How to test α-amylase activity with DNS method

Take 1ml enzyme liquid in the test tube, heat up at 70 ° C for 15 minutes, quickly cool down, preheat 15min at 40 ° C, then add 2ml40 ° C to preheat 1%soluble starch, react to 5min at 40 ° C after shaking, quickly cool down and cool down and cool down quickly.Add 4ml0.4m sodium hydroxide to terminate the reaction, then take a 1ml reaction solution in the test tube, add 1mldns, boil water bath for 5min, quickly cool, set the OD value to 15ml, and test at 520nm.
Reference: To live with sodium hydroxide with sodium hydroxide, and then add starch, other steps are the same.
Blank: Use 1ml distilled water instead of 1ml reaction solution, other steps are the same.
The definition of enzyme vitality unit: Under the above conditions, the substrate is defined as an enzyme vitality unit U (unauthorized) under the 1min catalytic production of 1 μmol.

I think the steps I use are small, and the enzyme degradation reaction is small, which is convenient for large -scale operations.

The formula of the DNS reagent I used is as follows (1L):
200g of potassium sodium sodium sodium, 20g of sodium hydroxide, 0.5g of sodium sodium sodium, 2g crystalline phenol, DNS 10g, room temperature avoiding light and calmness. It can be used after 7 days.

The steps of my operation are as follows:
2.2.6.1.4 Measurement of the vitality of amylase
(1) Collect fermentation upper liquid and make appropriate dilution.
(2) Add 350 micro-lift 0.1m-soluble starch to 0.1m citric acid-0.2m hydrogen phosphate buffer to 2ml EP tube, preheat 3min in a water bath pot that has been adjusted to temperature; [[[[[[[[[[[ 123] (3) Take the amylase crude enzyme liquid 50 microsexation to the buffer and mix well;
(4) After the setting time is reached, the reaction of the 0.8ml DNS reagent is added with each tube;
(123] ((123] ((123] ( 5) Cold water for 5 min boiled in boiling water. The instant centrifugation will be thrown off the liquid attached to the wall of the tube;
(6) The surplus liquid of boiling 5 minutes of boiling water is also treated as a control;
(7) Take 200 micro -ascending to the serum board and measure the light inhalation value at 540nm.

There are three repetitions for each experiment. The definition of enzyme vitality: Under the measured pH and temperature conditions, hydrolysis 1%soluble starch, the amount of enzymes required for 1 microcoltanin per minute is an enzyme vitality required for one enzyme vitality. unit.

As for the enzymes that interference, in addition to the 3.2.1.1 required by the landlord, there are also 3.2.1.2 that you want 70 degrees of extent on the upstairs, as well as 3.2.1.3 and 3.2. 1.10, the products that degrade the starch can be determined by the DNS method, and they will interfere. The DNS method is not a dedicated measurement method. Many of my fungi can produce 3.2.1.1, the most suitable temperature is less than 50 degrees, and it will be lost in 70 degrees for so long.

The DNS method is actually a commonly used method to determine the content of reducing sugar, and amylase can hydrolyze starch to become restored sugar (glucose), so you only need to take a certain amount of starch, add a certain amount of amylase, and control the effect of enzymes to control the effect of good enzymes.Conditions, then extinguish enzyme activity, and then measure the restore sugar content with DNS method, thereby calculating the enzyme activity size

There is a biochemical experimental book

What does your standard curve use