Investigating+the+Effect+of+Temperature+and+pH+on+Enzyme+Action

Experiment Title: Investigating the effect of temperature and pH on enzyme action.

Aim: To explore how environmental factors such as temperature and pH affect enzyme (amylase) reaction on starch.

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. 4 250 ml beakers 7. 2 white tiles 8. 2 droppers 9. I ce 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


 * __Part A__**

Hypothesis: **Set up** || **Colour change in contact with iodine** || **Explanation** || A || Black, dark blue || A is immersed in ice. With a surrounding temperature of 0°C, amylase molecules are not in a mobile phase, and hence will be unable to physically interact with starch to break it down into maltose. Hence there will be an abundance of starch. || B || Brown/Yellow || B is immersed in water at 37°C. This is the temperature closest to the optimum temperature at which amylase functions at the maximum rate. Hence it will be able to break down significant amounts of starch into maltose, leaving little or no amounts of starch in the solution. || C || Black, dark blue || C is immersed in boiling water at 100°C. The high temperature denatures the enzyme structure, causing permanent irreversible damage. Hence there will be little/ no enzymes remaining to break down starch into maltose. Hence there will be an abundance of starch. || D (control) || Black, dark blue || D is the control set up in this experiment. Given the presence of enzymes in this set up is absent, hence there is zero reaction with starch. || Independent variable: The temperature of surroundings of which the set ups are immersed in.

Dependent variable: Reaction between amylase and starch, impact on amylase.

Constant variables: Amount of starch used in each set up, amount of amylase used in each set up, the amount of time set ups were immersed in respective environments / given to react, the start and end time for all set ups.

Precautions: White tile must be dry when testing for iodine, when testube is immersed in environment, the liquid in the tube must at least be fully immersed /covered by the surrounding substance.

1. Label 8 test tubes **A1, A2, B1, B2, C1, C2, D1 and D2**. 2. Add 5cm 3 of starch solution to tubes A1, B1, C1 and D1. Add 3 cm 3 distilled water to D2, and then 3 cm 3 of amylase solution into tubes A2, B2 and C2. 3. Place A1 and A2 into a beaker of ice water, B1 and B2 into a water bath at **37** **0** **C**, and D1 and D2 into another water bath at **37** **0** **C**. 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 and D2 into the starch tube, A1, B1, C1 and D1, next to it. Record the time immediately. Keep the tubes A1, B1, C1 and D1, containing the mixture in the respective water bath 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. **
 * PROCEDURE

Results:

**Set up** || **Colour change in contact with iodine** || **Analysis** || A || Black, purple || A is immersed in ice. With a surrounding temperature of 0°C, amylase molecules are not in a mobile phase, and hence will be unable to physically interact with starch to break it down into maltose. Hence there will be an abundance of starch. || B || Brown || B is immersed in water at 37°C. This is the temperature closest to the optimum temperature at which amylase functions at the maximum rate. Hence it will be able to break down significant amounts of starch into maltose, leaving little or no amounts of starch in the solution. || C || Black, dark blue || C is immersed in boiling water at 100°C. The high temperature denatures the enzyme structure, causing permanent irreversible damage. Hence there will be little/ no enzymes remaining to break down starch into maltose. Hence there will be an abundance of starch. || D (control) || Black, dark blue || D is the control set up in this experiment. Given the presence of enzymes in this set up is absent, hence there is zero reaction with starch. || General analysis: Enzymes like amylase are able to function best at fixed temperatures. Temperatures too high will denature the enzyme structure - an irreversible process. Temperatures too low will result in immobile amylase enzymes which can be restored to their mobile state by increasing the temperature. The closer the surrounding temperature of the environment is to the optimum temperature at which the enzyme functions at its maximum potential, the higher the reaction rate with the carbohydrate molecule. For amylase, its optimum temperature is 40 °C, given this is close to the average human body temperature of 37 °C.

Conclusion: Amylase functions best when immersed in surroundings with a temperature of 37°C given it is the closest temperature value in this experiment to the optimum and ideal temperature.


 * __Part B__**

Hypothesis: **Test tube** || **Contents** || **Iodine Test** || **Explanation** || W || 2cm3 of starch solution + 2cm3 of HCL+ 2cm3 of amylase || Black, dark blue || Digestion has not occurred / occurred at the minimum level, given immersed in a solution with a relatively low pH. Hence there are high amounts of starch. || X || 2cm3 of starch solution + 2cm3 of distilled water+ 2cm3 of amylase || Brown/ Yellow || Digestion has occurred given immersed in distilled water with a pH of 7.0. Hence there are low levels of starch given high break down into maltose. || Y || 2cm3 of starch solution + 2cm3 of NH4OH+ 2cm3 of amylase || Black, dark blue || Digestion has not occurred / occurred at the minimum level, given immersed in a solution with a relatively low pH. Hence there are high amounts of starch. || Z || 2cm3 of starch solution + 4cm3 of distilled water || Black, dark blue || Digestion has not occurred given no enzyme present. Hence there are high amounts of starch. || 1. To each of the 4 test tubes, W, X, Y, Z, add 2 cm 3 of starch solution. Place the tubes in a water bath at **37** **0** **C.** 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. 4. Record your observations and conclusions in table 2 below.
 * PART B**

Results:

**Test tube** || **Contents** || **Iodine Test** || **Analysis** || W || 2cm3 of starch solution + 2cm3 of HCL+ 2cm3 of amylase || Black, purple || Digestion has not occurred / occurred at the minimum level, given immersed in a solution with a relatively low pH in comparison to the optimum pH at which amylase best functions at. The low pH level has denatured the enzyme, hence it cannot undergo catalysis as starch cannot bin to the active site. Little/no amounts of starch are broken down into maltose. Hence there are high amounts of starch. || X || 2cm3 of starch solution + 2cm3 of distilled water+ 2cm3 of amylase || Reddish brown || Digestion has occurred given immersed in distilled water with a pH of 7.0 which is the pH value in this experiment closest to the optimum pH value of amylase. Hence there are low levels of starch given high levels have been broken down into maltose. || Y || 2cm3 of starch solution + 2cm3 of NH4OH+ 2cm3 of amylase || Black, light blue || Digestion has not occurred / occurred at the minimum level, given immersed in a solution with a relatively high pH in comparison to the optimum pH at which amylase best functions at. The high pH level has denatured the enzyme, hence it cannot undergo catalysis as starch cannot bin to the active site. Little/no amounts of starch are broken down into maltose. Hence there are high amounts of starch. || Z || 2cm3 of starch solution + 4cm3 of distilled water || Black, dark blue || Digestion has not occurred given no enzyme present. Hence there are high amounts of starch. ||   General analysis: Too high or low a pH value in comparison with the optimum pH will denature the enzyme, making it lose its shape/change its properties and hence unable to undergo catalysis. The optimum pH level of each enzyme differs. For amylase, the optimum pH level is 7.0, given it can be found in a biologically neutral environment - the human mouth.

Conclusion: Amylase functions best when immersed in surroundings with a pH of distilled water at 7.0 given it is the exact same pH as the optimum and ideal pH at which amylase functions best.