Required Practical: Enzymes

Practical Investigation of Amylase Activity

• This experiment is carried out to see the effect of pH on enzyme activity.
• The enzyme used is AMYLASE which breaks down STARCH into MALTOSE.
• The reaction will involve the amylase reacting with starch solution
• This reaction can be monitored using IODINE solution, which changes from brown-orange to blue-black in the presence of starch.

  

• If the enzyme does it’s job and the reaction is SUCCESSFUL, the starch will turn into maltose and the iodine solution will be BROWN-ORANGE.
• If the enzymes DENATURE and the reaction is NOT CARRIED OUT, starch will remain present and the iodine solution will be BLUE-BLACK.
• A BUFFER solution is used to change the pH of the reaction

 

Steps for Enzyme Activity Experiment

To examine the impact of pH on amylase, you can set up a practical experiment using the following steps:

• Add iodine solution to each well of a SPOTTING TILE.
• Set up a WATER BATH at 35°C using a beaker of water and a thermometer for consistent temperature control during the experiment.
• Combine 1 cm³ of amylase solution with 1 cm³ of a buffer solution with a pH of 5 in a test tube.
• Place the tube in the water bath for five minutes using test tube holders.
• Add 5 cm³ of starch solution to the amylase and buffer mixture and start timing the reaction using a stopwatch.
• Every 30 seconds, use a DROPPING PIPETTE to add a drop of the mixture to the iodine on the spotting tile.
• Note the color of the iodine
• Record when the iodine no longer turns BLUE-BLACK, indicating starch breakdown.
• Repeat with buffers of different pH values to see the effect of pH on amylase activity. 

Understanding the Results

• The sooner the color of the iodine solution stops turning BLUE-BLACK, the FASTER the rate of reaction, and the better the enzyme works.
• The pH at which the iodine stops turning blue-black the FASTEST, will be the OPTIMUM pH
• If the iodine remains BLUE-BLACK at a particular pH for the full amount of time, the amylase is assumed to have DENATURED, meaning the starch has NOT been broken down
• Comparing rates across different pH levels will highlight the pH sensitivity of amylase.
• CONTROLLED VARIABLES: Ensure a FAIR TEST by keeping other variables constant, such as amylase concentration and temperature.