Investigating Factors Affecting Enzyme Action

Section 1 of 7Method

1

Put 10 cm³ of starch solution in one test tube and 2 cm³ of amylase solution in another.

2

Place both tubes in a water bath at 20 °C for about three minutes so that they reach the same temperature.

3

Put a drop of iodine in each well of the spotting tile.

4

Pour the amylase into the starch, mix and start the stopwatch at once.

5

At the start and then every minute, use a pipette to transfer one drop of the mixture into a fresh well of iodine and note the colour.

6

Stop when the iodine stays orange-brown and record the time taken.

7

Repeat at 30, 40, 50 and 60 °C, keeping everything else the same.

Section 2 of 7Variables and safety

Type	Detail
Independent	Temperature of the water bath
Dependent	Time for the starch to disappear (iodine stays orange-brown)
Controlled	pH, concentration and volume of both the amylase and the starch
Safety	Wear eye protection; take care with hot water and iodine, which stains skin and clothes

Section 3 of 7Interpreting results

1Low temperature

• Low temperature Few successful collisions between enzyme and substrate, so the starch takes a long time to break down.

2Rising temperature

• Rising temperature Particles move faster and collide more often and with more energy, so the time falls and the rate rises.

3Optimum

• Optimum The shortest time is found at the optimum temperature, often around 40 °C for human amylase.

4Too hot

• Too hot The active site changes shape, the enzyme is denatured and the starch is not broken down.

Section 4 of 7Calculating and extending

Rate

• Rate Rate can be calculated as 1 ÷ time (in s⁻¹), so a shorter time gives a bigger rate.
  • Plot rate against temperature to show the bell-shaped curve.

Other factors

• Other factors pH: use buffer solutions of different pH and keep the temperature constant.
• Enzyme or substrate concentrationdilute the solution with water to give a range of concentrations and keep the other factors the same.

Section 5 of 7Key terms

Term	Meaning
Optimum	The temperature or pH at which an enzyme works fastest.
Denatured	Having an active site that has permanently changed shape so the enzyme no longer works.
Amylase	An enzyme that digests starch into maltose.
Buffer solution	A solution that holds a steady pH.
Rate of reaction	How quickly a reaction happens, often calculated as 1 ÷ time.

Section 6 of 7Syllabus checklist

Reaction

• Reaction Amylase is an enzyme that breaks down starch into the sugar maltose.

Indicator

• Indicator Iodine solution is orange-brown but turns blue-black with starch.
  • When the drops stop turning blue-black, all the starch has been digested.

Apparatus

• Apparatus Starch solution, amylase solution, test tubes and rack, measuring cylinders, a water bath at different temperatures, a spotting tile with iodine, a dropping pipette and a stopwatch.

Section 7 of 7Exam technique

1

Saying the enzyme is killed at high temperature; it is denatured.

2

Forgetting to bring the enzyme and substrate to the same temperature before mixing.

3

Not using a fresh well of iodine for each drop, which contaminates the readings.

4

Concluding that rate is slow when the time is short. A short time means a fast rate.

5

Changing more than one variable at once, such as using different amounts of amylase at different temperatures.