Investigating How the Rate of a Reaction Involving Amylase Differs as the Temperature Changes

Investigating How the Rate of a Reaction Involving Amylase Differs as the Temperature Changes
Planning: Our aim is to observe what effect varying the temperature will have on the rate of a reaction involving an enzyme. I predict that as the temperature increases, from 5oC, the rate of a reaction will increase till it reaches the optimum rate of reaction. After this the rate of reaction will decrease. This is due to the structural format of the enzyme. An enzyme is a globular protein structure which has a very specific primary structure and because of that can fold up into a very specific shape, if shape or amino acid is missing or replaced by another in primary structure the enzyme cannot function. As the temperature increases the enzyme will have more energy so will be able to collide more often with a substrate and bind.This will increase until optimum temperature which is around 37OC - 40oC. After it has reached this temperature the heat energy causes the molecules to vibrate because of the kinetic energy from the heat, this will cause bonds to break particularly hydrogen bonds which are not the strongest bonds made. This will therefore change the globular protein shape, and so the active site, therefore no substrate will fit into the site and the enzyme is no longer useful and is called denatured. We have chosen to vary the temperature, there are three other variables to change and see what effect they will have on an enzyme, therefore all other variables need to be controlled. We will need to control the pH because the pH scale represents the percentage of hydrogen ions in the solution; these H+ will react with globular protein and make hydrogen bonds with its side chains. Therefore each molecule has its own pH it works best at. At the optimum pH the active site of an enzyme will accept the substrate. Amylases? optimum pH is around 10. Another variable needed to be controlled is the concentration of the substrate. We will need to use a set concentration, and volume to ensure test is fair. The concentration and volume of the enzyme is another variable that will need to be controlled to be fair. Sins we as a class has decided to vary temperatures, each person will do the experiment for each temperature. We all decided to have the temperatures 10oC, 20oC, 30oC, 40oC, 50OC, 60OC. we decided to continue taking a sample every 1 minute until it reached the aromatic point, where the solution had changed the iodine completely from blue-black toe its natural colour orange-red. We all had to also ensure that we equilibrated the enzyme and substrate before we mix the two together to start the reaction. We decided to use amylase as the enzyme and starch as the substrate which amylase works on. Method: The temperature which I was assigned to was 50oC. Firstly I had to wash all the equipment which I was going to use with distilled water, and then the solution which was going to be in it. Then I raised my water baths temperature to 50oC, and the used 5cm2 of amylase into one test tube and 5cm2 of starch into another, and put both test tubes into the water bath. We also all agreed on using our own saliva to see what effect the temperature would have on them. So I added my saliva to a different test tube and diluted it with distilled water. I also had another test tube filled with 5cm2 of starch. I left the starch and amylase in the water bath for 2 minutes then added them together and started the reaction. I then recorded if starch was present of not at every min. I started off taking readings every 30 seconds but saw that this was not necessary as the starch did not disappear quickly. I recorded all my results for my own personal amylase and the laboratory provided amylase in a results table. Apparatus:
Test tubes
Test tube rack
bunson burner
potassium iodide
fire proof tile
dimple tray
Risk assessment: The risks involved in this experiment was mainly the fact that care should be taken e.g. lab coats and goggles, when using a bunson burner to avoid burning, and when using enzymes as a person could be allergic to them and this could cause an outbreak. Observation: As our own results were not very accurate I will be using these simulated results found in a text book to analyse. Simulated results found in text book: Temperature (oC) Average Time (min) 1 / Time (x10-2) 10 No change after 30 min 12 24 0.0417 15 16 0.0625 20 11 0.0909 25 7 0.1429 30 6 0.1667 35 5 0.2000 40 5 0.2000 45 5 0.2000 50 5 0.2000 55 10 0.1000 58 28 0.0357 60 No change after 30min Analysis: As the temperature increased the rate of reaction increased fairly regularly this is because at low temperatures the particles do not have enough kinetic energy to move around as much so there is a less change of any collision between a enzymes? active site and the substrate. However at 35oC the rate of reaction reached its optimum temperature and the increase was slowed down. The optimum temperature for this particular amylase was between 35oC and 50oC. Between these two temperatures the enzyme worked at its best. After 50oC the rate of reaction dropped quite suddenly. This is because the enzyme became denatured because of the heat. Heat energy increases the amount of kinetic energy each particle has so if heat energy increases the particles will have more kinetic energy so will move around more and vibrate. This will cause weak bonds to break like hydrogen bonds. If hydrogen bonds are broken the globular structure of an enzyme will no longer be wrapped up in a precise tertiary structure which is needed for its job and the active site will be changed so enzyme can no longer function. Evaluation: Our own results were not very accurate because we were working in a laboratory where accuracy is not always possible because
Cross contamination could occur very easily as we used equipment
which has been used for many different experiments and solutions
this could have contaminated our solutions.
The reading taken at each minute. It was very difficult to be able
to take a reading at each minute, as we had two solutions to test,
therefore this was inaccurate
The temperature was difficult to control, because we used a bunson
burner and cold water to change and keep temperature the same
throughout the experiment.
The volumes of liquid where also difficult to control. There was
no time to accurately measure what volume you took out each min to
test with the iodine for starch. The volume of iodine also varied.
When we equilibrated, we did not know if the two solutions were
accurately equilibrated so one solution could have been warmer
than another, this could also have affected the results and rate
of reaction.
From the above you can see that our results were of a tentative nature because even though we tried to be as accurate as possible by controlling all the other variables, the things listed above still made our experiment very inaccurate. However due to the circumstances our results were the most accurate results we could have achieved. If I was to do this experiment again I would think about control or overcoming more of the problems listed above. I would also have my range of temperatures especially at the high temperatures more closely together i.e. between 55oC and 58oC there is a large ?jump? between the times, I would like record another temperature between those two, perhaps 56oC or 57oC.

Investigating How the Rate of a Reaction Involving Amylase Differs as the Temperature Changes 7.6 of 10 on the basis of 3407 Review.