Investigating the effect of temperature on the enzyme amylase.

Biology coursework – Investigating the effect of temperature on the enzyme amylase

Introduction

Enzymes are vital; they control and catalyse all of the chemical reactions inside living cells. The enzymes speed up reactions, which would not otherwise happen rapidly enough to maintain the essential life processes.

Amylase is the enzyme found in our Saliva. It is a catalyst, which works by breaking down substrate into smaller pieces: increasing the surface area, allowing the enzyme to work on the starch quicker and therefore speeding up the reaction. The aim of this experiment is to see how temperature affects the amylase and its ability to effectively break down starch and form maltose. I will be investigating at which temperature the enzyme becomes irreversibly de-natured and at which temperature it becomes inactive.

Starch mixed with water on its own reacts very slowly, taking years too react but because we have the enzyme amylase in our saliva the enzymes catalyses the reaction so well that it can break down starch into sugar in minutes or seconds.

Variables

The variables in the experiment will be the temperature of the saliva and starch solution, the time at which I record the results, the pH of the starch solution and saliva and the quantities of the solutions. The temperature will be the manipulated, Independent variable as this is what I will be changing. The dependant variable will be the iodine solution turning brown indicating when the amylase has successfully broken down the starch, this being the rate of reaction.

I will control the pH, the quantities and the concentration of the substances. I will keep the temperatures we choose below 70*c as the enzyme could be denatured (the active site changing irreversibly by the sheer heat) and above 0*c as the amylase would become dormant (not an irreversible change – can become active by being brought back up to its optimum temperature). In my preliminary work, I found out the optimum pH for amylase to work in is pH7, which is neutral so I shall control the pH using pH7 starch solution each time. I shall use the results from my preliminary work to decide on the concentrations of the saliva and starch solutions.

I cannot control the pH of my saliva however, I can predict that it will be at pH7 as it is found in the mouth and will presumably work in neutral conditions best as opposed to enzymes in the stomach which work best in acidic conditions. Therefore the pH will hopefully be the same in both the solutions as I am choosing a pH7 starch solution as well.

In an acidic pH the enzyme amylase would become de-natured: the extreme acidity changing the shape of the active site irreversibly. The same goes for a too alkaline pH which would also become de-natured becomes of the extremes in pH.

So the variables are:

Dependant variable: time taken for the iodine solution to turn brown (indicating the absence of starch and showing it has been broken down into maltose by the amylase)

Independent variable: Temperature of solution (starch+saliva)

Controlled variable: The pH of the solution, volume of saliva solution, volume of starch solution, concentration of starch solution, how often I pipette the solution into the iodine (timing), strength of saliva.

Prediction

I predict that the saliva solution and the amylase at 40c will work best because that is the closest of my five chosen temperatures to the human body temperature of 37c and since the enzyme amylase is found in human saliva, in the mouth I think this will be its optimum temperature. My highest temperature of 60c will perhaps be too far above the amylase’s optimum temperature and the enzyme may become de-natured. By this, I mean the enzyme will change shape because of the high temperature. The temperature increases kinetic energy breaking the enzyme’s bonds and so the active site will have changed shape and will not work and fit with the starch particle to react. Enzymes are specific and so the ‘lock and key’ method will not work. In the same way if my lowest temperature of 20c is too far below the optimum temperature the enzyme will become dormant. This, unlike de-naturing, is not an irreversible change and once brought back up to the optimum temperature the amylase will work again.

I also predict that with the increase in temperature will come an increase in rate of reaction because, supporting the Brownian motion’s ideas, when a substrate is heated the kinetic energy causes the molecules to move around faster and therefore the enzyme and starch molecules are more likely to collide and then react efficiently – increasing the reaction rate. This will not be the case when the temperature reaches the enzymes de-natured temperature, as the enzyme will then become useless: it will have changed shape irreversibly and be unable to fit to the starch molecules using the lock ...

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Preliminary work

In my preliminary work I investigated the effect of pH on enzymes. I did a similar experiment to the one I am doing now but where the manipulated variable was the pH instead of temperature and I used different pH’s to find out which was the optimum temperature, controlling the temperature by bringing the two solutions (saliva solution and starch solution) to equilibrium in a hot water bath before starting the experiment.

My preliminary work showed me several things. Firstly it told me that the amylase’s optimum working pH is pH7 so I will use this in my current experiment as a controlled variable – controlling the pH by having a pH7 starch solution. I cannot control the pH of my saliva but I can presume that because amylase is found in the mouth, in the body it will work best at the general body’s optimum pH of a neutral pH 7. Of course in the stomach there will be an optimum pH of about 2 which is acidic because the body contains acids to break down food and the enzymes found in the stomach work best in acid conditions.

I also found out that I need to strengthen my saliva solution from the 1:4 (1cm saliva 4 cm of distilled water) ratio I had last time as this was too diluted and did not work quick enough to show results in the limited time I had to do the experiment. I shall use a 2:3 ratio of saliva solution in this experiment and shall use a higher concentrated starch solution for 2% to 5%. Hopefully these changes will increase the reaction rate and speed up the experiment. In one case it took up to 10 minutes for the iodine solution to turn brown and show results at the optimum of pH7 and so these increases in concentration should mean a faster reaction and speed up the experiment because according to the ‘collision theory’ a higher concentration results in more frequent collisions - and therefore more reactions.

Also, in my preliminary experiment I used 3 drops of iodine solution and only one drop of saliva/starch solution in the dimple tiles and this could have delayed my results as it would have taken longer for the iodine solution to turn brown indicating the lack of starch than if I had used 1 drop of iodine and 1 drop of the solution. The other thing I learnt from my premilinary work was to label test tubes as they could easily get mixed up, presenting a health and safety risk. This time I shall label all the test tubes I am using for my saliva solution.

Finally I realised while evaluating my preliminary experiment that I had not been completely accurate in putting the fresh drop of solution into the dimple tray exactly on every 15 seconds, especially zero seconds. It is hard to do this as I need to shake up the solution, start the timer and pipette the solution into the dimple tray all at zero seconds as soon as the mixture touches. In my preliminary work I decided that mixing up the solution was more important than to pipette the solution onto the dimple tile at zero seconds as it would probably not show results and the iodine show brown at even the optimum pH till well after zero seconds. I still think is relevant with the optimum temperature and it did not seem to disrupt my results last time so I shall do this again.

Method

I will measure out 5cm3 of a 5% starch solution in a 10cm3 measuring cylinder and pour it into a test tube. Then I will fill another test tube with 5cm3 of dilute saliva solution (2:3 ratio – 2cm3 saliva and 3cm3 of distilled water). Using the pipette, i will then put 3 drops of iodine solution into each of the ‘dimples’ on the dimple tile. I will put both test tubes into a water bath heated to one of the temperatures I have chosen (20*c, 30*c, 40*c, 50*c and 60*c) so they can come to equilibrium. I shall leave them in there for 5 minutes then mix the two solutions and start the timer as soon as the two solutions make contact. Then, starting at zero seconds, I will pipette two fresh drops of the solution into each dimple with iodine every 15 seconds. I will then stop the timer when the iodine solution turns brown; indicating that there is no starch left in the solution and the amylase has worked and turned it to maltose. If the solution does not turn brown after 10 minutes, I will stop the timer.

I will do 5 different temperatures and repeat each one 3 times for reliable results so I can find an average and plot it on a graph and then to see the rate of reaction I will use 1/time to produce a rate graph. I will identify anomalous results and re-do them to get results that are more accurate. I will have a range of 20c to 60c, which is sensible as it should be below de-naturing temperature and above the in-activating temperature.

Equipment

Dimple tile
10cm3 Measuring cylinder
Test tubes
Pipettes
Timer
Test tube rack
Iodine solution

Risk assessment

I will be using hot water baths to heat the solutions so it is important I am careful when putting the test tubes in the hot water bath so I do not burn myself. I need to be careful when handling glass not to break the test tubes or the glass pipettes so I do not cut myself. It is very important that health and safety is observed considering the saliva. Test tubes should be labelled so they are not mixed up and picked up by accident. Aprons will be worn to stop the saliva splashing onto our clothes and I realised from my preliminary work that ideally we should wear safety goggles and gloves so we do not come into contact with other people’s saliva as it could be carrying diseases. When I have finished the experiment the plastic pipettes will be thrown away and the rest of the equipment will be soaked in bleach to be properly cleaned. It would be safer and easier to use powdered saliva, as it is quite hard to produce 5cm3 of your own saliva, but it would not be practical. The powdered saliva come from plants and has an optimum temperature of 70c, which is nothing like amylases optimum of the human body temperature 37c. In addition, I need to be careful not to get the iodine solution onto my skin, as it will stain it.

Results table

Because of limited resources I had to change the temperatures I had chosen of 20, 30, 40, 50 and 60 (*c) to 0, 20, 30, 40 and 60 (*c) this however, will give me a wider range than I had originally planned and therefore more accurate results. These are the results from the experiment. The results in the time in seconds column is the time it took the iodine solution to show that the starch was gone by turning brown. The number in brackets is the set temperature of the water baths and the number outside of the brackets is the accurate temperature that the water baths really were when measured with the thermometer

Rate table (1/time): Rate of change in drops per second

These are the results I worked out showing the rate of reaction. I worked them out by dividing the times of my results by 1. This allowed me to be more accurate, see a trend and put the results that were infinity onto the new rate graph that I drew.

Conclusion

I can see from my rate graph that there is a general trend showing the optimum temperature at 40*c which is what I predicted as it is close to amylases natural optimum temperature working environment of 37*c. The enzyme shows evidence from the rate graph that it was denatured at 60*c, the heat intensity being too much and causing the enzymes active site to change shape. Similarly, though not completely inactive the enzyme working at 0*c had a very low reaction rate with an average of 0.0047. My results reflect my prediction. My second set of results are much higher than the other two sets of results taking a much longer time for the iodine to turn brown and this pulled the average down considerably on the rate graph. They were anomalous results that could be explained by either that particular pupil having a very weak saliva solution or perhaps they put in a large amount of iodine solution into the dimple tiles and only a small amount of saliva/starch solution so it would take longer for the iodine solution to turn brown indicating the lack of starch.

The results show, as I predicted, that the enzyme efficiency decreases with the extremes of temperature. This is because enzymes have a 3D structure with an active site. Even the slightest change in the shape of the active site can decrease enzyme efficiency because enzymes are specific catalysts – they only catalyse and speed up one reaction. The enzyme works by the lock and key method: the substrate and enzyme are specific and fit together for a short time (like a key in a lock) before the enzyme releases a new substrate. The enzyme does not get used up in the experiment. This is how it works:

It has been suggested in particle theory that a rise in 10*c will double the rate of reaction. However, by doubling the average of 20*c It does not come to the average of 30*c which this theory suggests it should (0.0061 x 2 = 0.0122 not anywhere near 0.074. This could be because of my anomalous second set of results. There is no noticeable relationship between the input and output variable according to the particle theory: just the basic trend that with extremes of temperature enzymes deficiency increases.

I think my results are quite accurate and give me the general idea of what the optimum temperature is (around 40*c), around what point the enzyme becomes de-natured (about 60*c) and at what point the enzyme becomes inactive (0*c). All my results support my prediction except that I expected the enzyme to be de-natured completely at 0*c.

Evaluation

Overall I think the experiment went well. I measured everything carefully and tried to be as accurate as possible in putting 2 drops the solution into the dimples every 15 seconds. I was inaccurate in putting the solution into the dimple tile at zero seconds because I shook the solution and could not pipette it till it was mixed so it was more like 5 seconds. But, as I said before this should not have given me inaccurate results as none of the temperatures, not even the optimum of 40*c showed results that early on.

I did not have time to do a repeat so I took other peoples results and this was obviously not as accurate as it would have been had I done the experiment with my own saliva each time as people have different strengths of saliva. Therefore I was not controlling this variable and, had I had time I would have remedied this by repeating the experiment another 2 times to find an accurate average. However this was not possible but despite the anomalous second set of results my results and graph did show the basic idea that in extremes of temperature the enzyme is less efficient.

My results are not very reliable as the saliva strength was not controlled as it should have been (I should have repeated with my own saliva) but they are accurate - I did measure everything out carefully with precision and timed to.0 of a second.

To improve the experiment I would repeat it with my own saliva. Another thing I would do is let the froth from the saliva go down before measuring it because it was hard to tell where the liquid level was. It would have been a good idea to in a test tube do a sample of iodine solution with no starch present so I could have known exactly what colour to expect the solution to be once I had put the saliva/starch solution in it and the enzyme had catalysed the reaction and the starch had been broken down.

To extend the investigation I could test the final solution with Benedict’s solution to check if the starch had gone and there was maltose present. Also having seen the effects of temperature and pH on enzyme activity I could do the experiment but this time with the manipulated variable being the concentration to see how that affected the enzymes efficiency