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For this investigation I am going to investigate the effect of the concentration of an enzyme, in this case Amylase, on the time taken for the enzyme to fully breakdown the substrate, which in this case is starch. The reason why these two enzymes have to be used is because each enzyme is designed specifically to break down only one substrate as each enzyme is made of a protein that causes it to be a specific shape, in this case the enzyme Amylase can only break down starch to dextrin. I am going to do this by planning an experiment, carrying out the experiment, recording any relevant results and plotting graphs from which I will be able, hopefully to gain a strong conclusion. I will finally evaluate the whole investigation. Before the experiments start we will be given a bottle containing a 1 % solution of starch and another bottle containing a 1 % solution of the enzyme Amylase.
From these bottles I will be able to take out as much of either solution as I wish. I cannot start he experiment until I know which factors I am going to change and which I am going to keep constant. Because the thing I am trying to test in the effect of the concentration of the enzyme, the only thing that I should be changing in each experiment is the concentration of the enzyme and nothing else. This will make all of the tests identical (apart from the enzymes concentration) which means the experiment should be accurate and fair.
Because of this any differences in the results of my experiment I will be able to put down to the concentration of the enzyme. If I was to use two variables, and there were a difference in the results, e. g. I changed the temperature as well as the concentration of the enzyme and the reaction sped up, I would not know which of the factors had done this and therefore could not reach a valid conclusion. Things that have to be kept constant throughout all of the experiments Temperature: - It is vital that the temperature remains the same in all the experiments as this will have a great impact on the results if not. This is because if the temperature increase the amylase and starch molecules will begin to move faster due to the kinetic theory.
Because of this amylase molecules will? bump into? and come into contact with the starch molecules more often causing the starch to be taken up by the active sites of the amylase, broken down quicker and the product dextrin released. This will lead to an increase in rate of reaction. If however the temperature rises too much (above about 60 degrees) the ionic and hydrogen bonds holding the amylase together will break causing the active sites on the enzyme to become denatured, this will completely stop the enzymes working and therefore the breakdown of starch will stop thus causing the rate of reaction to stop. pH Level: - This is again like temperature in the way that every enzyme has an optimum temperature the same as they have an optimum pH.
Optimum means the? best? or in this case the best conditions for the enzyme to break down the starch the quickest. If the pH level is at an extreme (in this case strong alkali) the enzyme will be denatured and work at a slower rate or even stop. For this reason the pH will remain the same throughout the experiments so as not to change the rate of reaction.
In a more complicated experiment a pH buffer may be used to be certain the pH level would remain constant. Concentration of substrate: - If there are more enzymes (amylase molecules) than substrate (starch) molecules and then more substrate is added the rate of reaction will increase because there will now be more active sites available for the starch to be broken up in but if there is enough substrate to occupy all the active sites on the enzymes then the rate of reaction will not change and for this reason I will use the same amount and the same concentration of substrate for all of the experiments. This meaning all the concentrations of amylase will be working on the identical in every way starch. Inhibitors: - Competitive inhibitors fight with the substrate for the same active sites on the enzyme this means the reaction will be slower as the starch will have less active sites to go into, where as non-competitive inhibitors compete for different sites but change the shape of the enzyme they attach to thus making the substrates active site inactive as the starch molecules now cannot fit into the new shape of the active site. In both cases the rate of reaction will be slowed down. This should not be a factor in my investigation as no inhibitors will be used in any way.
Volume of amylase and starch solution: - has to be the same in each experiment as differences would cause there to be a different number of either starch or enzyme molecules which would have the same effect as a different concentration of substrate. The thing that I will be changing in order to make the investigation work is the concentration of the enzyme solution. This is done so any change in the time taken for the amylase to break down the starch can be said to be because of the concentration which reinforces the aim of the investigation. I will do this by diluting the 1 % amylase solution to the desired concentrations. The concentrations I am hoping to use are 0. 2 %, 0. 4 %, 0. 6 %, 0. 8 % and 1 %. Method Equipment list Reason 15 x 10 mm diameter These will be used for diluting the solutions and for Clean test tubes mixing the amylase with the starch in the water bath.
The test tubes have to be clean to prevent any unwanted contaminants getting into the experiment. 3 x 10 ml syringe Used for very accurate measuring of both the amylase, starch solution and water. This will be vital for getting the correct volume of the 3 substances into the test tubes. Thermometer The most accurate way of measuring the temperature of the water bath and therefore the temperature of the enzyme (see earlier) and substrate. Stopwatch accurate To measure accurately the time it takes for each to 10 th of a second concentration of amylase to break down the starch. 250 ml beaker Used for the water bath because it is large enough to hold the water, 2 test tubes and the thermometer at the same time.
It also conducts heat well. Bunsen burner, heat Used as a unit to heat the water bath. Proof mat, tripod Test tube rack To hold the test tubes before and after the reaction has Tongues been carried out. Iodine solution in To test for the starch as iodine turns from a reddish potassium iodide orange to purple black if starch is present. (See later).
With dropper. Pipette Used to take a tiny amount of the solution out of the test tube during the time when the enzyme will be breaking down the starch. Making the amylase concentrations As the amylase solution that is given is already at 1 % I will not be able to concentrate the solution any higher without high difficulty for this reason I have decided to lower the concentration of the enzyme to my desired levels. To do this I will have to have, depending on the concentration needed a ratio of water amylase solution. I am wanting to make 10 cm cubed of all the above values.
E. g. To make a 0. 5 % concentration of amylase I will need 5 cm cubed of water and 5 cm cubed of amylase solution. The volumes needed for the concentrations I will use are shown below. (TABLE) These will be made by using the syringes to collect the amylase and water.
Different syringes for different substances is important o the concentrations are accurate. They will then be put into a test tube which will be slightly shaken to make the solution mixed equally... ? First the amylase solution has to be mixed to the needed concentrations. (See above)? Then taking the test tube with the 0. 2 % amylase in it suck up 2. 5 cm cubed into the syringe.
Making sure to hold the syringe at 180 degrees to the test tube so the measurement is accurate. This can then be put into a test tube. ? Using the same method with a separate syringe draw up 2. 5 cm of starch solution which can then be placed into a test tube. ? Both the test tubes have to be placed in a water bath so that both the enzyme solution and the starch are at the same needed temperature of 35 degrees Celsius.
To make the water bath place roughly 125 cm of water into the beaker. Place the beaker onto a tripod that will be on the heat proof mat. Into the water place the two test tubes with the solutions in and a thermometer. Heat the water in the beaker using a Bunsen burner. Until the temperature is at a constant 35 degrees. Keep the temperature at this for about one minute so the starch and amylase solutions have time to reach this temp.
Then being vitally careful not to spill any solution add the enzymes solution to the starch and slightly shake the test tube. ? Start the stopwatch as soon as the solutions are mixed. ? Now the solutions are mixed slight help may be needed to keep the temperature at 35 degrees while I will be checking the solution to see when the amylase has completely broke down the starch. I will do this by taking a very small amount of the solution out of the test tube, using the pipette, and dripping it onto a spotting tile. I will then add a tiny drop of iodine to the starch solution using the provided dropper. If starch is still present the iodine will turn a blue / black colour which shows to me that the amylase has not completely broken up the starch.
I will repeat this process every 20 - 25 seconds until eventually the starch does not change colour this will mean the amylase has broken the starch up fully and the stopwatch can be stopped. The results will need to be recorded in a suitable results table so I will be able to analyse them later. ? At the start of the experiment a control will have to be set up. A control is something that is used to prove that the thing that is being investigated in this case the concentration of amylase is the factor that is breaking down the starch and not the starch is breaking down on its own or in some other way. To do this I will place 2. 5 cm of the 1 % amylase solution into a test tube using the syringe. I will then heat this using the water bath to about 70 degrees.
This temperature should be hot enough to break the hydrogen and ionic bonds of the enzymes and therefore denature them making them inactive. This solution then can be added to 2. 5 cm of starch solution that will already be in a test tube from earlier prepared in the same way. The test tube can then be left and checked at 5 min intervals throughout the lesson using the iodine in the same way. The iodine should always turn blue / black as the starch should not have been broken down due to the amylase being denatured. This will prove it is the amylase breaking the starch down.
The syringes can then be washed and the process repeated three times for each of the chosen concentrations. I am getting three results with each concentration as it will give me a better average to work with when analysing the results. As I will have multiple lessons to do the practicals and am making 10 cm of each concentration of amylase it will be easier to do the three reading with the 0. 2 % solution before moving onto the 0. 4 % solutions. The reason for using the water bath is to make sure that the solutions for all the experiments are at the same temperature. If I did not use the water bath they may differ as a different area of the room may be different to another area or as the practical will be carried out over a number of different days if one day is warmer or colder than the other there may be quite a temperature range.
I have used the range of concentrations (0. 2 % etc) because when I come to plot a graph of the results a wider range of readings will mean that the graph will show any pattern better and I have used 3 readings so a better average can be gained again making for a better more accurate graph from which my analysis can be based. The graph I will be plotting will be of concentration of amylase against time taken for the starch to be broken down. I believe that the graph will show that as the enzyme concentration increases the time taken decreases. Safety aspects Goggles must be worn and especially while using the iodine as it can be irritable to the eyes. Stand up while using the Bunsen burner so you can move away quickly should the water bath fall. Any long hair tied back to keep out of way of flames
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