water, add 2.54g of iodine, and after dissolution, fix the volume to 100ml.

2.3 0.025 mol/L sodium thiosulfate:

Accurately weigh 6.2g sodium thiosulfate, add distilled water, and adjust the volume to1l.

2.4 0.5% soluble starch indicator:

Accurately weigh 0.5g of soluble starch and boil it in boiling water until it is transparent.

2.5 1M sodium carbonate solution:

Accurately weigh 10.6g sodium carbonate and put it into 100ml water.

2.6 2N sulfuric acid:

Absorb 10ml concentrated sulfuric acid and pour it into 170ml water.

2.7 Preparation of enzyme samples

Accurately weigh 1.000g of solid enzyme or remove 1ml of liquid enzyme sample, fix the volume to 100ml, soak in a water bath at 50℃ for 1 hour, and filter, and the filtrate is the enzyme solution to be detected. The enzyme was diluted 100 times.

3. Procedures

3. 1 Take 1% pectinase 10ml, add 5ml of enzyme solution and 5ml of distilled water (PH3.5), and react in a water bath at 50℃ 1 hour.

3.2 Take out and cook for 2~3min. After cooling, replenish water to 20ml.

3.3 Take 5ml of reaction solution in a 100ml iodine volumetric flask, add 5ml of 1M sodium carbonate solution 1ml and 0. 1N iodine solution, shake well, and keep it in the dark for 20min at room temperature.

3.4 After taking it out, add 2ml of 2n sulfuric acid, immediately titrate it to light yellow with 0.05N sodium thiosulfate solution, add1ml of 0.5% soluble starch solution, and continue titration until the blue color disappears.

3.5 In the blank test, titrate with boiled inactivated enzyme solution or distilled water instead of enzyme solution.

3.6 Prepare at least two parallel samples for each enzyme sample.

calculate

4. 1 Find the average of the measured OD values of parallel samples.

4.2 Calculate the activity unit of the enzyme according to the following formula

Enzyme activity = [(b-a) * n * 0.5 *175 * 20 * n *1000]/[51* 52 * w * 60].

Unit: Microgram (mL)

Where: a: the number of milliliters of sodium thiosulfate consumed in sample titration.

B: the number of milliliters of sodium thiosulfate consumed in blank titration.

N: molar concentration of sodium thiosulfate.

0.5: 1 equivalent sodium thiosulfate is equivalent to 0.5 equivalent galacturonic acid.

20: Total volume of reaction liquid