Principle:

If amylase is added to a solution of starch, starch will be digested. As the

reaction proceeds, the progress of the reaction can be visualized by testing:
1. the disappearance of the substrate (starch).
2. the appearance of one of the products (the reducing sugar, maltose).
Two simple tests, iodine test for starch detection and Benedict's test
for maltose detection are used for this purpose.
Procedure:
1. In a test tube, put 2 ml of 2% starch solution and carry out iodine test
as follows: add 3 drops of iodine solution to the content of the test tube;
a blue-black color is formed (N.B. shake starch solution well before
withdrawing any volume).
2. In a test tube, put 2 ml of 2% starch solution and carry out Benedicts
test as follows: add 2 ml of Benedict's reagent to the content of the
test tube and put in a boiling water bath for 5 minutes. The test gives
negative result where Benedict's reagent retains its clear blue color and
no precipitate is formed indicating the absence of maltose (starch did
not undergo any hydrolysis yet).
3. In a test tube, put 2 ml of the previously prepared enzyme solution and
add 2 ml of 2% starch solution; mix well and leave for 10 minutes.
4. Divide the enzyme-starch mixture in 2 clean test tubes and carry out
Iodine test to the first and Benedict's test to the second as follows:
a. To the first test tube, add 3 drops of iodine solution; the test still
gives positive result (blue-black color) indicating the presence of a
remaining part of starch that did not undergo hydrolysis by salivary
amylase.
b. To the second test tube, add 2 ml of Benedicts reagent and put
in a boiling water bath for 5 minutes. The test gives positive result
(development of a precipitate with a color ranging from green,

through yellow and orange to red) indicating the formation of

maltose; the test depends upon the ability of the reducing sugar
maltose to reduce cupric ions (blue) in Benedict's reagent to
cuprous ions (green to red).
II- Effect of temperature on the activity of salivary amylase:
Principle:
Salivary amylase is protein in nature; the optimum temperature for its
activity is 37C. In extreme temperatures (cold and heat), the activity of the
enzyme is inhibited (high temperature causes denaturation of the protein
structure of the enzyme).
Procedure:
1. Get 3 test tubes and number them 1, 2 & 3.
2. Put 2 ml of the previously prepared enzyme solution in each of the
three test tubes.
3. To each test tube, add 2 ml of 2% starch solution and mix well.
4. Immediately put test tube 1 in an ice bath, test tube 2 in a 37C
water bath and test tube 3 in a 70C water bath, each for 20 minutes.
5. Remove each tube to room temperature and carry out Benedicts test
to check for the presence of the starch hydrolysis product maltose as
follows: add 2 ml of Benedicts reagent to the enzyme-starch mixture in
each tube and put in a boiling water bath for 5 minutes; Benedicts
reagent will retain its clear blue color in test tubes 1 & 3 indicating
the absence of maltose as a result of inhibited enzyme activity at cold
and hot temperatures, while a green to red precipitate is formed in test
tube 2 indicating the formation of maltose as a result of a retained
activity of salivary amylase at 37C.

III- Effect of pH on the activity of salivary amylase:

Principle:
The optimum pH for the activity of salivary amylase is the neutral pH (about
7). In the acidic pH of the stomach, the activity of salivary amylase stops.
Pancreatic amylase, on the other hand, has an optimum activity in alkaline
medium.
Procedure:
1. Get 3 test tubes and number them 1, 2 & 3.
2. Put 2 ml of the previously prepared enzyme solution in each of the
three test tubes.
3. To each test tube, add 2 ml of 2% starch solution and mix well.
4. Add 2 ml of acidic solution to test tube 1, 2 ml neutral solution to test
tube 2 and 2 ml alkaline solution to test tube 3. Put the 3 test tubes

in a 37C water bath for 10 minutes.

5. Remove each tube to room temperature and carry out Benedicts test
to check for the presence of the starch hydrolysis product maltose as
follows: add 2 ml of Benedicts reagent to the content of each tube and
put in a boiling water bath for 5 minutes; Benedicts reagent will retain
its clear blue color in test tubes 1 & 3 indicating the absence of
maltose as a result of inhibited enzyme activity in acidic and alkaline
pH, while a green to red precipitate is formed in test tube 2 indicating
the formation of maltose as a result of a retained activity of salivary
amylase in neutral pH.
IV- Effect of time on the activity of salivary amylase:
Principle:
When a specified quantity of salivary amylase is added to a specified
quantity of starch, starch is gradually digested until, by time, all starch is
consumed.
Procedure:
1. Put 5 ml iodine solution in a number of test tubes and number them.
2. In another test tube, put 2 ml of the previously prepared enzyme
solution and add 2 ml of 2% starch solution and mix well.
3. Quickly add 5 drops of the enzyme-starch mixture to the iodine solution
in the first test tube; a deep blue color is formed (zero time).
4. At one minute interval, add 5 drops of the enzyme-starch mixture to
iodine solutions in successive test tubes.
5. Record the time at which iodine retains its yellow color indicating the
full digestion of starch by salivary amylase; this point is known as the
achromic point.