Diluting an Enzyme

Investigating Whether Diluting an Enzyme Affects the Rate at which it Breaks Down its Substrate

Introduction

I am doing an experiment to investigate whether diluting an enzyme affects the rate at which it breaks down its substrate. The enzyme I am using is amylase and the substrate is starch.

Enzymes are large protein molecules and are essential for the body to function; they speed up reactions and are therefore called biological catalysts. They enable the different products of the reactions to be absorbed and assimilated quickly, without them reactions would take place too slowly for the body to sustain life. Enzymes break down their respective substrate using the lock and key theory; each enzyme has a differently shaped active site, or ‘lock’, which only one substrate, or ‘key,’ can combine with. Enzymes are therefore called substrate-specific. Each enzyme breaks down a different substrate. For example, amylase breaks down starch to maltose, pepsin breaks down protein to amino acids and lipase breaks down lipids to fatty acids and glycerol. After an enzyme has broken down its substrate into simpler substances, it remains unchanged. This is very useful because it means that the enzyme can continue this process with a new substrate. Enzymes are also very sensitive to heat. Enzymes have an optimum temperature of 40oC. This means that at 40oC an enzyme works fastest, but above 40oC the active site of the enzyme starts to denature, meaning that it cannot lock onto its substrate so effectively. As the temperature increases above 40oC the active site of the enzyme begins to denature more and more, so less and less of the substrate is broken down, until eventually the enzyme’s active site is completely denatured and no substrate can be broken down at all. As the temperature increases up to 40oC the enzyme starts to move around more quickly, meaning that there is more chance of it colliding with its substrate. As well as temperature, enzymes are also sensitive to pH; they will start to denature if the conditions are too acidic or alkaline. Enzymes are adapted for their conditions; their optimum temperature is 40o and this is the temperature of the body. Because, for example, pepsin is found in the stomach, it works best under acidic conditions, and amylase, which is found in saliva in the mouth, works best under neutral/alkaline conditions. The actual optimum pH of amylase is 7.5.

For my experiment, I am going test different strengths of amylase and starch together and see how long it takes the amylase to break down the starch and whether this varies when the amylase is diluted. I am going to test amylase with strengths of 1%, 0.8%, 0.6%, 0.4%, 0.2% and 0.1%. I am also going to test water. The different strengths of amylase will be made up, and then the first one will be mixed with the starch. A dropping tile with iodine will be prepared and at regular intervals the amylase/starch mixture will be tested with the iodine. If the mixture goes black/blue then I will know that starch is still present. If the mixture goes brown then I will know that all the starch has been broken down. The time when the mixture is no longer black/blue but brown will be recorded as the amount of time it took that strength of amylase to break down the starch. The experiment will be repeated three times for each strength of amylase and an average will be calculated. Using this average the rate that the amylase broke down the starch at will be calculated

Preliminary Work

Before I conduct this experiment, I will carry out some preliminary work. I am going to test 1% and 0.1% amylase with the starch. This will indicate how long I need to leave the amylase and starch together to react and how often I ought to test them with iodine. I have chosen to test 1% and 0.1% amylase because this will give me both ends of the spectrum I am going to use. I am also going to test different ratios of amylase to starch to see which is the most appropriate to use with the amount of time that is available to me. I will test a 1:1 ratio and a 2:1 ratio, where I will have 2 parts amylase to 1 part starch. This will enable me to decide which ratio to use given the amount of time I have.

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I found from my tests that in a 2:1 ratio the 1% amylase solution reacted with the starch too quickly to make it practical to test. I therefore tried a 1:1 ratio and found that the 1% amylase solution reacted with the starch in roughly twenty seconds. In a 1:1 ratio the 0.1% amylase solution reacted in about seven and a half minutes. From my preliminary work I have been able to conclude that a 1:1 ratio would be appropriate to use and that the 1%amylase solution would need to be tested every two second and that the 0.1% amylase solution would need to be tested every thirty seconds. Because I now knew that the weakest amylase solution would take no longer than ten minutes to break down the starch, I could judge how many different strength amylase solutions I would have time to test. I decided I could test 1%, 0.8%, 0.6%, 0.4%, 0.2% and 0.1%. From my preliminary work, I could also gather how much of the amylase and starch I would need to mix together to ensure that I had enough to last for the duration of the experiment. I found that I would need to have a volume of eight millilitres, so four millilitres of each, to have enough to test with the iodine.

Prediction

My preliminary work also enabled me to make some predictions as to the results of my experiment. I could see from the results of my tests with 1% and 0.1% amylase that the amount of time needed to break down the starch was much greater when the amylase was weaker, in the 0.1% solution. I can make an informed prediction, then, that as the amylase solution gets weaker, the amount of time needed to break down the starch will be greater, and so the rate will be slower. This is because the volume of amylase solution mixed with the starch will be the same every time, regardless of the strength of the solution. For example, with the 0.8% solution, I will mix the solution using 2mls of water and 8mls of amylase, and then take 4mls out of this ten to mix and react with the starch. With the 0.6%, I will mix the solution using 4mls of water and 6mls of amylase, so the volume is the same in each case, it is just the proportion of amylase within the solution that varies. It is logical, then, that as the solution gets weaker, and the amount of amylase within the solution is less, it will take longer for the amylase to break down the starch.

List of Apparatus

To investigate whether diluting an enzyme affects the rate at which it works, I will use the following apparatus;

The enzyme amylase, at a strength of 1%- the other strengths will be made up using this and water.

The substrate starch, which will be mixed with the enzyme amylase.

Iodine solution, to test the amylase/ starch solution with. Iodine turns blue/black if starch is present and remains its own colour if starch is not present.

Water, to dilute the amylase with.

Two dropping tiles, to test the amylase/starch solution with iodine.

Beakers and test tubes,

Pipettes,

Syringes, to accurately measure the volume of the various solutions.

An electric stopwatch, to accurately measure the amount of time it took the different strength amylase to break down the starch.

Method

A beaker of 1% amylase was taken and from this the amylase solutions of strengths 0.8%, 0.6%, 0.4%, 0.2% and 0.1% were made up. 10mls of each was made up; so, for the 0.8% amylase, 8mls out of the ten were amylase and 2mls were water. For the 0.6% solution, 6mls were amylase and 4mls water, and so on for the other strengths. Then two dropping tiles were taken and prepared with a drop of iodine in each dip. 4mls of the first solution to be tested was put in a test tube using a syringe and then 4mls of starch was added. As soon as the starch was added to the amylase solution, the stopwatch was started and at regular intervals a drop of the amylase/starch solution was added to the drop of iodine in the dropping tile, until the iodine no longer went blue/black and stayed brown. The time at which this happened was recorded. This was repeated three times for each strength of amylase, an average was calculated, and from this the rate was calculated by dividing the average time taken by one. For the 1% amylase solution, I tested it with the iodine every two seconds. For the 0.8% solution I tested it every three seconds. For the 0.6%, every five seconds, for the 0.4%, every five seconds and for the 0.2% and 0.1% solutions I tested it every thirty seconds.

Variables and Controls

The independent variable in this experiment is the strength of the amylase solution, which is being reacted with the starch and then tested with the iodine solution. The dependent variable is the length of time each strength of amylase solution took to break down all the starch. As a control, I tested water with starch as well as the amylase solutions. I mixed 4mls of water with 4mls of starch and left it for forty minutes, testing it with iodine every ten. This was to check that the liquid I was using to dilute the amylase didn’t react with starch itself, at least not in the amount of time that I would be leaving the amylase solution and starch together. I found from this that water did not react with starch, because the iodine solution stayed very black. Because the iodine solution did not get at all lighter in colour as I tested the water and starch with it, I can safely assume that it would not break down the starch at all.

I tested each strength of solution at least three times, if there was a slightly anomalous result then I tested it four times. By doing this and then calculating an average, I was ensuring that the impact of any anomalous results on my overall results would be minimised.

To ensure that this experiment is a fair test, certain steps have been taken. These are;

As far as it was possible, each test was conducted under the same temperature; the starch solution was cooled under cold water before it was reacted with the amylase, and we tried to keep the temperature in the laboratory the same. This is very important because temperature affects the rate at which enzymes work; up to 40oC, as the temperature gets higher, enzymes work faster and break down their substrate more quickly. At 40oC the active sites of enzymes start to denature, so the rate at which they work starts to slow down.

The same ratio and volume of enzyme to substrate, amylase to starch, was used each time, so they would all be equal.

The volume of amylase solution that was mixed up was always the same, 10mls, and the same proportions were used.

All the apparatus; dropping tiles, pipettes, beakers, syringes and test tubes, were washed thoroughly before the experiment was begun and after each different strength of amylase solution was tested and the same syringe was used for the starch, the same for the amylase and the same for the water, to ensure that nothing was contaminated with any stray substance.

The same stopwatch was used for each test.

As far as I am aware, the pH was the same in each test. I can assume this because the pH of amylase and the pH of starch won’t have changed, and the pH of water is neutral so the addition of water shouldn’t have affected the overall pH value.
The starting strength of the amylase, before I mixed it with the water, was always the same, 1%.
Each test was stopped when the iodine turned the same shade of brown, so it was fair.

Results

It is obvious from the results above and the graphs, that as the amylase solutions get weaker the time it takes to break down the starch increases, and therefore the rate at which the amylase breaks down the starch decreases. This is because the volume of amylase solution mixed with the starch was the same every time, regardless of the strength of the solution. For example, with the 0.8% solution, I mixed the solution using 2mls of water and 8mls of amylase, and then I took 4mls out of this ten to mix with the starch. With the 0.6%, I mixed the solution using 4mls of water and 6mls of amylase, so the volume is the same in each case, it is just the proportion of amylase within the solution that varies. As the solution got weaker, the amount of amylase in the solution was less, so it is perfectly logical that it would take longer to break down the same amount of starch than a stronger solution, simply because there is less of the amylase present. This matches the prediction I made that as the amylase solution gets weaker, the amount of time needed to break down the starch will be greater, and so the rate will be slower.

We can see in the table above that there were a few anomalous results. With the 0.6% amylase solution, the first two results were 15 and 25 seconds. These were done on the same day. The third time I tested that strength, was on a different day, and it took 45 seconds. Because this result seemed anomalous, I tested it a fourth time, on the same day as the third time, and it took 50 seconds. Because the first and second tests, which are very similar in results, were conducted on the same day, and the third and fourth tests which are also very similar, were conducted on the same different day, I can only conclude that there must have been some difference in temperature in the laboratory, which would affect the results because as the temperature increases, up to 40oC, the rate of the enzyme also increases. Because this experiment took a very long time, it had to be conducted over several days, so this difference would probably explain any anomalous results. Also, because amylase does not keep very well, a fresh batch had to be made up each day, and though in theory this shouldn’t have made a difference because all the amylase was 1% strength, there might have been a very small difference, which could have affected the results.

Conclusion

From the results of my experiment, I can conclude that the rate at which an enzyme works is related to its strength; as an enzyme gets weaker its rate decreases.

Evaluation

The results of my experiment support my predictions, and also the reasons of logic. My results showed that as the amylase solution got weaker, the rate that the amylase broke down the starch decreased. This is logical because as the amylase solution became weaker, there was less amylase present so it was to be expected that it would therefore break down the starch more slowly. Because my results support this I can conclude that they are reasonably valid. However, there were a few anomalous results, which I believed to have occurred due to some inevitable inaccuracy in the method used. Some of the problems with the experiment were;

A great deal of time was needed to conduct this experiment, and as a result of this, the tests were carried out over a period of two weeks. Also, the tests were carried out at different times of day. This meant that the temperature in the laboratory could have varied, and as enzymes are sensitive to temperature, this could have affected the results. If it was warmer than the amylase would have had more kinetic energy, so moved around faster and therefore collided and combined with the starch more quickly, thus breaking it down at a faster rate.

Amylase doesn’t keep very well, so a new batch had to be made up each day, and though in theory this shouldn’t have made a difference to the results because all the amylase was 1% strength, there might have been a very small difference, which could have affected the results.

Because the weaker strengths, 0.1 and 0.2%, took a very long time to break down all the starch, I tested them with iodine solution every thirty seconds. This meant that all the starch might have been broken down before the next thirty seconds had elapsed, but I wouldn’t know. This meant that there was some degree of inaccuracy with all the results, because practically I could not test the solutions every second.

Despite these problems, many steps were taken to ensure the experiment was a fair test. These were;

As far as it was possible, outside the temperature of the laboratory, each test was conducted under the same temperature; the starch solution was cooled under cold water before it was reacted with the amylase. This is very important because temperature affects the rate at which enzymes work; up to 40oC, as the temperature gets higher, enzymes work faster and break down their substrate more quickly. At 40oC the active sites of enzymes start to denature, so the rate at which they work starts to slow down.

The same ratio and volume of enzyme to substrate, amylase to starch, was used each time, so they would all be equal.

The volume of amylase solution that was mixed up was always the same, 10mls, and the same proportions were used.

The same stopwatch was used for each test.

As far as I am aware, the pH was the same in each test. I can assume this because the pH of amylase and the pH of starch won’t have changed, and the pH of water is neutral so the addition of water shouldn’t have affected the overall pH value.

The starting strength of the amylase, before I mixed it with the water, was always the same, 1%.

Each test was stopped when the iodine turned the same shade of brown, so it was fair.

The experiment was repeated three or four times for each strength of amylase solution and an average was calculated, thus minimising the impact of any anomalous results.

To improve this experiment I would try to improve the accuracy of it. I would like to have had one complete day to do the whole practical experiment, so that there wouldn’t be any difference in temperatures, or in the batches of amylase. I would also improve the accuracy of the measurements. I could have spent more time on the preliminary work and tested all the solutions I was going to use, and have worked out roughly how long each strength solution would take. I could have then, in the real experiment, have left the solutions and the starch for nearly that amount of time, and then tested it every second until all the starch was broken down. This might have given me more accurate results.

If I was going to continue this experiment, I would also like to test whether the temperature affects the rate at which amylase breaks down starch. I know that it does, but I would like to find the exact temperature that an enzyme is wholly denatured. I could also test it the other way, by trying to find out at what temperature an enzyme becomes dormant, and stops reacting with the substrate because it is too cold. I would also like to test how far the pH affects the rate at which an enzyme breaks down its substrate, and how far the pH can be from that enzyme’s optimum pH, and still enable the enzyme to work. I could also test amylase with starch, after the amylase has been left for different periods of time. I know that amylase does not keep very well, so it would be interesting to see how soon the amylase stops working at its optimum, and whether this could have been a contributing factor to the results of this experiment.