Investigate the effect of temperature on amylase activityIntroduction Amylase is an enzyme that catalyses the breakdown of starch into sugars. Amylases are foundin almost all plants, animals and microorganisms. Large amounts of amylase occur in germinatingcereals, and in the pancreas and saliva of higher animals.AimThe aim of this experiment is to find out the rate of reaction between amylase and starch in arange of different reaction temperatures.Hypothesis As the reaction temperature of amylase solution and starch solution increase, the reaction rateof amylase and starch will increase. After reach the optimal temperature of amylase, the reactionrate of amylase and starch will rapidly decrease. The lock and key model explains my hypothesis. Most enzymes are very specific for a certainsubstrate, the active site on the enzyme molecule forms a "keyhole" into which the substrate fitslike a key, the substrate molecule is then broken up into many smaller pieces. The higher reactiontemperature, the more kinetic energy the substrate molecules will have. It means that themolecules can move faster and have a greater chance to collide and react. However, if thereaction temperature is too high, enzyme may become denatured. Denaturation is a structuralchange in a protein that alters its three-dimensional shape and causes the loss of its biologicalproperties1. As the temperature rises the amount of active enzyme progressively decreases, andthe rate of reaction is slowed. So there should be an optimal temperature for each enzyme.Research QuestionHow does a range of different reaction temperature (0°C, room temperature during theexperiment, 40°C, 60°C, 80°C) effect on the rate of reaction between amylase and starch?VariablesDependent variables: the required time for the disappearance of blue-black color (color of Iodinesolution mix with starch solution ).Independent variables: reaction temperature (0°C, room temperature during the experiment,
40°C, 60°C, 80°C).Controlled variables Significance/effect if not controlled Method of controlVolume of amylase solution in each test tube The volume of amylase solution ineach test tube needs to be a constant number, because we do not know if this variable will affectresult. To ensure that the result is accurate, it is necessary to maintain same volume of amylasesolution in each test tube. So that the reaction temperature will be the only independent variableinfluence the required time for the disappearance of iodine color. Using same measuringcylinder and read accurately.Volume of starch solution in each test tube The volume of starch solution in each testtube should be the same. If not, the rate of reaction between amylase and starch will be affected.Some test tubes which have more starch solution than others may decrease the time required toreach the reaction temperature. And also, the rate of reaction between amylase and starch willbe affected because it may take more time to completely break down starch molecules if there ismore starch solution. The required time for the disappearance of blue-black color in test tubeswill become inaccurate. Using same measuring cylinder and read accurately.pH value Change in pH can have a dramatic effect on the rate of amylase –catalyzed reaction.This effect of pH is because the structure of a protein is maintained by various bonds within thethree-dimensional structure of the protein. A change of pH from the optimum values alters thebonding patterns, progressively changing the shape of the molecules. If the molecules aredenatured, the reaction cannot happen, the result will become unreliable. pH value will be a newindependent variable if it is not controlled. Using pH test strips to test the pH value of amylasesolution and starch solution before the experiment.Concentration of amylase solution in each test tube Concentration of amylasesolution in each test tube should be the same. If the volume of amylase solution is same ineach test tube, the lower concentration means that there are less amylase molecules in that tube.If the amylase molecules are not enough to combine with starch molecules, starch cannot becompletely broken down, the mixed solution may still appear blue-black color. Therefore, it willaffect our result if it is not controlled. Using amylase solution in the concentration level.Concentration of starch solution in each test tube Concentration of starch solutionis the substrate concentration. As the substrate concentration increases, the rate of reactionincreases until all active sites engaged in catalysis. To prevent Concentration of starch solutionfrom being a factor of changing the rate of reaction, it must be controlled. Using starch solution inthe concentration level.Uncontrolled variables Significance/effect if not controlled Method of controlMicroorganism Microorganism will slowly pollute the solutions and may cause someunpredictable results. Sanitize laboratory before doing this experiment.Light Sunlight in different stages carry different amount of heat. It may cause the roomtemperature changes slightly. Avoiding sunlight shooting straight.
Apparatus100cm3 beakers X2Test tubes X1050cm3 1% starch solution50cm3 1% amylase solutionThermometers X1115cm3 Iodine solutionPipetteStopwatch X5Test tube rack x3Marker pen10cm3 measuring cylinder X2Water bath X3 (40°C, 60°C, 80°C)150g crushed icepH test stripsLab coatSafety glovesSafety gogglesRisk Assessment1. Starch solution and Iodine solution are required for this experiment.Wear safety goggles to prevent any starch solution or iodine solution getting in your eyes.
2. Some water bathes are very hot.Don’t directly touch the water inside the water bathes. Using a rack to put test tubes into thewater bathes.3. Crushed ice can damage your hands.Wear safety gloves to protect your hands from cold and stiff.Method1. Before start the experiment, lab coat, safety glasses and safety gloves must be worn. Cleanup your hands as well.2. Measure the room temperature with a new thermometer. Use marker pen to name beaker 1and beaker 2. Divide 10 test tubes into 2 groups, name them from 1-5 in both of the group. Youwill need five stopwatches to record the time for the disappearance of blue-black color in testtubes, name them from 1-5 as well.Stopwatch 1 is corresponding to the test tubes 1 in beaker1(0°C ),stopwatch 5 is corresponding to the tst tubes 5 in 80°C water bath. Record the roomtemperature.3. Add crushed ice into beaker 1 and fill it with tape water. Beaker 2 only fills with tape water.Put a thermometer in both of the beakers. After a few minutes, check the thermometers inbeaker 1 and beaker 2 to see if they have reached 0°C and the room temperature. Once thetemperatures reach our reaction temperatures, the experiment may start.4. Make sure that the amylase solution and starch solution you used in this experiment musthave a , you may test the pH values by using pH test strips. Add about 3cm3 of amylase solutioninto a 10cm3 measuring cylinder,add more drops or pour away more drops until it reaches the3cm3 mark. And pour the solution into test-tubes 1-5 in group 1. Repeat the step to the starchsolutiona and pour them into test-tubes 1-5 in group 2.5. Put test tubes 1 from group 1 and group 2 into beaker 1, and put test tubes 2 from group 1 andgroup 2 into beaker 2. Place test tubes 3 in a test tube rack and put it into water bath(40°C ). Testtubes 4 and 5 should be puttrd into water bath 60°C and 80°C with racks.6. Check the temperature of solutions in each container every 30 seconds by using thermometersto determine the current temperature of the solutions. Be careful not to mix thermometers up,they are single-use items in this experiment. 7. If the two types of solution in one container have already reached the reactiontemperature, mix them up and start the corresponding stopwatch as fast as you can. Add a dropof iodine solution immediately into the mixture of amylase solution and starch solution. Thesolutions cannot be taken out of the beakers or water bath during your operation, it will preventthe solutions loss of heat in air temperature. Observe the color change in test tube, stop thestopwatch when the blue-black color completely disappears, record the time. Repeat it five times,observation of color changes in the five test tubes can proceed at same time. 8. Repeat step 2-7 to get accurate results. 9. The experimental place and the tools should be cleaned up as the experiment is
completed.ObservationThere is no obvious color change in the five test tubes, the blue-black doesnt disappear. No otherphenomena occured. After waiting for 25 minutes, The test tubes appear blue-black color, thereis still no color change in any of the test tubes.ConclusionThe experiment is basiclly not successful, the test result is not essential.Evaluation of resultsThe result doesnt show a lot of valuable information to me. No any color changes,the test tubesstill appear blue-black color, it means that the experiment is a failure. If starch reacts withamylase, the color should turn to brown-yellow(color of iodine solution). Only thing I receivedfrom the result is there must be some mistakes in my method.Evaluation of the methodI am not able to prove my hypothesis correct. I fell that my method could be improveddramatically as stated below.Weakness/limitation Reason ImprovementsToo many drops of iodine solution. I didnt control the drops of iodine solution should beadded into each test tubes. Too many drops may darken the color, and it would become difficultto see some small changes. Use a smaller pipette and only add one drop of iodine solution ineach test tube.The heat has already broken down starch before mixing with amylase solution. Starch moleculeswill be broken down by heat. I leave the starch solution in water bathes for too long, the heataccelerated the decomposition of starch. And there is no starch anymore when I added it intoamylase solution. Therefore the reaction wouldnt occur, the color wouldnt disappear. Checkthe temperature more frequent; dont leave the solutions in the water bathes for too long.temperature of iodine solution. The iodine solution I used in this experiment is in airtemperature. When I added iodine solution, the reaction temperature between starch andamylase would be greatly affected. The result will become unreliable. Prepare another 5 testtubes for iodine solution, and put them in beakers and water bathes to achieve a same reactiontemperature with amylase solution and starch solution.It may cause loss of heat when I open the water bath to check the temperature of the solutions.
Because the loss of heat of the solutions, the temperature may be lower than it should be. Itwill increases the time of the solution soaking in hot water, which is not good for the result. Usebeaker and burner to instead water bath or use electronic temperature.References1. "Biology for IB Diploma" by C.J.Clegg, published by Hodder Education. 2007.BibliographyRamasubbu, N.; Paloth, V.; Luo, Y.; Brayer, G. D.; Levine, M. J. (1996). "Structure of Human Salivaryα-Amylase at 1.6 Å Resolution: Implications for its Role in the Oral Cavity".Campbell, Neil, J. Reece, and L. Mitchell.Biology 5th Edition. Menlo Park, CA.Benjamin/Cummings, 1999.