Enzymes: amylase and starch

Method

1. Put one drop of iodine solution into each well of a spotting tile.
2. Put 2 cm³ of amylase and 1 cm³ of buffer solution in one test tube, and 2 cm³ of starch solution in another.
3. Stand both tubes in a water bath for 5 minutes so they reach the bath temperature.
4. Add the starch to the amylase and buffer, mix and start the stopwatch.
5. Every 30 seconds, use a pipette to put one drop of the mixture into a new well of iodine.
6. Record the time when the iodine stays orange-brown. This is when all the starch has been broken down.
7. Repeat with buffers of different pH (or water baths at different temperatures). Do three repeats of each.

Safety: wear eye protection. Iodine solution is an irritant and stains skin.

Variables

• Independent: pH of the buffer (or temperature of the water bath)
• Dependent: time taken for the starch to be broken down
• Control: volume and concentration of amylase and starch; volume of buffer; temperature (in the pH investigation) or pH (in the temperature investigation)

The iodine test

• Iodine is orange-brown. It turns blue-black when starch is present.
• Amylase breaks starch down into the sugar maltose, so the blue-black colour fades as the reaction goes on.

How enzymes work

• Enzymes are biological catalysts. They speed up reactions without being used up.
• The substrate (starch) fits into the enzyme’s active site, like a key in a lock, forming an enzyme–substrate complex.
• Each enzyme works best at its optimum pH and temperature. Amylase from saliva works best at about pH 7.

Why the rate changes

• pH too high or too low: the bonds holding the enzyme’s shape are disrupted. The active site changes shape, so starch no longer fits.
• Temperature rising to the optimum: molecules move faster, so there are more collisions between amylase and starch.
• Above the optimum: the active site changes shape and the enzyme is denatured. The reaction slows and then stops.