Investigating Factors Affecting Enzyme Action (WJEC GCSE Biology)

• It is possible to investigate the effect of various factors on the rate of an enzyme-controlled reaction, e.g.
  • Temperature
  • pH
  • Substrate concentration
  • Enzyme concentration

Investigating the effect of temperature on amylase activity

• Amylase is an enzyme that digests starch (a long-chain carbohydrate) into maltose (a short carbohydrate)

Apparatus

• Starch solution (1 %)
• Amylase solution (10 %)
• Six test tubes and a test tube rack
• Measuring cylinders (25 cm³ and 10 cm³)
• Temperature-controlled water bath
• Iodine solution
• Spotting tile
• Pipette
• Stopwatch

Method

1. Add 10 cm³ starch solution to a test tube 
2. Measure 2 cm³ amylase into a second test tube
3. Add both test tubes to a water bath at 20°C for 3 minutes
4. Add a drop of iodine to each of the wells of a spotting tile
5. Remove both test tubes from the water bath, mix the contents of the two test tubes and start the stopwatch
6. Immediately after mixing, and then at 1 minute intervals, transfer a droplet of solution to a new well of iodine solution; record the colour of the solution
   • Blue-black will indicate the presence of starch
7. Repeat this transfer process until the iodine solution stops turning blue-black, or to the end of the spotting tile
   • When the iodine no longer turns blue-black there is no starch left in the solution; the amylase has broken it all down
8. Record the time at which the iodine stopped turning blue-black
9. Repeat steps 1-8 over a range of temperatures, e.g. 30 °C, 40 °C, 50 °C, 60 °C

Investigating temperature & enzyme activity diagram

A water bath and iodine can be used to study the effects of temperature on amylase activity

Variables

• The independent variable is the temperature of the water bath
• The dependent variable is the time taken for the iodine to stop turning blue-black
• Control variables include:
  • The pH of the solutions
  • The concentration of the starch solution
  • The volume of starch solution
  • The concentration of amylase solution
  • The volume of amylase solution

Results and analysis

• Amylase is an enzyme which breaks down starch
• The less time that it takes for the iodine to stop turning blue-black, the faster the rate of reaction
• This investigation should show:
  • The iodine will take longer to stop turning blue-black at temperatures below the optimum
    • There will be few successful collisions between the amylase and the starch at lower temperatures and the starch will be broken down more slowly
  • The iodine will stop turning blue-black very quickly at its optimum temperature
    • The enzyme breaks down all the starch in a short period of time
  • The iodine will remain blue-black throughout the investigation at temperatures too far above the optimum
    • The amylase enzyme has become denatured and so can no longer bind with the starch or break it down

Limitations

• Students without access to a temperature-controlled water bath may need to use a beaker and a Bunsen burner; this is likely to be less accurate than a water bath as the temperature may increase above the desired level
  • A thermometer would need to be used to monitor the temperature of the water bath
  • The beaker may need to be removed from and replaced over the Bunsen burner several times to maintain the correct temperature
• The starch and amylase solutions may not be left in the water bath for long enough to reach the desired temperature
  • A thermometer could be used to check this before mixing the solutions
• Determining the point at which the iodine stops turning blue-black could be subjective
  • A piece of equipment called a colorimeter can be used to measure the colour of the solution more accurately
• Only carrying out the experiment once at each temperature makes it impossible to identify anomalies
  • The experiment would ideally be repeated at least twice more at each temperature
  • This may be unrealistic under the time constraints of a science lesson

Risk assessment

• A hazard in an experiment is something that could potentially harm you
• A risk is the harm that could occur as a result of the risk
• A control measure is the action taken to reduce the chance of the hazard causing a problem

Factors affecting enzymes risk assessment table