Amylase, Temperature and a Confident Conclusion
Devon and Akeil investigate the effect of temperature on the breakdown of starch by amylase. For each temperature they put 5 cm³ of 1% starch solution in one test tube and 1 cm³ of amylase solution in another, stand both in a water bath for five minutes, then pour the enzyme into the starch and start a stopwatch. Every 30 seconds they remove a drop and test it on a spotting tile with iodine solution, recording the time until the iodine stops turning blue-black. Their results: 20 °C - 11.0 min; 30 °C - 5.5 min; 40 °C - 2.0 min; 50 °C - 1.0 min; 60 °C - 1.5 min; 70 °C - 2.5 min; 80 °C - still blue-black after 20 min. They conclude that the optimum temperature for amylase is 50 °C. Their classmate Ravi says the 80 °C result must be wrong because hotter particles move faster, so the reaction should be fastest at the highest temperature.
1. Do the results justify the conclusion that the optimum is 50 °C? Explain your reasoning, and state one change the students should make to the temperatures they chose.
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Only partly. The data show that activity rises between 20 °C and 50 °C and falls again above 60 °C, but they do not pin the optimum to 50 °C. Readings were taken only every 10 °C, so the true peak could be anywhere from about 45 °C to 60 °C, and the 60 °C result (1.5 min) is close to the 50 °C result (1.0 min). That 0.5 min difference is the same size as their 30-second sampling interval, so the method cannot reliably separate those two temperatures. Each temperature was also tested once only, giving no evidence of repeatability.
To justify 'optimum = 50 °C' they should test narrower intervals - 45, 50, 55 and 60 °C - take readings more frequently (every 10–15 s), and repeat each temperature at least three times. A safer conclusion from the present data is that the optimum lies between 40 °C and 60 °C, with a sharp loss of activity above 70 °C.
2. Explain the results at 70 °C and 80 °C, and evaluate Ravi's reasoning.
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Amylase is a protein, and its activity depends on the precise shape of the active site, held by hydrogen bonds and other weak interactions. Above the optimum these bonds are broken, the tertiary structure unravels and the active site loses its shape, so starch molecules can no longer bind and few or no enzyme–substrate complexes form. Denaturation is progressive, which is why the time rises from 1.0 min at 50 °C to 1.5 min at 60 °C and 2.5 min at 70 °C; by 80 °C almost all the enzyme molecules are denatured, so no clearing is seen within 20 minutes.
Ravi is applying collision theory without thinking about protein structure. Higher temperature does give particles more kinetic energy and more successful collisions, and that is exactly why the rate increases from 20 °C to 50 °C. But this only holds while the enzyme's shape is intact; once denaturation begins, the loss of active sites outweighs the extra collisions. The iodine test itself is not affected by temperature, so the 80 °C result is a real failure of starch digestion, not a faulty test.
3. Describe two changes the students should make to improve the reliability and validity of their results, and state what each change would improve.
Read the worked answer
Repeat every temperature at least three times and use the mean, investigating any obvious anomaly before discarding it. This improves reliability: at present a single reading at each temperature could be a one-off error, and the 50 °C and 60 °C results are only 0.5 min apart.
Control pH by using the same buffer in every tube, and use the same batch, volume and concentration of enzyme and starch throughout. This improves validity: amylase activity is pH-sensitive, so if pH or enzyme concentration varied between runs, temperature would not be the only factor affecting the rate and the comparison would be unfair.
Further refinements worth mentioning: narrower temperature intervals (45, 50, 55, 60 °C); sampling every 10–15 s to sharpen the endpoint; and measuring the temperature with a thermometer placed in a control tube of starch rather than trusting the water-bath dial.