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

  • Prepare solutions
  • Set up water bath at 370C

Advance chemical preparation

(a) Starch solution:

  • Shake 1 g of soluble starch (Analar) on to100 ml of water and bring to boiling point to obtain a clear solution. Cool.
  • Make up to100 ml of water.
  • Starch solution should be freshly prepared every time it is used.

(b) Amylase solution

  • Add 1g of amylase solution to a little distilled water.
  • Make up to 100 ml with distilled water.

Safety precautions

  • See MSDS for further information

Expected outcome of experiment

  • The enzyme works best at a pH of 7.
  • At this pH, the blue black colour will fade the quickest.

Disposal and post-experiment work

  • Amylase – mix with excess sand (100g: 5L) and put in refuse. Flush solutions with excess water to foul drain
  • Buffers – dissolve small quantities (50g) in 5L of water and flush to foul water drain.

4.3 (a) To investigate the effect of pH on amylase activity

Student Notes

Apparatus required per group

  • Amylase solution
  • Starch solution
  • Range of Buffer solutions
  • pH paper
  • 6 x Boiling tubes
  • 1 x Thermometer
  • Labels
  • 1 x Timer
  • 1 x Boiling tube holder
  • 1 x Boiling tube rack

1

Biology SLSS 2009

Assembled apparatus

Method

  1. Set up a water bath at 37OC.
  2. Into each of 3 boiling tubes put 5ml starch solution and 3 drops of iodine (each will go blue / black in colour).
  3. Into each of 3 more boiling tubes put 2ml of amylase solution.
  4. To each amylase solution add 5ml 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 solution / Time taken for colour to change

Conclusion/Comment

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Biology SLSS 2009