# The Effects Of Temperature On Enzyme Activity

Introduction We are trying to discover what effect temperature has on enzyme activity. Hopefully my results will show me that temperature does have some effect on the activity of the enzymes and show me at what temperature enzymes become denatured. I will use 5 different temperatures from 10ºC to 50ºC which will hopefully be enough to supply me with the results I want, these may not show when enzymes become denatured but I may do 1 other temperature once to show when they become denatured . Variables I could change many variables for this experiment; I could change temperature, volume of milk or volume of enzymes, the concentration of milk or enzymes, those could all be used to show an effect on the enzyme activity, however, I am investigating the effects of temperature on enzyme activity so henceforth my variable will be temperature
Apparatus: ? 2 x Test tubes, 1 for the milk and one for the trypsin. ? 1 x test tube rack. ? 2 x plastic syringes with measuring marks on them, one for the trypsin and one for the milk so they don?t mix before you even start the experiment. ? 3 x water baths, 30 ºC, 40 ºC and 50 ºC (A fifth may be needed if room temperature is below 20 ºC in your classroom.) ? 1 x Ice Bath, 10 (A second may be needed if room temperature is above 20 ºC in your classroom.) ? 1 x timer to time how long it takes for the cross to appear. ? 7 x thermometer, 1 for the trypsin test tube, one for the milk and the others for the water baths and ice bath(s) ? Beaker of trypsin and beaker of milk Plan I shall be using 5 different temperatures of 10 ºC, 20 ºC, 30 ºC, 40 ºC and 50 ºC to find out what effect temperature has on enzymes and maybe one 60 ºC-70 ºC temperature to see what happens when the enzymes are denatured. The optimum enzyme working temperature is 37 ºC which is body temperature; I feel my temperatures that I am going to use fit well around the optimum as I have some cold and some hot temperatures. The temperatures I have chosen go up in equal blocks of 10 ºC which should make them easy to distinguish between them on the graph. The enzymes will be in a substance called trypsin, and will be mixed together when at the acquired temperature with the substrate which is 2% milk. As long as the enzyme hasn?t become denatured at too hotter temperature then the substrate will enter the active site and cause the milk to turn clearer, we will time how long it takes for the cross we will have drawn on the other side of the test tube to appear to give a more accurate test, or we could use a light sensor but that if extremely complicated but more accurate if you can get it to work, People walking past through the light will cause shadows to fall on the light sensor and cause dips in the line and make for inaccurate results. Fair Test To make this experiment fair we should: 1. Make sure the amount of enzymes or milk is precise. 2. Use the same person to confirm when the substance mixture is clear. 3. The temperature of the Trypsin and milk should be the same when mixing them. 4. Make sure you stop the timer as soon as the confirming person says clear or stop. These are all very important factors, if you do not keep these the same then all the times will be less accurate and will not be able to prove at what temperature it goes quickest as all the results will be random. Method First of all make sure you have all of the apparatus set up and ready to use before you start to do anything else. Once you have done this collect 3 ml of trypsin and 3 ml of milk substrate using your 2 plastic syringes, carefully squirt these into you two test tubes, and insert two of the thermometers into the test tubes carefully, trying not to spill any. Take these and insert them into a water bath or ice bath if necessary, if you are using either of these you will need to either hold them up in it or use your test tube rack to support them. Once you have heated or cooled both the milk substrate and the trypsin, remove them when they are the same desired temperature and quickly remove the thermometers and tip the substances into one of the test tubes, I suggest that you add the enzymes to the milk substrate because the enzymes are what you are supposed to be adding to the milk substrate but it doesn?t matter either way. You must mix these quickly so the substances don?t heat up or cool down before they are mixed, and then start the timer immediately. Once the person who decides it clear says it is the person timing stop the timer immediately. Write the time down, temperature and amount of milk substrate and the amount of trypsin. Repeat 3 times or more for each temperature to record accurate results. remember: Wash out the test tubes, wash the thermometers and the plastic syringes before the next experiment to prevent the substances mixing before the timer has started. Prediction The supposed optimum temperature for enzymes to work at is 37 ºC so I think that when I test the enzymes and milk at 40 ºC it will be the quickest to reveal the black cross drawn on the back of the test tube. Any colder than this i.e. 10 ºC, 20 ºC and 30 ºC, I predict that these will be slower than the 40 ºC, but any higher than this, I think should be slower though we will have to do the test first to find out. I feel this prediction should prove to be correct because the temperature enzymes work at in the body is 37 ºC and 40 ºC is the closest temperature to this so the trypsin and milk should react quickest at this temperature. The scientific explanation of this is that when an enzyme becomes too hot it becomes denatured and changes its active site therefore cannot react because the substrate will not fit. No reaction occurs so the milk cannot become clear. There is a diagram showing this below. At the other extreme of an enzyme, at its coolest, the activity now relies on the collision theory. As the temperature of a particle increases, the more it vibrates. The cooler a particle gets, the less it vibrates, and this is what happens to an enzyme below its optimum temperature (37 º C). What Happens When An Enzyme Becomes Denatured? [image] [image] Results Temperature (ºC) Time Taken for cross to appear (s) Average Time Taken for cross to appear (s)(2d.p) Reaction Rate (4d.p) 10 314.16 313.17 0.0096 314.07 311.29 20 182.73 187.86 0.0160 192.58 188.27 30 72.90 77.50 0.0387 75.38 84.24 40 142.29 131.71 0.0228 120.99 131.87 50 29.39 27.06 0.1109 [image] 27.87 23.94 Conclusion These results are very peculiar, as you can see from the results table, 50 ºC was faster to react than any of the other temperatures, and this is very unusual as the optimum temperature for trypsin is 37 ºC so we expected it to react quickest at 40 ºC, of course there are many factors which may have caused this defect, by the time it took for us to start the timer and mix the two substances the 50 ºC mixture may have cooled down to around 40 ºC which might explain why it reacted so quickly. All of this evidence however, does come to the conclusion that 50 ºC is the optimum temperature for trypsin and milk substrate to react. Evaluation This investigation has shown me that the temperature of an enzyme can significantly change how the enzyme behaves. My results disagree with my prediction in that the time for the cross to appear decreased at 50ºC, and that the enzymes then became denatured and the process slowed down as the temperature continued to rise. However there were a couple of anomalies, both of them had faster reaction rates than the other equivalent results, I repeated these in my results and they became similar to the others. If I were to repeat this investigation, I would try to eliminate these anomalies by working efficiently and concentrating on the task through out by making sure I did the same each time and not have to repeat any results, this may get more accurate results.. Other things I would change if I were to repeat the investigation would be the amount of set temperatures used. I would use an experiment at either 0ºC or at 70ºC so I could discover when the enzymes would become denatured, this could prove my theory of enzymes becoming entirely denatured or my collision theory theory right or wrong.

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