4.3 (a) To investigate the effect of pH on amylase activity
Teacher Notes
Apparatus required per class group of 24 students
Enzyme source eg. Amylase
Starch
Range of buffer solutions
Ph paper
Water bath
1
Boiling tubes
60
Thermometer
12
Disposable gloves
Labels
36
Timer
12
Dropper
12
Boiling tube rack
12
Boiling tube holder
12
The apparatus for this experiment lends itself to being pre-prepared in a ‘box of
equipment’ – see Section 1.
Advance preparation
o Prepare solutions
o Set up water bath at 370C
Advance chemical preparation
(a) Starch solution:
o Shake 1 g of soluble starch (Analar) on to100 ml of water and bring to boiling point to
obtain a clear solution. Cool.
o Make up to100 ml of water.
o Starch solution should be freshly prepared every time it is used.
Biology SLSS 2009
1
(b) Amylase solution
o Add 1g of amylase solution to a little distilled water.
o Make up to 100 ml with distilled water.
Safety precautions
o See MSDS for further information
Expected outcome of experiment
o The enzyme works best at a pH of 7.
o At this pH, the blue black colour will fade the quickest.
Disposal and post-experiment work
o Amylase – mix with excess sand (100 g: 5 L) and put in refuse. Flush solutions with
excess water to foul drain
o Buffers – dissolve small quantities (50 g) in 5 L of water and flush to foul water drain.
Biology SLSS 2009
2
4.3 (a) To investigate the effect of pH on amylase activity
Student Notes
Apparatus required per group
o Amylase solution
o Starch solution
o Range of Buffer solutions
o pH paper
o 6 x Boiling tubes
o 1 x Thermometer
o Labels
o 1 x Timer
o 1 x Boiling tube holder
o 1 x Boiling tube rack
Assembled apparatus
Biology SLSS 2009
3
Method
1. Set up a water bath at 37OC.
2. Into each of 3 boiling tubes put 5 ml starch solution and 3 drops of iodine (each
will go blue / black in colour).
3. Into each of 3 more boiling tubes put 2 ml of amylase solution.
4. To each amylase solution add 5 ml of one of the buffer solutions. Label each
tube with the appropriate buffer, e.g. pH 4, 7, 9 etc.
5. Place all the tubes into the water bath at 37OC and leave for 5 minutes.
6. For each pH pour the amylase into the starch tube. Gently shake the tube to mix
the contents.
7. Replace the tubes in the water bath and measure the time taken for the solution
to go colourless – the blue/black colour will fade as the amylase changes the
starch into sugar.
8. Test for sugar at the end by adding Benedict’s solution to the boiling tube and
heating for 5 minutes – a brick red colour indicates the presence of sugar.
9. Repeat the entire experiment with boiled amylase. This will act as a control.
10. Draw a graph of your results.
11. At the end of the experiment, clean all of the equipment and replace it in its
correct place.
Results:
pH of buffer
Time taken for
solution
colour to change
Conclusion/Comment
Biology SLSS 2009
4