Effect of Temperature on Reaction Catalyzed By Amylase.
A substance that alters the rate of a reaction but remains chemically unchanged is called a catalyst. The catalysts that are present in living organisms and which participate in biochemical reactions are called enzymes. These are proteins and are present in all processes such as photosynthesis, respiration and digestion. Amylase is one example of an enzyme which catalyses the breakdown of starch into sugars (Mohanty, Ramasubbu and Gooley, 2015, p.1014). The enzyme is found in animals, plants and microorganisms. There is high presence of the enzyme in the saliva and pancreas of animals. In plants it has a high presence in germinating cereals.
Enzymes like other proteins can be affected by external factors such as temperature and pH. High temperature results in high rates of reaction. The temperature should however be within the tolerable range. Since the chemical bonds in the active bonds are affected making them less or more suited to bind to other substrates (Encyclopedia Britannica, 2016). High enzyme temperatures can cause enzymes to denature or them losing their shape. Hence affecting their ability to bind to specific active sites.
The experiment investigates the effect of temperature on the digestion of starch by amylase.
As the temperature of the reaction increases the reaction rate of amylase increases. After reaching a certain optimal temperature of about 40oC the reaction rate will decrease rapidly. The hypothesis is explained by the lock and key model. Whereby enzymes are specific to a substrate. Their active site forms a keyhole where the substrate fits like a key. During the reaction, the substrate is broken into small pieces. Higher reaction temperature means that the particles have more kinetic energy hence the molecules have a high rate of movement and hence a greater chance for collisions and react. When the temperature is high the enzymes become denatured. Denaturing is a change in the enzymes structure that alters their dimensional shape leading to the loss of their biological properties.
Independent Variable is the temperature of the reaction.
The dependent variable is the time that it takes for the starch to break down due to amylase
The controlled variable is the iodine solution.
Apparatus and experiment materials
• water baths at 40°C,45°C,50°C and 70°C
• 1% starch solution
• 1% amylase solution
• Iodine solution
• A Thermometer
• Test tube rack
• 3 Graduated pipettes
• 96 well plates
• Six test tubes
• Marker pen
• Dropping pipettes
• pH 7 buffer solution
5 cm3 of amylase solution were taken and put in three different test tubes. A 1 cm3 of the buffer solution was then added to each test tubes. A 5 cm3 of the starch solution was then put in other 3 different test tubes. The six test tubes were then placed in a test tube rack and in a water bath at 40oC. The test tubes were left in the water bath for 10 minutes. In the meantime one drop of iodine solution was placed in 10 different wells in the 96 well plate. More wells were also added with iodine on a need basis. The Amylase solution was added to the starch solution in each tube and mixed. The test tubes with the mixtures were then returned to the water bath and timing was then started.
One drop of the mixture was then added to a well of iodine solution at time zero. After every 30 seconds a drop of mixture was added to a new well containing iodine solution. This step was repeated until addition to the mixture in a well that contained iodine solution showed that the starch had been digested. The time that the starch had taken to be digested was recorded. This process was repeated on other test tubes in the experiment. The procedure was used to determine how long it takes for the starch to be digested at room temperature of about 23oC and at other experimental temperatures of 45oC, 50oC and at 70oC. The results for the experiment were then recorded.
One should wear safety googles to prevent the starch solution or the iodine solution from splashing to the eyes. Secondly, racks should be used to place the test tubes in water. This is because some water baths are hot and result in burns.
Temperature/°C Time taken for colour change to take place
Trial 1 Trial 2 Trial 3 Mean
20 60 51 56 55.66
38 22 20 18 20.00
53 240 340 280 286.67
59 No colour change No colour change No colour change No colour change
69 No colour change No colour change No colour change No colour change
The graph shows that the mean time taken for a colour change in iodine to take place decreases from 20o C which is the room temperature to 380C. After which the time taken for the colour to change increases after that to the final temperature of 690C.
The protein Amylase has an active site whose complementary shape is to that of the active site of the starch.
From the plotted graph at 20o C the mean time that starch takes to be digested in the reaction was 55.66 seconds. The results indicate that the substrate and enzyme have a high kinetic energy. Hence there is high kinetic energy conversely the number of collisions in the reaction is high resulting in formation of enzyme substrate complexes. As the reaction continues, more and more starch is digested.
The optimum temperature for the reaction is at 38oC. This is because was the least time that the starch took to be digested. At this temperature, the substrate and enzyme have high kinetic energy which results in an increase in the number of collisions (Encyclopaedia Britannica, 2016). Consequently, the number of starch that bind to the amylase active sites increases resulting in more enzyme-substrate complex being formed.
From 53oC onwards the time taken for starch to react and be digested increases. This is as a result of denaturing of amylase resulting in its structure changing. Hence the shape of its active site changes and less and less starch is able to bind to the structure of amylase. This is explained by the breakdown of the ionic bonds and hydrogen bonds in amylase.
At 59oC the time taken for starch to be digested increases significantly. This is because of the deterioration in the structure of amylase which denatured further resulting in further change in its active site’s structure. Hence less starch are able to bind to the active sites resulting in decreased rate of starch digestion.
Beyond 590C there were no further colour changes or the colour changes took too long. This is because the amylase is completely denatured because of the break down if the ionic and hydrogen bonds in the structure. The breakdown in the bonds is due to high temperatures.
The test tubes were allowed to remain in the bath for 10 minutes so that the temperatures of all the solutions were the same hence the results would be reliable.
Amylase is added to starch rather than the other way round because the starch particle have large molecular structure as compared to the former. Hence if the opposite is done some of it would accumulate at the bottom because they have high density. Resulting in less starch would bind to amylase. In the opposite adding amylase to starch the reaction will take place at higher rate because amylase has small molecules hence have a large surface area for reaction.
Experiment timing began once the solutions were mixed and only stopped at the point of colour change. Colour change from blue black to yellow, indicates that amylase has completely digested the starch.
Sources of error in the experiment.
1. The time measured was not precise because the timers were not synchronized to measure even milliseconds.
2. The time period between mixing starch and amylase as well as the time for adding the solution to iodine was not measured. This time is significant because it allowed the original temperature to change hence modifying the results that were obtained.
3. The amylase and starch solution was not homogeneous due to differences in their structure.
The experiment only utilised three trials which in this form of experiment were not sufficient hence the reliability of the results could not be ascertained. This can however be improved by increasing the number of trials. Furthermore, the number of trials between 38 and 530C should also be increased so that the results were reliable.
The hypothesis of the experiment was supported in the results. The optimum temperature for amylase digestion by starch was found to be 38oC. The temperature is close to optimum temperature levels for enzymatic reactions. The number of trials between 38 and 53oC should be increased so that the accuracy of the experiment increases.
Encyclopedia Britannica. (2016). Starch | chemical compound. [Online] Available at: https://www.britannica.com/science/starch [Accessed 14 May 2018].
Sethi, A., Mohanty, B., Ramasubbu, N. and Gooley, P. (2015). Structure of amylase-binding protein A ofStreptococcus gordonii: A potential receptor for human salivary α-amylase enzyme. Protein Science, 24(6), pp.1013-1018.