The Effect of Temperature on Amylase

Results

1.)

2.) A graph was plotted showing rate as 1/T against temperature. (See Appendix 1).

Experiment 2

Aim

The aim of the investigation is to measure the time taken for amylase to completely break down starch.  Samples will be withdrawn at 10 second intervals and noting the time at which the solution no longer gives a blue-black colour with iodine solution and the iodine solution remains orange. Buffers will be used to provide solutions at different pHs. 

Apparatus and Chemicals.

• Amylase 1% (or 0.5%)
• Starch 1% (or 0.5%)
• Buffer solutions covering a range of pH, each with a labelled syringe/ plastic pipette
• Syringes, 5 cm3, 2 (1 for starch, 1 for amylase)
• Iodine solution in a dropper bottle
• Test tube rack
• Test tube, 1 for each pH to be tested
• Dimple tile or white tiles
• Teat pipette
• Stop clock
• Marker pen

Health & Safety

• Wear eye protection must be worn when handling iodine solution.
• Hazards of buffers are varied.

Method

1.  A fresh starch suspension was made.
2. A solution of 5 g soluble starch was produced in cold water. 500 cm3 of boiling water was poured in and stirred.
3. This was boiled until a clear solution was produced. Modified starch was not used.
4. Sodium hydroxide was within the universal buffer at approximately 0.25 M was labelled an irritant.
5. Reference to relevant Hazcards was always carried out.
6. A 0.01 M solution was suitable for starch testing. This was produced by a 10-fold dilution of 0.1 M solution. The solution was  a low hazard but could stain skin or clothing if spilled.
7.  Single drops of iodine solution were placed in rows on the tile.
8. Label a test tube with the pH to be tested was labelled.
9.  The syringe was used to place 2 cm3 of amylase into the test tube.
10. Add 1 cm3 of buffer solution was added to the test tube using a syringe.
11.  Use another syringe was used  to add 2 cm3 of starch to the amylase/ buffer solution,
12. The stop clock was started and left on throughout the test. The solution was mixed using a plastic pipette.
13. After 10 seconds, the plastic pipette was used to place one drop of the mixture on the first drop of iodine. The iodine solution turned blue-black.
14. The rest of the solution in the pipette was squirted back into the test tube.
15. This was left for another 10 seconds . Remove a second drop of the mixture was removed to add to the next drop of iodine.
16. These steps were repeated until the iodine solution and the amylase/ buffer/ starch mixture remained orange.

Results

Analysis

When the pH is changed, or the concentration of the hydrogen ions is modified, the reaction rate decreases rapidly. This is because the shape of the enzyme molecule is altered which in turn changes the shape of the active site.  Enzymes are not denatured by minor changes in pH. If the pH is changed then it returns to its optimum then the bonds that have been broken can be re-formed. Therefore the chemical formula of the enzyme molecule is also affected by the pH.

Evaluation

This is a straightforward practical that gives reliable, unambiguous results. The main errors will be in the order of mixing the enzyme/ substrate/ buffer, or a delay in sampling so that the reaction time is underestimated.