Experimental effects of temperature and pH on salivary amylase activity

This experiment takes the action of salivary amylase on starch at different temperatures and pH as an example to observe the effect of temperature and pH on the enzyme activity, and the degree of hydrolysis of starch is distinguished by its coloring reaction with iodine solution. (Source: Laboratory instruction of biochemistry and molecular biology, Shao Xueling et al, Wuhan University Press, 2003)

Operation method

iodine color development method

Principle

Enzymes are proteins, and their activity is significantly affected by the pH of the environment. Usually all kinds of enzymes only in a certain pH range to show its activity, an enzyme performance of its highest activity when the value of pH, known as the enzyme's optimal pH.

Materials and Instruments

Starch
Iodine Potassium iodide Disodium hydrogen phosphate Citric acid
Test tubes Suction tubes Test tube holders Suction ear balls

Move

Solution Preparation:

0.5% starch solution (containing 0.3% NaCl) (freshly prepared), iodine-potassium iodide solution (4 g of iodine and 6 g of potassium iodide dissolved in 100 ml of distilled water and kept in a brown bottle), 0.2 mol/L disodium hydrogen phosphate solution, 0.1 mol/L citric acid solution.

1. Sample collection.

Saliva was collected after gargling, filtered through a funnel with a small amount of skimmed cotton wool, and the filtrate was diluted 100-fold and set aside.

2. Effect of pH on enzyme activity

(1) Preparation of buffer solution



(2) Preparation of substrate

Six dry test tubes were numbered and 3 ml each of buffer solutions of different pH were added sequentially, the 6th tube was the same as the 3rd. Then add 2 ml of 0.5% starch solution to each test tube and shake well.

(3) Enzymatic reaction time measurement

Add 2 ml of 100-fold diluted saliva to test tube No. 6, shake well and put into a 37 ℃ constant temperature water bath. Take 1 drop of the mixture on a white porcelain plate every minute, add 1 drop of potassium iodide-iodine solution, remove the test tube when it turns orange-yellow and record the time.

(4) Optimum pH determination

At 1 min intervals, add 2 ml of 200-fold diluted saliva to test tubes 1 to 5 sequentially, shake well, and also at 1 min intervals, place 5 test tubes into a 37 ℃ thermostatic water bath to keep warm, and react until the desired time. Remove them in turn and immediately add 2 drops of potassium iodide-iodine solution and shake well. The color was observed to show the degree of starch hydrolyzed at different pH values, the effect of different pH values on salivary amylase activity, and to determine its optimum pH.

3. Effect of temperature on enzyme activity

(1) Take three test tubes and follow the table below:



After adding, respectively, continue to leave for 10 min according to the above conditions.

(2) Remove 1 drop of solution from the three test tubes on the reaction plate, add 1 drop of iodine solution, observe the coloration phenomenon, record the results and explain the reasons.

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