The Effect Of Temperature On Plant and Fungal Amylases

The Effect Of Temperature On Plant and Fungal Amylases
Enzymes are biological catalysts responsible for catalysing metabolic reactions. They increase the rate of reaction. Amylase is an enzyme which catalyses the hydrolysis of poly/disaccharides into monosaccharides. Temperature affects how quickly the enzyme catalyses the breaking up of the substrate (what the enzyme is breaking down). It increases the rate of reaction (amount of substrate which is converted to product per unit time). The reason for this is because at higher temperatures the molecules have increased kinetic energy than at lower temperatures. With increased energy, they are more likely to bump into each other and it is more likely that the substrate and active site will collide and react to produce a product. When the molecules do collide, there is increased chance they will overcome the activation energy barrier. This is the energy needed to start the reaction. At very high temperatures the enzyme ceases to work. This is because enzymes are proteins and become denatured at high temperatures. The high temperatures cause the hydrogen bonds and hydrophobic interactions to break, which changes the shape of the enzyme.At a different shape the enzyme can no longer hold the substrate. As the temperature increases even more the whole protein becomes completely denatured. The reaction stops. At the optimum temperature, the maximum rate of reaction occurs, the maximum amount of product produced per unit time. Plant enzymes have an optimum temperature of around 25 degrees C. Some other plant enzymes can withstand even higher temperatures. Enzymes in fungi have very high optimum temperatures. Rises of 10 degrees approximately double the rate of reaction until the optimum temperature is reached. Enzymes have a particular shape. The active site of the enzyme is designed to enable the substrate to fit in exactly. In this investigation the substrate is starch. Lining the active site are amino acids, which form temporary bonds with the starch molecule, as it slots into the active site. A hydrolysis reaction occurs between the enzyme and substrate, which splits the starch molecule into smaller molecules of monosaccharide. Hypothesis == I predict that as the temperature increases, the rate of reaction will increase. This is because the molecules have more kinetic energy and the chances of the enzyme and substrate colliding will be greatly increased. At lower temperatures, the molecules have less kinetic energy and there is decreased chance that enzyme and substrate will collide and react. When the molecules have more energy there is more chance they are able to overcome the activation energy barrier and form a product. I predict that at an increase of 10 degrees, the rate will double. This is because the molecules have double the amount of energy. This means there is twice as much chance of enzyme and substrate to collide and react. I predict that at very high temperatures the rate of reaction will be greatly decreased and at a certain point the reaction will stop altogether. This is because the enzyme will become denatured at the higher temperatures. This is what happens when hydrogen bonds and hydrophobic interactions break in the protein and it loses its original shape. Enzymes catalyse only certain substrates. When the enzyme loses its shape, the substrate can no longer fit into the active site and cannot be catalysed. The reaction stops. Due to the enzyme?s unique active site it can only convert one kind of substrate into one kind of product. I predict that there will be differences between plant and fungal amylases. I predict that the plant amylase will have a higher optimum temperature than the fungal, as it is more robust and can withstand higher temperatures. Materials & Methods =======
10 test tubes
3 pipettes
3 dimple trays
2 glass rods
Test tube rack
Electronic water bath
Marking pen
Distilled water
Stopwatch
Bunsen burner
Gauze
400cm3 beaker
Lighter
Tripod
Heatproof mat
Fungal amylase solution
Diastase
Starch solution
Iodine
Thermometer
Bung
Goggles
Tongs
Benedict?s solution
Pipette
The reason for using a pipette is to accurately control the amount of substrate to be used in the investigation. I will use an electronic water bath to control the lower temperatures, as it will be difficult to maintain these using a Bunsen burner. Pilot -? 1. Pour amylase solution into a test tube (3cm) 2. Pour starch solution into a test tube (6cm) 3. Place a drop of iodine into each of the dimples of the dimple tray 4. Add a drop of starch to the 1st dimple a blue/black stain appeared to show starch was present. 5. Pour amylase into the starch solution and mix 6. Add a drop of the mixture to the next dimple. 7. Repeat at 30-second intervals until the colour no longer develops. 8. Add Benedict?s solution to the mixture 9. Place in an electronic water bath. A change from blue to orange occurred. Iodine is used to show the presence of starch. Benedict?s is used to detect the presence of a reducing sugar. This test is carried out at the end of the pilot to prove that digestion has occurred. This pilot was adopted to investigate the effect of temperature on diastase and fungal amylase. After carrying out the pilot experiments I decided to change these things: 1. Add the amylase every 20s instead of 30s 2. Dilute the fungal amylase using distilled water 0.1% instead of 0.2%. 3. Leave the liquids at correct temp for 3mins before mixing. Diastase 1. Pour diastase into test tube (3cm) 2. Pour starch into test tube (6cm) 3. Place them both in a test tube rack and place in a water bath set to 21 degrees. 4. Maintain temperature and leave solutions for 1 min 30 s unmixed. 5. Add 2cm3 distilled water to starch. 6. Place drop of iodine into each of the dimples. Mark each dimple. 7. Place drop of starch into the 1st dimple (control) 8. Pour diastase into starch and shake. 9. At 15s intervals add a drop of the solution to a dimple. 10. Repeat 1-9 using 30, 40, 50, 60, 70, 80, 90, 100 degrees using a Bunsen burner and beaker to reach the temperatures above 40 degrees. 11. Record the time it takes for the starch to be digested. Fungal amylase The experiment for diastase will be followed using fungal amylase instead and the time taken for the starch to be digested will be recorded. Results Temperature (degrees C) 21 30 40 50 60 70 80 90 Diastase 9 mins 30 s 8 mins 30 s 6 mins 3 mins 30 s Fungal amylase 2 mins 20 s 2 mins 1 min 40 s 1 min 20 s 1 min 40 s 30 s The optimum temperature for both amylase enzymes is 80 degrees C. After this temperature both enzymes denatured. The diastase enzyme did not work until 50 degrees C was reached. Discussion == From the table above and the graph the results show that the plant amylase does not start to digest until a high temperature, whereas the fungal amylase starts to digest even at room temperature. Thinking of the structure of both the plant and fungus, it would make sense the larger of the two would digest at a higher temperature. The structure of fungi is only cell thick. At the temperature increased by 10 degrees, the rate did not double. The molecules did not have double the amount of energy. As temperature increased, the rate did increase but it did not double. This may be because I am not sure exactly how accurate some of the temperatures were when using the electronic water bath and Bunsen burner. If the temperatures were inaccurate then the results would have also been inaccurate. At 90 degrees the enzyme ceased to work. At very high temperatures the enzyme ceases to work. This is because enzymes are proteins and become denatured at high temperatures. The high temperatures cause the hydrogen bonds and hydrophobic interactions to break, which changes the shape of the enzyme. At a different shape the enzyme can no longer hold the substrate. As the temperature increases even more the whole protein becomes completely denatured. The reaction stops. If I had unlimited time I would like to repeat the experiments several times to check the accuracy of the results. I would also like to see the difference between human amylase and the plant and fungal amylase. Conclusion == I predicted that as the temperature increased, the rate of the reaction would also increase. Looking at the graph, the hypothesis has been proven. As the temperature rose, the time taken for the starch to be digested decreased. I predicted that at every increase of 10 degrees C, the rate would double. Looking at the graph and the table, this has not been proven. Even though I believe my results were accurate, if I had unlimited time I would like to repeat each experiment several times. I would also use an increased amount of the enzyme to see if the enzyme does digest at lower temperatures. I predicted that at very high temperatures the enzyme would not digest the starch at all as it would be denatured. Looking at the table, the hypothesis has been proven. At 90 degrees the enzyme had been denatured and no longer worked. To make sure that the enzyme had definitely been denatured I waited double the time that had taken for the enzyme to digest the starch at 80 degrees.

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