Required Practical: Enzymes

Enzyme Rate

• Aim: To investigate the effect of pH on the rate of reaction of amylase
• You will:
  • Use the enzyme amylase to breakdown starch at a range of pH values, using iodine solution as an indicator for the reaction occurring
  • Use a continuous sampling technique to monitor the progress of the reaction
• Amylase is an enzyme that digests starch (a polysaccharide of glucose) into maltose (a disaccharide of glucose)
• Starch can be tested for easily using iodine solution

Iodine can be used qualitatively to indicate the presence or absence of starch from a sample

Method

• Place single drops of iodine solution in rows on the tile
• Label a test tube with the pH to be tested
• Use the syringe to place 2cm3 of amylase in the test tube
• Add 1cm3 of buffer solution to the test tube using a syringe
• Use another test tube to add 2cm3 of starch solution to the amylase and buffer solution, start the stopwatch whilst mixing using a pipette
• After 10 seconds, use a pipette to place one drop of the mixture on the first drop of iodine, which should turn blue-black
• Wait another 10 seconds and place another drop of the mixture on the second drop of iodine
• Repeat every 10 seconds until iodine solution remains orange-brown
• Repeat experiment at different pH values - the less time the iodine solution takes to remain orange-brown, the quicker all the starch has been digested and so the better the enzyme works at that pH

Investigating the effect of pH on enzyme activity

Improvements to method

• The above method can be adapted to control temperature by using a water bath at 35℃
• All solutions that need to be used (starch, amylase, pH buffers) should be placed in a water bath and allowed to reach the temperature (using a thermometer to check) before being used
• A colorimeter can be used to measure the progress of the reaction more accurately; with a solution containing starch being darker and glucose light (as a result of the colour-change of iodine) - this will affect the absorbance or transmission of light in a colorimeter