Amylase and Starch: Timing Digestion

Chew a piece of plain bread for a minute and it starts to taste faintly sweet. That’s the enzyme amylase in your saliva breaking starch into sugar. In this practical you’ll measure how fast amylase digests starch, using iodine solution to track when the starch has gone. It’s a standard required practical in many courses because it’s safe, cheap and gives clear results — and it’s an excellent way to learn about rates, variables and the effect of pH or temperature on enzymes.

Purpose

To investigate how a chosen factor — pH (or temperature) — affects the rate at which amylase breaks down starch.

Principle

The reaction

Starch is a polymer of glucose units (see polysaccharides). Amylase catalyses the hydrolysis of the glycosidic bonds in starch, producing the disaccharide maltose and short fragments:

starch + water → maltose (catalysed by amylase)

Amylase is a protein with an active site that fits the α-glycosidic bonds in starch. Its activity depends on the shape of that active site, which in turn depends on conditions such as pH and temperature (see factors affecting enzymes).

Tracking the reaction with iodine

Iodine solution (iodine in potassium iodide) is orange-brown. With starch, it forms a blue-black complex, because iodine chains fit inside the helical coils of amylose (see the starch–iodine test). Maltose and short fragments don’t give this colour.

So if you take a drop of the reaction mixture every 30 seconds and add it to iodine:

• Blue-black → starch still present.
• Orange-brown (no colour change) → starch has all been broken down.

The time taken for the starch to disappear is a measure of how fast the enzyme works. A shorter time means a faster rate.

Rate

rate = 1 ÷ time (units: s⁻¹)

Using 1/time lets you plot a graph where a higher value means a faster reaction, which is easier to interpret than time alone.

Variables

Variable	In this experiment
Independent (you change)	pH, using buffer solutions: e.g. 5, 6, 7, 8, 9
Dependent (you measure)	time for starch to disappear (then rate = 1/time)
Control variables (keep the same)	temperature (water bath), volume and concentration of amylase, volume and concentration of starch, volume of buffer, sampling interval, source of amylase

Equipment

• Spotting tile (white, with wells)
• Iodine solution in a dropping bottle
• 1 % amylase solution
• 1 % starch suspension
• pH buffer solutions (5, 6, 7, 8, 9)
• Boiling tubes, test tube rack
• 10 cm³ measuring cylinders or syringes
• Water bath at 35 °C (or a beaker of water with a thermometer)
• Dropping pipettes, glass rod
• Stopwatch
• Eye protection

Safety

• Wear eye protection.
• Iodine solution stains skin and clothes and is an irritant.
• Amylase can cause allergic reactions if inhaled or on the skin: avoid spills and wash hands. Don’t use saliva as an amylase source in a shared lab unless your teacher says it’s allowed.
• Take care with hot water.

Method

1. Place one drop of iodine solution into each well of the spotting tile.
2. Put 2 cm³ of amylase solution into one boiling tube and 2 cm³ of starch suspension plus 1 cm³ of pH 7 buffer into a second tube.
3. Stand both tubes in the water bath at 35 °C for 5 minutes, so they reach the same temperature before mixing.
4. Pour the amylase into the starch–buffer tube, mix with the glass rod, and start the stopwatch immediately.
5. After 30 seconds, use a clean pipette (or the glass rod) to transfer one drop of the mixture to the first iodine well. Note the colour.
6. Repeat every 30 seconds, using the next well each time, keeping the tube in the water bath.
7. Stop when the iodine stays orange-brown. Record this time.
8. Repeat the whole experiment with each of the other pH buffers.
9. Repeat each pH three times and calculate a mean time.

Tip: rinse the pipette between samples, or use a fresh one, so starch from an earlier sample doesn’t contaminate the next.

Results

Example data

pH	Trial 1 / s	Trial 2 / s	Trial 3 / s	Mean time / s	Rate (1/mean time) / s⁻¹
5	300	330	300	310	0.0032
6	150	180	150	160	0.0063
7	90	90	120	100	0.0100
8	180	180	210	190	0.0053
9	> 600	> 600	> 600	—	~0

(Illustrative values. With 30-second sampling, your times will always be multiples of 30 s.)

Calculation example (pH 7): mean = (90 + 90 + 120) ÷ 3 = 100 s. Rate = 1 ÷ 100 = 0.010 s⁻¹.

Graph

Plot rate (y-axis) against pH (x-axis). Join points with a smooth curve or straight lines between points, as your teacher prefers. The graph should show a peak around the optimum pH, which for salivary amylase is close to 7 (roughly 6.7–7.0).

Explaining the results

• At the optimum pH, the charges on amino acid side chains in the enzyme are arranged so the active site has exactly the right shape and chemistry to bind starch. Many enzyme–substrate complexes form, and starch is broken down quickly.
• Away from the optimum, the concentration of H⁺ or OH⁻ changes the charges on groups such as –COO⁻ and –NH₃⁺. This disrupts the ionic bonds and hydrogen bonds holding the enzyme’s tertiary structure. The active site changes shape, so fewer substrate molecules fit — the rate falls.
• At extreme pH, the enzyme may be denatured: the active site is permanently changed and no reaction occurs in the time available (see denaturation).

This also explains why salivary amylase stops working in the stomach (pH around 2) — and why starch digestion restarts in the small intestine with amylase from the pancreas (see the chemistry of digestion).

Sources of error

• Sampling interval: with 30-second intervals, the true end time could be anywhere in the 30 seconds before. This limits resolution — sampling every 10 or 15 seconds would help.
• Judging the endpoint: the change from blue-black to orange-brown passes through intermediate shades; different people may judge it differently. Compare with a control well containing iodine and water.
• Contamination: a dirty pipette carries starch between samples, delaying the apparent endpoint.
• Temperature drift: if the tubes cool during sampling, the enzyme slows. Keep the tube in the water bath.
• Delay before timing: starting the stopwatch late makes times too short.
• Amylase variability: enzyme solutions lose activity over time; use the same batch for all runs.

Improvements

• Use a colorimeter to measure the blue-black colour intensity at fixed times, giving continuous, objective data.
• Sample at shorter intervals.
• Test more pH values around the optimum (6.0, 6.5, 7.0, 7.5) to locate it more precisely.
• Include a control with boiled (denatured) amylase to show that the enzyme is responsible for starch breakdown.

Variation: temperature

The same method works for temperature. Keep the pH at 7 and run the reaction at 20, 30, 40, 50 and 60 °C. You should see the rate increase up to about 35–45 °C (more collisions with enough energy) and then fall as the enzyme denatures. You can also calculate the temperature coefficient, Q₁₀ — see enzymes practice questions.

Key takeaways

• Amylase hydrolyses starch to maltose; iodine stays orange-brown once the starch has gone.
• Measure the time for starch to disappear; rate = 1/time.
• Control temperature, volumes and concentrations; vary pH with buffers.
• The rate peaks at the optimum pH (~7) and falls as ionic and hydrogen bonds in the active site are disrupted.
• The main limitation is the sampling interval; a colorimeter improves precision.