Investigating the effect of temperature and pH on enzyme action

Part A:

Apparatus & materials

  1. Bunsen burner, tripod stand and gauze
  2. 3 water baths
  3. Thermometer
  4. 12 test tubes and rack
  5. Measuring cylinder
  6. 4 250ml beakers
  7. 2 white tiles
  8. 2 droppers
  9. Ice cubes
  10. Distilled water
  11. Test tube holder
  12. Stop watch
  13. Goggles
  14. 2% starch solution
  15. 1% amylase solution
  16. 1 M Hydrochloric acid solution
  17. 1 M Ammonium Hydroxide solution
  18. Iodine solution

Procedure
  1. Label 8 test tubes A1, A2, B1, B2, C1, C2, D1, D2.
  2. Add 5cm3 of starch solution to tubes A1, B1, C1 and D1. Add distilled water to tubes D2 and then 3cm3 of amylase solution into tubes A2, B2, C2.
  3. Place A1 and A2 into a beaker of ice water, B1 and B2 into a water bath at 370C, and D1 and D2 into another water bath at 370C.
  4. Place C1 and C2 in boiling water.
  5. Leave the tubes for 10 minutes to allow the solutions in the tubes to reach the temperature of the water bath.
  6. Pour the contents of A2, B2, C2, D2 into the starch tube, A1, B1, C1, D1 next to it. Record the time immediately. Keep the tubes A1, B1, C1, D1, containing the mixture in the respective water baths throughout the experiment and ensure that the temperatures are maintained.
  7. After 10 minutes, test the mixture in each tube with iodine solution on a white tile. Draw table 1 with appropriate headings and record the results and conclusions.










Results

Test Tube
Contents
Iodine test
Conclusion
A1 & A2
5cm3 of starch solution + 3cm3 of amylase solution
Placed in a beaker of ice.
Blue black
Starch is present.
B1 & B2
5cm3 of starch solution + 3cm3 of amylase solution
Placed in a water bath of 37 deg.
Brown
Starch is not present.
C1 & C2
5cm3 of starch solution + 3cm3 of amylase solution
Placed in boiling water.
Blue black
Starch is present.
D1 & D2
5cm3 of starch solution + 5cm3 of distilled water.
Placed in a water bath of 37 deg.
Blue black
Starch is present.


Interpreting results

  1. Which tube shows that starch digestion has occurred? Give your reasons.

Tube B1 and B2 shows that starch digestion has occurred. When it was tested with iodine, the iodine had no change in colour, which meant that starch was not present since iodine turns blue-black in the presence of starch.

  1. Which of the test tubes contained mixtures that gave a blue-black colour?

Test tubes A, C and D.


  1. Why did the mixtures in tubes stated in (2) remain blue-black?

The mixtures of the above mentioned tubes still contained starch and thus remained blue-black. Starch was present as it had not been broken down by the enzymes.

  1. What purpose does the tube D1 serve?

D1 served as a control for the experiment to show that any changes were due to the temperature and not other factors.

  1. Using your knowledge of the structure of starch, describe the test you would use to detect the presence of starch produced as a result of the reaction in the test tube stated in (1) above?



  1. What conclusion(s), based on the observations in this experiment, can be made about the effect of temperature on enzyme actions?

Temperature affects the enzyme action. The ideal temperature for enzyme action is at 37 deg, as starch was fully broken down at this temperature based on the results obtained. This temperature is our body temperature, which explains why our body temperature is 37 deg and why our body is not able to function properly when we exceed this temperature.

Part B

Procedure

  1. To each of the 4 test tubes, W, X, Y and Z, add 2cm3 of starch solution. Place the tubes in a water bath at 37 deg.
  2. Make up the contents of the 4 test tubes as shown in the table below.
  3. Test the solution with iodine solution at the beginning of the experiment and after 10 minutes. Record your observations and conclusions in table 2 below.

Results

Test Tube
Contents
Iodine test
Conclusion

Before
After

W
2cm3 of starch solution + 2cm3 of HCl + 2cm3 of amylase
Blue-black
Blue-black
Starch present
X
2cm3 of starch solution + 2cm3 of distilled water + 2 cm3 of amylase
Blue-black
Brown
Starch not present
Y
2cm3 of starch solution + 2cm3 NH4OH + 2cm3 of amylase
Blue-black
Blue-black
Starch present
Z
2cm3 of starch solution + 4cm3 of distilled water
Blue-black
Blue-black
Starch present


Interpreting the results

  1. Which tube shows that starch digestion has occurred? Give your reasons.

Tube X. The iodine did not turn blue black which meant that starch was not present, and had been digestion.

  1. Which of the test tubes contained mixtures that gave a blue-black colour?

Test tubes W, Y, Z.


  1. Why did the mixtures in tubes stated in (2) remain blue-black?

Starch digestion had not occurred as the conditions were not favourable.

  1. What conclusion(s), based on the observations in this experiment, can be made about the effect of pH on enzyme action?

Enzyme action only takes place when the pH is neutral (7.0), as starch was not digested when the conditions were acidic or alkaline.

  1. How does temperature and pH affect enzyme action?

Temperatures both lower and high than 37 deg, and pH more or less than 7.0 will result in no digestion of starch, showing that there is no enzyme action. Hence, the ideal temperature and pH for enzyme action is 37 deg and pH 7.