Dextranase powder

1 Principle

Dextranase powder hydrolyzes 1,6-α-D-glucan glycoside bonds and releases reducing sugar groups to react with 3,5-dinitrosalicylic acid (DNS reagent). The content is directly proportional. The light absorption value is measured at 540nm, and the standard curve (calculated as glucose) can be used to obtain the amount of reducing sugar, and the glucanase activity can be calculated based on this.

2 Instruments and equipment
2.1 Analytical Balance: Accuracy 0.0001g
2.2 Constant temperature water bath: Accuracy ± 0.2 ℃
2.3 Chronograph
2.4 Spectrophotometer
2.5 boiling water bath
2.6 Oscillating Mixer

2.7 pH meter: accuracy 0.01pH unit

3 Reagents and solutions

3.1 Preparation of pH 5.5 Disodium Hydrogen Phosphate-Citric Acid Buffer: Solution A: 20.369g disodium hydrogen phosphate dodecahydrate, dissolved with about 200mL water; Solution B: 4.530g citric acid monohydrate, with about 200mL water Dissolve; mix liquid A and liquid B and make up to 500mL. Sterilize at 121 ℃for 20min.

3.2 Preparation of 2% dextran (T-2000) solution: accurately weigh 2g dextran (T-2000) into a 100ml beaker, add pH = 5.5 disodium hydrogen phosphate-citrate buffer to dissolve, wait until After complete dissolution, transfer to a 100ml volumetric flask and make up to 100ml with buffer.

3.3 DNS solution preparation:
(1) Weigh 3.15 grams of 3,5-dinitrosalicylic acid and dissolve it with about 500mL of water. It can be dissolved by heating but the temperature cannot exceed 45 ℃.
(2) Weigh 20 grams of sodium hydroxide and dissolve it in about 200 mL of water.
    Add (2) to (1), and then add 91 g of potassium sodium tartrate tetrahydrate, 2.5 g of phenol, and 2.5 g of anhydrous sodium sulfite. After it is completely dissolved, make up to 1000 ml, put it in a brown bottle, and place in a cool and dark place After 10 days, it was filtered through filter paper for use. .
3.4 Preparation of standard glucose solution

Glucose solution (1 mg / ml): Accurately weigh 0.100 g of glucose and dissolve to a final volume of 100 ml with a buffer solution of pH = 5.5.

4 Analysis steps

4.1 Drawing of standard curve
(1) Take about 1 gram of glucose, bake it at 60 ℃ for 30 minutes, then raise the temperature to 80 ℃, then bake for 30 minutes, then raise the temperature to 104℃, bake for 1 hour, and cool down for later use.
(2) Preparation of anhydrous glucose standard solution: 0.1000 grams of glucose is accurately weighed, dissolved and made up to 100 mL.
(3) Take 6 colorimetric tubes with 25mL graduation and test according to the following table:

4.2 Sample determination
4.2.1 enzyme-like preparation
The fermentation broth was centrifuged at 8000 rpm for 10 minutes, the supernatant was taken, and the enzyme solution was appropriately diluted by a certain multiple, so that the final OD540 was in the OD540 range of 0.1-0.6 mg / mL glucose content.

4.2.2 enzyme-like assay
(1) Take 900μL of pH 5.5 buffer to dissolve dextran T-2000 to make a 2.0% solution, add it as a substrate to four suitable test tubes, and place each test tube in a 55℃thermostatic water bath to incubate 5min.
(2) Add 0.1 mL of the appropriately diluted enzyme solution to three of the pre-heated test tubes, and add 0.1 mL of distilled water as the blank for the precise reaction for 10 minutes.
(3) Immediately add 2 mL of DNS to each of the above four test tubes and place in a boiling water bath for precise reaction for 5 min, then quickly cool down, and then make up to 25 mL with distilled water.
(4) The spectrophotometer is used to zero the instrument with water, and the absorbance of each sample tube and blank at 540 nm is measured in a cuvette with an optical path of 1 cm. Record data.
4.2.3 Calculation
Measure the average of the OD values of each measured parallel sample, and calculate the unit of enzyme activity according to the following formula

Dextranase activity (U / mL) = 1000 n (A-b) / (aMt)

Where A is the average value of sample OD540
 b and a are coefficients of the standard curve
 n: dilution ratio of enzyme solution
 t: enzymatic reaction time = 10
 M: Molar mass of glucose = 180.16

CERTIFICATE OF ANALYSIS

Packaging and storage

Solid food packaging bag, 25 kg / barrel.

Dextranase powder should be stored in dry, cool and well ventilated places away from the sun, heat.

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