An Experiment to investigate the affect different temperatures have on the rate of an enzyme controlled reaction. In this case the reaction between Starch and Diastase to form Maltose.

Research

This experiment will test the affect temperature has on the rate of an enzyme controlled reaction. In this experiment the enzyme is Diastase, the substrate is Starch and the product which is produced is Maltose.

The equation for what is happening in this experiment is-

Diastase

Starch Maltose

The prediction for the results of the experiment is that as the temperature increases, so too does the rate of reaction. However this prediction is rather vague so for a more scientific explanation the theory behind the function of enzymes needs to be looked at and how temperature affects their functions and structures

Firstly, how enzymes work. They can work in two ways, the first of which is the lock and key system. This is that enzymes are specific and that generally any one enzyme will only work on one substrate. This is because only one substrate will fit exactly into the active site of the enzyme. Therefore that enzyme will not break down any other substrates. It has the name lock and key because only one key will fit into a lock to open the door. The other way that enzymes work is induced fit, which is where the substrate may not fit exactly into the active site, so the enzyme changes the shape of the active site slightly. This results in a much closer fit so that the activation energy for the reaction is lowered, then the reaction can take place to break down the substrate into the products. In this case starch into Maltose.

The Kinetic Theory is something else that needs to be looked into, and how it affects the enzyme and substrate molecules. The main principle of the kinetic theory is that as temperature increases, particles of matter vibrate more vigorously and start to separate. This is how a liquid turns to a gas. This theory has the same affect on the molecules of substrate and enzyme in this experiment. Therefore as the temperature increases the enzyme and substrate molecules will vibrate more, resulting in more collisions. Hence the reaction should happen over a shorter period of time. This theory is also used for the idea that as the temperature becomes lower the reaction will slow down. This is because the molecules of enzyme and substrate will vibrate less or possibly even stop, if the temperature gets too low for them. This temperature may be different for different enzymes. However kinetic theory can work against the rate of a reaction. This is because if the temperature rises too high the enzymes vibrates too much causing the bonds within the enzyme to begin to break. This causes the active site to change shape. This is known as Denaturisation. Therefore because the shape of the active site has changed the substrate molecule can no longer fit in, resulting in the reaction slowing down or stopping.

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So, by looking into the workings of enzymes and the affect of the kinetic theory on the enzyme and substrate molecules, the prediction for the experiment is that, as the temperature increases so too will the rate of reaction. However this will only happen up to a certain point, which is known as the optimum point. The optimum temperature for this enzyme (Diastase) is unknown, as different enzymes have different optimum temperatures depending on the surrounding environments they work in. I.E. enzymes within humans will have a higher optimum temperature than that of deep-sea creatures. Once the optimum temperature has been reached the rate of reaction will decrease as the enzyme molecules start to denature. However the prediction for when the temperature is at 0ºC is that the reaction will either be very slow or have stopped, as the enzyme and substrate molecules will hardly be moving.

Plan/Method

For this experiment a Colorimeter is going to be used, in this case it is going to measure how much Starch is present at different stages of the reaction. This is done by taking samples of the enzyme-substrate solution at regular intervals throughout the experiment, (every minute). These samples are then put in a curette and tested with iodine. A positive result for the presence of starch is a blue/black colour change. The level of colour change can be used to show the amount of starch present in the solution, as the darker the blue the more starch is present in the solution. Hence the use of the colorimeter. Each curette is placed into the colorimeter and the percentage of light transmission through the solution is recorded. Initially a sample of pure iodine would need to be tested and made to give the result of 100% transmission.

The way that the colorimeter works is by shining a light through the solution and recording the level of transmission through the solution on a scale; the lower the level of transmission, the more starch there is present in the solution. The percentage transmission through the solution will change because the solution will absorb some of the light. It would be useless using a blue light to test the solution because a blue solution would let all of the light through. Therefor a red light is used because a blue solution absorbs red light. This means that the denser the blue colour the more red light it will absorb, and the lower the lever of transmission will be recorded.

The method of the experiment is to heat the Starch and Diastase separately for five minutes at the relevant temperate. Once the five minutes are up to two are mixed together and the solution is tested instantly, (time 0). Then ever minute after that a sample of the solution is removed and tested with iodine. Samples are taken for ten minutes. Each curette of solution is kept and then they are all tested at the end of the experiment with the percentage transmission recorded. Whilst the experiment is being conducted the solution is kept in the water bath or ice so as to remain at the required temperature.

The experiment will be conducted at the temperatures- 0ºC, 30ºC, 40ºC, 50ºC and 100ºC. To get the temperature of the solution down to 0ºC the Starch and Diastase will be put into ice to cool to the temperature before being mixed in the same way as the other temperatures. The solution is then kept in the ice in the same way as the solutions being kept in the water baths.

Variables

The independent variable for this experiment is the temperature at which the solutions are getting tested; these are the temperatures which have been mentioned before. As a result of this the dependant variable is the level of starch in the solution, as the amount of starch produced depends on what temperature the enzyme-substrate solution is kept at. For the experiment to be fair there are some factors, which need to remain the same, which are-

The colorimeter- the same machine needs to be used to allow for a margin of error. If different machines are used they may have different margins or error which could cause the results to become inaccurate.
Time- if the time that it takes for the initial sample of the solution to be taken varies the first and subsequent results will be inaccurate. Therefor it is essential that the first sample be taken almost straight away. The time intervals at which the samples are taken need to be the same for each experiment I.E. every minute, on the minute.
Enzyme (Diastase)- if different volumes of Diastase were used in the experiment the results would change completely. This is because if there is more enzyme molecules added to a solution the rate of reaction increases, as there is a greater chance of collisions between enzyme and substrate molecules. Therefore the same concentration of enzyme must be used for each experiment.
Starch- if the amount of starch was increased and the enzyme level remained the same, the rate of reaction would change. This is because the enzyme may reach its Vmax, which is where the substrate molecules literally have to queue to get to an enzyme molecule. This would result in the reaction slowing down, which could affect the over all results for the experiment.
Iodine- the amount of iodine needs to remain the same because if a small amount of iodine were used for a sample it would give a different result compared to a larger quantity of iodine used for the same volume of enzyme-substrate solution.

Diagram

Results

Temp- 0ºC

Temp- 30ºC

Temp- 40ºC

Temp- 50ºC

Temp 100ºC

These results show that as the temperature increased so to did the rate of reaction. They also help to illustrate the denaturisation of the enzyme molecules. This is illustrated in the results for 100ºC where there was no break down of starch and therefore that the enzyme had denatured and the substrate molecule could no longer fit into the active site. Also for 0ºC the kinetic theory has been demonstrated, this is because the substrate and enzyme molecules would have slowed down so much or stopped all together resulting in the starch not breaking down into maltose.

Evaluation

The results from the experiment match the prediction, which was made previously. As the temperature increased so too did the rate of reaction. This was the case where 30ºC, 40ºC, and 50ºC rates of reaction were much quicker than 0ºC. 50ºC’s initial rate of reaction was much quicker than 40ºC and 30ºC, which would suggest that this enzyme works better at 50ºC. However 30ºC managed to break down all of the starch into maltose well within the 10 minutes, where as 40ºC and 50ºC didn’t which could suggest that the enzyme molecules might have denatured slightly after being at the higher temperatures for so long. If all of the different temperatures were left to finish breaking down the starch they would have eventually. Obviously the 100ºC experiment wouldn’t have because it didn’t break down any starch within the 10 minutes so it wouldn’t suddenly start to work. This temperature showed how temperature denatures the enzyme molecules, because no starch was broken down therefore the substrate molecules couldn’t fit into the enzyme’s active site.

There is a general trend as already has been stated that as the temperature increased so did the rate of reaction, this obviously wasn’t the case for the 100ºC experiment as it didn’t react at all so 100ºC must be higher than the enzyme’s optimum temperature. Something else that could be a general trend is that as the temperature increase the actual time for the entire break down of starch increased. The 30ºC reaction finished well within the 10 minutes but the 40ºC and 50ºC didn’t quite finish. This could have been if the enzyme molecules were denaturing slightly causing the reaction to slow down. The enzyme molecules at this temperature might not have been denatured at the start of the reaction but as the time went on the bonds within the enzyme started to break causing the active site to change shape slightly.

There is an anomalous result for the 50ºC experiment. The percentage transmission went from 68% to 66%. It is impossible for starch to be produced therefore either that result or the previous one was wrong. Later on in the experiment this happens again. This temperature would need to be completed again, as all of the results could be wrong. This could be due to bad timing or inaccuracy when reading the colorimeter.

There are many ways that this experiment could be improved. Firstly, the precision when taking the first sample. On some occasions the sample may have been taken quicker than others which would explain the 3% difference in the first reading. This may not seem like a big difference but it would be expected that all of the first readings would be almost exactly the same. This could be improved by having all of the appropriate equipment ready before mixing the solution and by making sure that the solution is mixed in the same way each time. I.E. pouring one into the other at the same rate and with the same force each time so that the enzymes mix in equally.

Also when measuring volumes of enzyme, substrate and iodine use the appropriate syringe needs to be used so as to use it the least amount of times to measure out a volume. So if measuring out 3cm³ don't use the small syringe (1cm³) use the 5cm³ syringe and measure upto 3cm³. This way there are fewer opportunities for bad measuring as only one measurement is made rather than three. In the same way if measuring small volumes such as 0.5cm³ don’t use a big syringe where guesswork is needed, use the smallest syringe possible, without it being too small

The colorimeter will have a slight margin of error so by using the same machine for each temperature that margin becomes irrelevant. Also each curette will either have two stripy sides or a V etched into one side. Ideally every curette should be the same kind, otherwise some results could end up being false. Therefore by using all of curettes the same all results should be true.

Rachel French 1.20