The Effect of Concentration of an Enzyme on the Time Taken for it to Fully Break Down Starch

The Effect of Concentration of an Enzyme on the Time Taken for it to Fully Break Down Starch
Aim For this investigation I intend to study the effect of concentration of an enzyme, in this case Amylase in two forms, standard and terminal, on the time taken for it to fully breakdown a substrate, which in this case is starch. Hypothesis The reason I have chosen these two enzymes is because each enzyme is designed specifically to break down only one substrate. Substances called catalysts speed up many chemical reactions. Catalysts called enzymes control the metabolic reactions in the body. Amylase is an enzyme; it is present in the digestive systems of many animals. Amylase speeds up the breakdown of long chain starch molecules in smaller chains of maltose. Enzyme molecules have a very precise three-dimensional shape. This includes a ?dent?, which is called the active site. It is exactly the right size and shape for enzyme?s substrate to fit into (in the case of amylase this is starch, which it breaks down into maltose). I hope to show with this experiment that the higher the concentration of Amylase the faster the rate of reaction, I also believe that the standard solution will use up the starch faster than the terminal amylase as the standard solution is closer to the concentration used in the body.
I am going to do this by planning an experiment, carrying it out, recording any relevant results and plotting a graph from which I will be able to gain a conclusion. Finally, I will 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. I cannot start he experiment until I know which factors I am going to change and which I am going to keep constant. Because I am trying to test 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 which means the experiment should be accurate and fair. Variables 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 changed. This is because if the temperature increases, the amylase and starch molecules will begin to move faster due to the kinetic theory. Because of this amylase molecules will vibrate 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 maltose 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 causing the rate of reaction to stop. pH Level:- This is again like temperature, in that every enzyme has an optimum pH. Optimum means the best conditions for the enzyme to break down the starch more quickly. 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. I will use a pH buffer at the level of neutral 7 to make sure it stays constant. Concentration of substrate:- the concentration of starch has to be kept a constant, because if the concentration of both amylase and starch increases then they will cancel out each others affect. Inhibitors:- Competitive inhibitors fight with the substrate for the same active sites, this means the reaction will be slower as the starch will have less active sites to go into. 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. 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 solutions. 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 solutions 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 3 × 10ml 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 To measure accurately the time it takes for each to 10th of a second concentration of amylase to break down the starch. 250ml 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 been carried out. Iodine solution 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. White tile Diagram of apparatus [image] Making the two types of Amylase concentrations As the standard 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 10cm cubed of all the above values. E.g. To make a 0.5% concentration of amylase I will need 5cm cubed of water and 5cm cubed of amylase solution. These will be made by using the syringes to collect the amylase and water. Having different syringes for different substances is important to make sure 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. Then taking the test tube with the 0.2% amylase in it suck up 2.5cm 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. I will then do the same with the terminal Amylase solution. Investigation practical Using the same method with a separate syringe draw up 2.5cm 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 125cm 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 temperature. 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 60seconds 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 2.5cm 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 2.5cm 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 will collect 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 practical and am making 10cm 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 the temperature could differ as a different area of the room may be a different temperature to another area, and as the practical will be carried out over a number of days, one day may be warmer or colder than the other causing a large 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. Risk assessment Risk Likelihood (out of 5) Outcome (out of 5) Total (out of 25) Irritation of eye from chemicals 1 3 3 Ingestion of chemical 2 2 4 Broken equipment 2 1 2 Scolding from hot water 2 3 6 Burning from Bunsen Burner 2 4 8 As all of the above risks have a total risk index of under 10, I have decided to proceed with the practical. But I understand that I have to take into account certain safety guidelines. Safety goggles must be worn to protect eyes, hand to mouth action must be banned after coming into contact with chemicals until hands have been thoroughly cleaned with a detergent. Broken equipment must be returned as soon as fault is detected to avoid accident. Care must be taken when handling hot water and naked flame such as that found on a Bunsen Burner. Long hair and flammable clothing must be tied back or removed. I will also make sure that I will stand up when taking part in my investigation , so as I can move out of the way quickly if there is a spillage. Modifications: In my plan I wrote that I would check the solution against the starch every 60 seconds. I did this for the first 5 minutes of each experiment and then I went onto checking them every 30 seconds until the desired result had been collected. Results: Results for standard Amylase Time in secs 60 120 180 240 300 330 360 390 520 550 580 610 640 670 700 730 760 790 820 0.2 % x x x x x x x x x x x x x x x x x x x 0.4% x x x x x x x x x x x x x x x x 0.6% x x x x x x x x x x 0.8% x x x x x 1.0% x x x Repeat 1 Time in secs 60 120 180 240 300 330 360 390 520 550 580 610 640 670 700 730 760 790 820 0.2 % x x x x x x x x x x x x x x x x x x x 0.4% x x x x x x x x x x x x x x 0.6% x x x x x x x x x 0.8% x x x x x x 1.0% x x x x Repeat 2 Time in secs 60 120 180 240 300 330 360 390 520 550 580 610 640 670 700 730 760 790 820 0.2 % x x x x x x x x x x x x x x x x 0.4% x x x x x x x x x x x x x x x x A 0.6% x x x x x x x x x x x 0.8% x x x x 1.0% x x x A Results for terminal Amylase Time in secs 60 120 180 240 300 330 360 390 520 550 580 610 640 670 700 730 760 790 820 0.2% x x x x x x x x x x x x x x x x x x x 0.4% x x x x x x x x x x x x x x x x x x x 0.6% x x x x x x x x x x x x x x 0.8% x x x x x x x x x x x x x 1.0% x x x x x x x Repeat 1 Time in secs 60 120 180 240 300 330 360 390 520 550 580 610 640 670 700 730 760 790 820 0.2% x x x x x x x x x x x x x x x x x x x 0.4% x x x x x x x x x x x x x x x x x x x 0.6% x x x x x x x x x x x x x x 0.8% x x x x x x x x x x x 1.0% x x x x x x x x x x Repeat 2 Time in secs 60 120 180 240 300 330 360 390 520 550 580 610 640 670 700 730 760 790 820 0.2% x x x x x x x x x x x x x x x x x x x 0.4% x x x x x x x x x x x x x x x x x x x 0.6% x x x x x x x x x x x x 0.8% x x x x x x x x A 1.0% x x x x x x x x x x Conclusion: From my graphs you can see that my hypothesis was correct. As concentration goes up, the time it takes for the Amylase to use up the starch decreases and the standard Amylase solution used up the starch faster. Unfortunately, during my analysis I have discovered some anomalous results which I have marked with an ?A? on the results tables and a circle on the graphs. I believe they were due to human error such as human reaction time. I may have failed to notice a slight colour change or I may have miscalculated the amount of seconds that had passed since I measured it last, thus giving me unexpected results. Evaluation: I believe that the experiment was designed well but there were a few problems. The concentration levels for amylase were far too low resulting in a long wait for results allowing human error over time and colour changes. I used a water bath to keep my solution at a constant temperature but because of time restraints they were only left in the water bath for 10 minutes before starting the experiment. They should have been left in the water bath for about 30 minutes so that the amylase had been completely affected by the temperature before the experiment was started. I decided to conduct the experiment at 30 second intervals instead of 60 second intervals because I thought my results would be more accurate, unfortunately I found that it was hard trying to keep water temperature at a constant and take readings in such a short space of time which could explain any anomalies. Anomalies may also be accounted for by the inaccuracy of the measuring syringes which could be rectified by using a computer to check exact volumes of solutions. Also, there is no way of checking how accurate the stopwatch I used to measure the time in between the checking of my results was, to combat this I could use more than one stopwatch to get times exactly right.. To make my experiment more accurate in the future I could engage in another person to keep the water bath at a constant temperature so I may concentrate on the reading of results and colour changes whilst keeping a constant solution temperature. Finally, as the Amylase solutions and buffers were made by human hands, it is impossible to give full reliability to them to be completely unadulterated and exactly at the percentages stated. All of the above are reasons for the anomalies found in my experiment For additional work, as well as using a different and more wide range of concentrations, an experiment could be carried out which investigates the effect of changing of temperature and pH level on the rate of reaction.

The Effect of Concentration of an Enzyme on the Time Taken for it to Fully Break Down Starch 7.6 of 10 on the basis of 1185 Review.