Equipments
Controlled variables
• Same temperature of amylase and starch solution
This can ensure that amylase can react with starch solution in the same temperature, a more accurate result can be received
• Same amount of amylase and starch solution in each time of the experiment
Fewer variables will be occurred as the solution was fixed in a certain volume, which will lead to a reliable result
• Water bath that letting the temperature stay constant
The stable temperature can ensure a stable reaction in the amylase and starch mixture, a more reliable result will be introduced
Method
1 Use a syringe to pump amylase into one test tube and starch solution into another.
2 Place the two tubes to equilibrate for 5 min.
3 Mix the amylase and iodine solution together and return the test tube to the water bath. Start the stopwatch immediately for 5 min.
4 Use droppers to drop 3 drops of iodine in each column of the spot-plate
5 Use droppers to drop 1 drop of the mixture in a column
6 Time the time taken for the iodine become light
7 Repeat this procedure for the other temperatures.
The color change is going to be observed by using spot-plates as the diagram below.
I will take 3 repeats to gain reliable data.
The rate will be calculated by 1/ average time taken.
Uncertainties
• There is insoluble particles are found in the spot-plate
Those particles may contain competitive inhibitors that may reduce amylases’ activity, affecting my results by decreasing the rate of reaction.
• Unknown substances in the syringes
Those substances may vary the concentration of the solution which pumped in, the solution may be having a bit diluted, and so my result may have a bit varied.
A result table to show the activity rate of amylase
*¹ In the third repeat first result of the time taken for starch to be digested, there is an anomalies which the time taken is 195 seconds. As it is very different compare with the first and second repeats, I redo this repeat.
The graph above support the hypothesis of my experiment (the reaction rate will increase as temperature increased, until a certain point at which the amylase would denature and no longer working) as the amount of time for the amylase to digest all the starch at 20°C was more than 600 seconds and the rate is 0.0017, and then the temperature was increased to 50°C the time decreased to 117 seconds and the rate is 0.0085, this showing the reaction was occurring at a faster rate when the temperature is at 50°C which show that 50°C is the optimum temperature of this amylase . However, when the temperature rose to 60°C the enzyme apparently denatured as we can see that the rate decreased.
Conclusion
In conclusion this experiment further confirmed that enzymes do increase/decrease their rate of reaction due to change in temperature. Each enzyme has its own individual optimum temperature, therefore if they are removed from these environments they will not function as well or possibly denature if it the temperature is too high. Although, if an enzyme’s optimal temperature is maintained then the enzymes will catalyze reactions at optimum rates.
The purpose of this experiment is to observe if the different temperatures affect the amount of starch broken down. My results had supported my hypothesis which is the amylase activity will increase as the temperature rise until a certain high temperature at which the amylase will denature and be non-functional.
Since enzymes are proteins, their secondary and tertiary structures will be easily affected by temperature, plus enzyme activity is closely related to the structure of an enzyme, therefore, changes in their secondary or tertiary structure will lead to changes in enzyme activity. Heat allows enzymes to speed up the process. However, if more heat is added on their optimum temperature, those enzymes will soon be loss their function and become denatured. Iodine forms dark brown/purple color with starch. This is due to amylase undergoes a process which broken down starch into glucose, the dark brown/purple color of the iodine solution and so disappears and form a lighter color. Therefore, loss of the dark brown/purple color can be used to measure of the extent of starch.
Evaluation
From the irrelevant results, I found that limitations either in the environment or the apparatus that I used had played an important role on affecting the results.
• use pipettes instead of syringes
Using pipettes are a more accurate way to measure a certain amount of liquid than using syringes, this is because pipettes have smaller diameter than syringes which can reduce the errors. Therefore, my result may not be the most accurate but still can show a general pattern.
• equilibrate iodine solution as well
As I only placed amylase and starch solution into the water bath to equilibrate but not iodine solution, the temperature differences of iodine solution may lower down the temperature of amylase-starch- solution, the enzyme activity may lowered down. This may vary my result and make it less accurate. To improve, I will equilibrate iodine solution as well. Therefore, every solution will be at the same temperature to give out more reliable result.
