The Effect of Temperature on the Rate of Amylase Breakdown

The Effect of Temperature on the Rate of Amylase Breakdown
Aim: To try to see if the temperature affects the rate in which Amylase breaks down starch into maltose. In this reaction starch is the substrate and maltose is the product. Amylase is an enzyme, Enzymes, also called catalysts, are in living things and there are thousand of them. Enzymes break down food by the active site on the Enzyme forming a chemical bond with a substrate and then water attacks the substrate until it is hydrolysed (split in 2). Equipment: Boiling tubes Timers/ stopwatch Starch Solution of Amylase ? colourless Thermometer Spotting tiles Pipette Water baths (at different temperatures) Iodine Measuring cylinder Method: 1. Collect equipment and set up as shown in diagram. 2. Fill a boiling tube with 10cm of starch and another with 5cm of enzyme. 3. Place both in a water bath at 20 degrees. 4. Wait until both test tubes reach equilibrium, test this with a thermometer. 5. Fill 30 spots on the spotting tile with 2 droplets of iodine. 30 spots are enough for 15 minutes, which is the maximum time the reaction will be allowed to run for. After this a result of no reaction will be recorded. 6. Place the 10 cm of starch and 5cm of enzyme in one boiling tube
Leave this tube in the water bath in order to maintain the current temperature. 7. Shake the tube once 8. Take 2 droplets of the starch/ enzyme solution and place in one spot on the tile. 9. Repeat this every 30 seconds until the solution turns the same colour as the control. In between the 30-second intervals wash the pipette in water, which is neutral in order to wash any remaining solution of the previous droplets. 10. Repeat this 3 times for reliability and accuracy. 11. Do this for 30 degrees and 40 degrees as these are an increase in 10 degrees and 40 degrees is also close to body temperature. Also repeat the experiment at 50 degrees and 100 degrees. The reason for testing 50 degrees is that above 50 degrees the enzymes are denatured and stop working because they are proteins and because the shape of enzymes are changed and can no longer combine with the substance. 100 degrees should be tested as a control. This will test whether a substance is an enzyme as if a substance can still carry out its reaction after it has been heated to boiling point, it cannot be an enzyme. Variables: the first variable is the type of enzyme used; this will always be amylase, as other enzymes, such as lipase, will react differently in different temperatures. Also, an enzyme, which normally acts on one substance, would not act on a different one because it would be the wrong shape. This means an enzyme that breaks down starch to maltose would not also break down proteins to amino acids. The volume of the solution should also be kept the same, 15cm, 10cm, of starch and 5cm of enzyme. The correct quantities should always be used in order to have a fair test, as an increase in the concentration of enzyme molecules would affect the reaction rates. As many chemical reactions can be speeded up by a substance called a catalysts. Catalysts speed up the rate of reaction without being changed itself. Almost, all the chemical reactions, which go on inside the body, are controlled by catalysts. The catalysts, which are found in living organisms, are called enzymes. The ph of the starch is also important, the optimum ph for the reaction is ph7, and this is clear from the preliminary work carried out, which shows that regardless of temperature, the starch at ph7 reacted quickest. All Enzymes have an optimum pH at which they work fastest. For most enzymes this is about pH 7-8 (physiological pH of most cells), but a few enzymes can work at extreme pH, such as protease enzymes in animal stomachs, which have an optimum of pH 1. The pH affects the charge of the amino acids at the active site, so the properties of the active site change and the substrate can no longer bind. For example a carboxyl acid R groups will be uncharged a low pH (cooh), but charged at high pH (coo-). The test for glucose should also be carried out fairly even though it is not part of the reaction. A control should be made showing iodine being used on the starch when fully digested to glucose. This would be essential as it enables comparison with iodine used on starch and the iodine used on glucose. The input variable, the one that will be changed, is the temperature of the starch/ enzyme solution. This is because as the temperature increases, the kinetic energy of the starch and amylase molecules increases and so they move faster. The faster the molecules move, the more often they collide with one another and the greater the rate of reaction. As the temperature increases, the more the atoms, which make up amylase molecules vibrate. This breaks the hydrogen bonds and other forces, which hold the molecules in their precise shape. The three dimensional shape of the amylase molecules are altered to such an extent that their active sites no longer fit the starch. The amylase is said to be denatured and loses its catalytic properties. Most of the chemical reactions, which happen inside a living organism, are controlled or catalysed be enzymes. Enzymes e.g. amylase is very sensitive to heat. Once, the temperature gets to about 50 degrees they begin to be damaged when this happens the reaction slows down. At higher temperatures, it will stop completely because the enzymes are destroyed. Preliminary work ==== Before we do this experiment we did preliminary work, which helped us with our investigation. Here?s what we did and the result outcome: Aim: to discover how the rate of reaction involving enzymes is affected by different PH?s. Method: 1. Fill a spotting tile with iodine each hole put in 3 squirts. 2. Fill a 25ml-measuring cylinder with 10ml of starch solution with a ph of 7 (neutral). 3. Fill a 10ml-measuring cylinder with 5ml of enzyme solution. 4. Put both solutions into a boiling tube and shake once. 5. Start the timer immediately and every 30 seconds put two drops of the solution in a part of the spotting tile so that it can mix with the iodine. 6. Do not stop timer until solution is brown. Do for max of 10 mins. 7. Repeat this with starch solution with a ph2 and ph10. Results === PH Time that solution turned brown PH 7 5 min 30 sec PH 2 Didn?t work within 10 mins PH 10 Didn?t work within 10 mins These results show that the ph does affect the rate of reaction and that enzymes work best as ph 7 as the other PHs didn?t work within 10 mins proving that enzymes work best a Ph 7 (neutral). ================= Prediction == I predict that the greater the temperature the less time it will take to digest starch to glucose. The reason for this is because the hot molecules move faster than colder ones; the high temperature causes more collisions per second. Also, when molecules move faster the collisions have more energy, therefore they are more likely to break bonds and cause a reaction. In addition, as the temperature increases, the kinetic energy of the starch and amylase molecules increases and so they move faster. The faster the molecules move, the more often they collide with one another and the greater the rate of reaction. All enzymes have an optimum temperature. Above the optimum temperature the rate decreases as more and more of the enzyme molecules denature. The thermal energy breaks the hydrogen bonds holding the secondary and tertiary structure of the enzyme together, so the enzyme (and especially the active site) loses its shape to become a random coil. The substrate can no longer bind, and the reaction is no longer catalysed. At very high temperatures this is irreversible. Remember that only the weak hydrogen bonds are broken at these mild temperatures; to break strong covalent bonds you need to boil in concentrated acid for many hours. Hydrogen bonds are caused by attractions between the positively charged nucleus of a hydrogen atom and a pair of electrons in the outer shell of another atom, either oxygen or nitrogen. These are very weak bonds and easily affected by temperature. Hydrogen bonds help to hold an enzyme into its proper 3D shape, and this will change if the enzyme is heated or cooled. The optimum temperature for the reaction would be around 37 degrees as this is they best temperature that digestive enzymes work at best that are found in the alimentary canal. In several parts of the alimentary canal, digestive juices are secreted. These digestive juices speed up the breakdown of large molecules o small ones. They are called digestive enzymes. This breakdown is called hydrolysis because water is also involved (lysis means splitting and hydro means water). Although, above 50 degrees the enzyme are change and can no longer combine with the substance, in this case starch. This is because enzymes are very sensitive to heat and as the temperature gets to about 50 degrees the enzymes begin to become damaged or said to be denatured. When this happens to an enzyme it cannot catalyse its reaction so well, so the reaction slows down. At higher temperatures, it will stop completely because the enzymes are destroyed. Additionally, if amylase gets too hot it will get denatured and will not be able to break down the starch because the active site will be a different shape not particular to starch. A more precise prediction is that for every 10 degrees increase in temperature the rate of reaction will double, this would only work up to 50 degrees, as after this it would become denatured. Also up to the optimum temperature the rate increases geometrically with temperature (i.e. it?s a curve, not a straight line). The rate increases because the enzyme and substrate molecules both have more kinetic energy so collide more often, and also because more molecules have sufficient energy to overcome the (greatly reduced) activation energy. The increase in rate with [image]temperature can be quantified as a Q10, which is the relative increase for a 10?C rise in temperature. Q10 is usually 2-3 for enzyme-catalysed reactions (i.e. the rate doubles every 10?C) and usually less than 2 for non-enzyme reactions Results -?? Results Table ===== Temp (Degrees) 1 2 3 Average 5 6.00 6.30 [image]11.00 6.15 23 (room) 4.30 4.06 3.00 3.78 40 2.00 1.30 2.00 1.76 60 6.30 8.50 8.30 7.70 80 N/a N/a N/a N/a [image] = Anomalous result My table of results prove that my prediction was right. However, I do have one anomalous result, which was not in included in the averages this could be due to many factors within the experiment. Conclusion My results prove that my prediction was correct and that the greater the temperature the quicker the rate of reaction that starch would turn and digest into maltose, although when the temperature gets above 50 degrees the reaction slows down and eventually the enzymes become denatured as my results show because at 80 degrees the reaction was still happening after 15 minutes therefore I?ve kept it as no result. The reason that the greater the temperature the quicker the rate of reaction and also the more precise prediction that for every 10 degrees increase in temperature the rate of reaction will double, this would only work up to 50 degrees. The reason for this is because as the amylase gets hotter and hotter, it will eventually become too hot and it will get denatured and will not be able to break down the starch because the active sites are a different shape not particular to starch. Also, because as the temperature increases, the kinetic energy of the starch and amylase molecules increases and so they move faster. The faster the molecules move, the more often they collide with one another and the greater the rate of reaction. As the temperature increases, the more the atoms, which make up amylase molecules vibrate. This breaks the hydrogen bonds and other forces, which hold the molecules in their precise shape. Hydrogen bonds are caused by attractions between the positively charged nucleus of a hydrogen atom and a pair of electrons in the outer shell of another atom, either oxygen or nitrogen. These are very weak bonds and easily affected by temperature. Hydrogen bonds help to hold an enzyme into its proper 3D shape, and this will change if the enzyme is heated or cooled. The three dimensional shape of the amylase molecules are altered to such an extent that their active sites no longer fit the starch. The amylase is said to be denatured and loses its catalystic properties. Most of the chemical reactions, which happen inside a living organism, are controlled or catalysed be enzymes. Enzymes e.g. amylase is very sensitive to heat. Once, the temperature gets to about 50 degrees they begin to be damaged when this happens the reaction slows down. At higher temperatures, it will stop completely because the enzymes are destroyed. Evaluation Overall, I think that the experiment went quite well and that that overall it was a fair test. Although I did have 1 anomalous results which could have been down to many factors within the experiment. These could have been not measuring the solution accurately and adding the wrong amount, also from not being accurate enough with the temperature of the solution and making the temperature too high or low, this would of effected the experiment because the molecules would of either of gained more or less energy meaning they?d collide quicker or slower than the correct temperature meaning that this would effect the results. If I was to do this experiment again they?re are a few things id change or do to enhance the reliability of my results and make them more accurate. Firstly, I would do more repeats of my experiment as this would increase the reliability of my results and also help to show if there is any anomalous results. Additionally, I would vary he temperatures that I used to see if the rate of reaction change if the temperature was lower and how the temperature affects how the molecules collide and vibrate. Also, I would continue you my experiment for longer than 15 minutes as this would mean id have more of an idea of the exact temperature where enzymes begin to become denatured and not have to write no result.

The Effect of Temperature on the Rate of Amylase Breakdown 9.5 of 10 on the basis of 1913 Review.