Enzymes have a very specific three-dimensional shape, held together by ionic and hydrogen bonds. If the amino acids are too vigorous in their motion, then, these bonds will brake. Once the bonds have been broken, the enzyme is said to have become denatured. As a result of becoming denatured, the enzymes’ rate of activity becomes less because the enzyme loses its specific three-dimensional shape. The enzyme will start to become denatured after around 40ºC as enzyme activity is usually at its optimum at this temperature. After this, the rate of reaction will probably deteriorate. After 60ºC, there is likely to be no reaction, as the enzymes would probably be completely denatured by then. *(Above from: Source, Page 47)
Methodology:
1. Wear goggles for protection, and lab coat if available.
2. Arrange apparatus.
3. Get 250cm3 beaker and fill with water to about the 150cm3 mark with water at a specific temperature. This temperature can be reached by either cooling, via the use of ice, or by heating, via the use of a Bunsen burner.
4. While the water is reaching the desired temperature, get potato and bore out a cylinder with cork borer. To do this, place potato on tile and stabilise by gripping potato by the sides with one hand. Then, press down firmly with cork borer. To retrieve potato cylinder from within cork borer, push it out with the flat end of a pencil.
5. Cut the cylinder of potato obtained, with a ruler so that it is three centimetres long. Then, cut this three centimetre long cylinder up into a further six equal pieces, each 5 millimetres wide. Make sure to keep eyes level with ruler so as to minimise the chance of parallax error. The diameter of the cylinder remains constant due to the use of the cork borer.
6. Put the potato pieces into a boiling tube and put aside.
7. Measure out 30ml of hydrogen peroxide, using a 50cm3-measuring cylinder. Pour this into a boiling tube.
8. Get the two boiling tubes containing one containing hydrogen peroxide and the other potato. Put them both into the beaker of water once the water has reached the desired temperature (this can be done by heating or adding ice to water). After this, wait until the temperature of the hydrogen peroxide and the water bath are equal.
9. Once the temperatures of the potato and hydrogen peroxide are equal, clamp the boiling tube containing the hydrogen peroxide to a clamp stand. Then add the potato into the test tube
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containing the hydrogen peroxide and put bung on top of the test tube, which now contains both potato and hydrogen peroxide, and make sure it is in the beaker of water.
10. Now, measure the water being displaced from the 100cm3 measuring cylinder every thirty seconds using a stop watch.
11. Take readings every thirty seconds for five minutes.
12. Repeat the above steps for different temperatures.
Risk assessment:
During this investigation, I will use catalase from potato cells to speed up the breakdown of hydrogen peroxide. Hydrogen peroxide is an oxidising agent, as oxygen will be given off during the reaction. This implies that it could help the burning of fires. Due to this, reasonable care must be taken to ensure that the oxygen is not directly exposed to the flame of the Bunsen burner while water is being heated. As well as this, hydrogen peroxide can be very harmful if it enters the eyes. Thus, goggles must be worn. Also, if available lab coats will also be worn.
The cork borer is also a potential hazard as it is quite sharp. When cutting out the piece of potato, it must be ensured that the potato is resting on the tile before it is cut. The methodology explains how it will be cut.
The blade which will be used to cut the potato, is also another hazard. It must be handled with appropriate care as it is sharp.
Glass apparatus must be handled with care so that they do not break. If they do, that is also another hazard, and should be cleared up and disposed of in an appropriate fashion.
Fair testing:
While conducting the experiment, it has to be taken into account that a few variables will have to be controlled so as to ensure that the test is fair.
1. The PH of the reactants should be kept constant. This will not be hard to ensure as the PH of the reactants does not vary significantly during the course of the reaction.
2. The temperature of the reactants will have to be kept constant during the reaction. This is a variable which will be harder to control. There will be used, a beaker of 150cm3 of water at the appropriate temperature into which will be immersed, the boiling tube containing the reactants once their temperatures have equalised and stabilised. This will ensure that the temperature of the reactants is stable for longer as the larger volume of water in the beaker will maintain its temperature for longer. Also, the same volume of water will be added to the beaker each time a new test is to be done, so as not have any differences in how long the water maintains its heat for.
3. It must be ensured that when taking readings off the measuring cylinders and thermometers etc, the readings are taken with the graduated markings at eye level, so as to minimise the risk of inaccurate readings.
4. All boiling tubes etc. must be washed thoroughly with water after their use, so as to minimise the chance of contamination.
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5. Making sure that all the potato pieces are cut to the same width, and have the same diameter will control the surface area of the potato.
While conducting the experiment, it must be ensured that the utmost attention is paid to accuracy due to the fact that accurate measurements will result in accurate results. Subsequently, the evidence for or against my prediction regarding the enzyme activity in relation to temperature will be more strong, and, more reliable. The above mentioned methods of accurate measurement will be used as they are most appropriate for the situation, with time factors being one of the major reasons for the augmentation of such methods, as well as the availability of equipment.
During the experiment, the following apparatus will be used:
Cork borer (size four)
Potato
Hydrogen peroxide
Water
Blade
Ruler (15cm)
Stop watch
Test tube rack
Boiling tubes
Delivery tube and rubber bung
Water container
Measuring cylinders (100 cm3 and 50cm3 )
Goggles
Cutting tile
Clamp and stand
Beaker (250cm3)
Graduated pipette (5ml)
ice
Thermometer (0 ºC-100 ºC)
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During the experiment, the apparatus will be set up in the way shown below:
During preliminary work, I found that the 100 cm3 measuring cylinder was ideal for the measurement of water being displaced by oxygen formed during the reaction. This is because it is graduated in millilitres. Thus, it will be possible to measure the displacement of water to the nearest 0.5 of a millilitre. Also, the measuring cylinder is not so small that the volume of oxygen produced will be greater than the cylinder can hold within the course of the reaction.
The 50cm3 measuring cylinder used to measure the volume of hydrogen peroxide was ideal as the amount of hydrogen peroxide was wasn’t too little that it would be inappropriate and also inaccurate to measure with a 50cm3 measuring cylinder, nor was it so large, that it would just about be measured.
Boiling tubes are ideal for the reaction to take place in, as the volume of oxygen produced is quite small. Thus, it will be quicker for the oxygen produced to be able to displace the water in the measuring cylinder. With a conical flask, it would take much longer.
I also found that the breakdown of hydrogen peroxide at room temperature is very slow (Without a catalyst). No oxygen was given off at all over the period of time I observed the hydrogen peroxide for any reaction. However, I did not test whether this was true for higher temperatures. If it was not, then there is the likelihood of major inaccuracies in the conducting of the experiment.
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Source:
Background information used during this investigation was obtained from Cambridge Advanced Sciences. Biology 1 – endorsed by OCR: Chapter 3. (Page 42 onwards.)
Also, information on variables was taken from concepts learned in AS chemistry: (Salters Horners Advanced Chemistry)
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Results:
A Table to show the Volume of Oxygen Produced due to the Catabolic breakdown of Hydrogen Peroxide in Relation to Temperature:
Key:
Time Volume of oxygen produced Temperature
(Seconds) (Cm3) (ºC)
A Table to show the Volume of Oxygen Produced due to the Catabolic breakdown of Hydrogen Peroxide in Relation to Temperature (repeat tests):
Key:
Time Volume of oxygen produced Temperature
(Seconds) (Cm3) (ºC)
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From the graphs, the gradients were taken for the graph of oxygen produced at each temperature. This gave the rate of reaction for the breakdown of hydrogen peroxide, at the different temperatures the experiment was carried out at, in volume of oxygen produced in cm per second. The rate of reaction was then converted to the volume of oxygen produced in minutes, by multiplying each gradient by sixty. The two sets of gradients obtained for the graph of each temperature (one for the initial experiment, and the other set of gradients for the graph drawn from the repeat tests) were matched according to temperature at which the hydrogen peroxide was catabolically broken down at, and their average was taken for a more true picture of what the rate of reaction really is at the different temperatures.
A table Showing the Rates of Reaction for the Initial Tests, the Repeat Test, and also the Average Rate of Reaction
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Conclusion: The results obtained during the course of the experiment seem to be quite conclusive. It possible to identify a pattern or trend in the results obtained. From the rate of reaction graph, we can see that the oxygen is produced more and more quickly via the breakdown of hydrogen peroxide (when the reaction is catabolic) with an increase in temperature. This is, however, only up to a certain point. We see that the rate of reaction keeps on increasing until 40ºC, after which it starts to fall. There is still some oxygen being produced after 70ºC, but only very little, almost zero.
With respect to the results obtained, I can now say that the prediction I made earlier on was more or less correct, although not as correct as I had hoped they would be, as I had predicted that the rate of reaction would be zero after 60ºC, and this is more true of enzyme activity at 70ºC. However, the rest of my prediction seems to be in support of my hypothesis. Thus, it would seem that the enzyme activity (in other words, the rate of reaction) increases with temperature up until around 40ºC as the enzyme and substrate molecules gain more and more kinetic energy. As a result, the reactants move around with increased vigour. This results in there being an increased number of effective collisions. Subsequently, the rate of reaction increases. After 40ºC, the rate of reaction deteriorates. Although the kinetic energy increases, and in essence, the rate of reaction should keep on increasing, this is however not true. This due to the fact that after 40ºC, the optimum temperature for catalase enzyme activity, the weak bonds that hold together the enzyme structure, start to break (this is especially true of hydrogen bonds), due to the increased kinetic energy. Resultantly, the rate of reaction deteriorates as the enzyme becomes denatured due to the fact that its active site, and ultimately its whole structure, is lost due to the breaking up of the bonds that hold it together. This means that the substrate molecule can no longer fit into the active site of the enzyme as the shape of the active site changes.
Evaluation: Although the results obtained from the experiment were more or less support in my prediction, I was not totally satisfied with the experiment. There were a lot of errors, both in the conducting of the experiment, and in the results obtained.
First of all the way I conducted the experiment was quite flawed. For example, the measuring cylinder used to measure the volume of oxygen produced, was quite inappropriate, and probably resulted in many of the readings being taken being quite inaccurate as it is easy to make a mistake in reading off the value while trying to hold the measuring cylinder straight, and also trying to keep the bottom of the meniscus of the water at eye level, all at the same time. Also, it was only possible to measure to the nearest 0.5 of a centimetre cubed, as the measuring cylinder was only graduated in 1cm3
The method used to keep the temperature of the reactants constant at certain temperatures was also improper, as water in the beaker which was used for the above purpose, was not in a large enough volume or quantity to retain its heat for more than a minute or two. Thus, the temperature of the reactants was fluctuating during the experiment. Also, the water in the beaker did not cover all the reactants in the boiling tube, and some of the reactants were not immersed by water. Thus, there were regions of unequal temperatures in the reactants.
The controlling of the surface area of the potato was also inaccurate as it was impossible to measure the lengths of potato to exactly 5mm throughout.
Also, it was assumed that the PH of the reactants would remain constant throughout. This may not have been the case
Considering the above, it is feasible to say that the results obtained during the experiment are neither likely to be very reliable nor very accurate.
Considering that the there was so much possibility for inaccuracy, there were not however, any major anomalies in the results obtained, although the results obtained in the repeats at
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20ºC and 50ºC, were on the doubtable side, as there was quite a difference when the volumes of oxygen obtained were compared with those obtained during the first test:
Volume of oxygen produced (in cm3) at:
20ºC 50ºC
1st test: 8.5 6
2nd test: 10.0 4.5
These slight anomalies may have arisen may have arisen due to a number of reasons:
a) Improper measurement off measuring cylinder
b) Improper surface area of potato, and effectively enzyme
c) Difference in temperature due to loss of heat (kinetic energy)
d) PH level may have altered
If I was to conduct the experiment again, I would make sure that it was more accurate overall. This would ensure that the results obtained were more reliable and accurate. I would do this in the following ways:
1) I would use a graduated syringe, instead of a measuring cylinder as this would ensure that it was easier to measure the volume of oxygen produced. It would also be more accurate as there would be a clear line to mark the amount of oxygen produced and there would not be a need to observe where thw meniscus of water touched on the graduation marks.
2) I would use a greater volume of water to ensure that the temperature of the reactants remained constant, due to the fact that water is such that, the greater the volume, the greater its ability to retain heat.
3) I would use a buffer to control the PH of the reactants as it would ensure that the Ph remained constant.
4) I would crush the potato to a paste, using a mortar ant pestle, and drain the filtrate for use as the catalase sample. A measure of the volume would be made.In this way, the exact amounts of enzyme could be calculated. If not, then I would use yeast as my enzyme, as it can be measured out to very precise quantities.
5) I would also use a graduated syringe to measure out the amounts of hydrogen peroxide, and other fluids. The reasons for this are the same as the ones mentioned above.
With the above taken into account, I would say that my conclusion is in fact, not very safe due to the fact that there were too many errors and uncertainties concerning the results obtained. Subsequently, the results do not provide a very stable evidence for support of my hypothesis. Although the percentage error of individual equipment may have been at first glance, small, they add up to large percentage errors which then render useless, the legitimacy of the results obtained.
Suma_83@hotmail
INVESTIGATING ENZYMESTo investigate how temperature affects the rate of reaction of the enzyme catalase on its substrate hydrogen peroxide.
Investigating Enzymes
Aim
To investigate how temperature affects the rate of reaction of the enzyme catalase on its substrate hydrogen peroxide.
Scientific Background
Enzymes are biological catalysts which increase the rate of reactions by lowering the activation energy needed for the reaction to tale place. The activation energy is the amount of energy needed for molecules to react when they collide. Molecules need to collide in order to react, this is known as the collision theory. When they collide they may not react as a certain amount of energy is required to break bonds, this energy is the activation energy.
Enzymes are made of a long amino acid chain, within this some molecules are attracted to each other, so the chain folds in on itself to form a 3D shape.
How enzymes are shaped.
An area on the surface of the enzyme is known as the active site. This is where reactions take place to form or break down substances. Enzymes are specific which means a particular enzyme only works on one substance known as its substrate. For example, the substrate of amylase is starch and the substrate of lipase is fats. They only have one substrate because the active site is formed in a different shape for each enzyme, where only one substance can fit. The ‘lock and key’ hypothesis states that the enzyme is like a lock which will only have one key.
‘Lock and Key’ hypothesis
The substrate shown is the only substance that fits the enzyme. An enzyme substrate complex is the compound formed when the substrate is attached to the active site, it is only in this form for a short time while the substrate is being broken down.
Enzymes can break own substances, known as catabolism, or can join substances together, known as anabolism. Together they form metabolism which is every chemical reaction in the body.
catabolism and anabolism.
Enzymes are affected by four factors which are
1. Temperature
2. pH
3. Enzyme concentration
4. Substrate concentration
A temperature increase gives ore energy to gives more energy to the substrate and the enzyme so they are more likely to collide and react. The frequency of the collisions with the right activation energy will increase so the rate of reaction will increase. The rate of increase is shown by a mathematical coefficient known as Q10, which states that a ten degree rise in temperture will cause the rate of reaction to approximately double. However at high temperatures enzymes will begin to denature. This means the attractions holding together the shape of the enzyme will begin to break so the active site loses its unique shape and is unable to react with its substrate. The optimum temperature for most enzymes is 37°C, after this they begin to denature. The enzymes in the body have this optimum temperature and the body has adapted to control its temperature so the enzymes are working at there best.
Enzymes also have an optimum pH level, where they work best, any changes to this level will cause the enzymes to begin to denature.
Pepsin works best in acidic conditions because it is used in the stomach along with stomach acid. Lipase works best in alkali conditions because it works with bile in the intestines.
Increasing the concentration of either the enzyme or the substrate will increase the number in the solution meaning there is more chance of collisions and reactions. There is a limit to the rate of reaction. For example if the enzyme concentration is increased from the same concentration of enzyme and substrate then the rate of reaction will not increase as there are not enough subsrate molecules to react with.
Hypothesis
I predict that a rise in temperature will cause a rise in the rate of reaction until 40°C, after which enzymes will denature so the rate will fall. This will happen because a rise in temperature will mean the are moving faster and are more likely to collide with the catalase on the potato resulting in a greater frequency of collisions. A higher temperature will also mean more hydrogen peroxide molecules will have an energy above the activation energy, so there will be more collisions with the right activation energy. This will result in the rate of reaction increasing.
After 40°C the rate will fall because catalase will denature. His means the attractions between amino acid molecules in the enzyme will break and the enzyme will lose its shape. The active site of the enzyme changes so it can not break down hydrogen peroxide. As the temperature rises further the catalase will denature more quickly and the rate of reaction will fall further.
A graph of rate of reaction against temperature may look like this.
The rise of rate of reaction is governed by the Q10 coefficient, which states that a 10°C rise will result in an approximate double of the rate of reaction.
Method
Preliminary work was undertaken to determine the amount of hydrogen peroxide and potato to use, and what temperature differences to use. The results are as follows;
1cm potato was too small to handle, and 2cm potato was not fully covered by 2cm3 hydrogen peroxide. So 2cm of potato was used with of hydrogen peroxide. There will not be enough time for all the experiments to be left for five minutes. So they will be left for four minutes, leaving enough time to complete all experiments.
Pour 3cm3 of hydrogen peroxide into a test tube, place this in a water bath of 20°C to warm up. Use a cork borer to retrieve a strip of potato from a potato, cut this to 2cm using a cutting board and a knife. Pour water into a beaker and place and measuring cylinder, full of water, into it, as shown below. Take care not to allow air into the cylinder. Place the end of a delivery tube into the measuring cylinder as shown below. Place the potato on the side of the test tube and close it with the bung of the delivery tube. Allow the potato to drop into the hydrogen peroxide, which should be the temperature of the water bath, and start a timer.
Set up of equipment
As oxygen is produced in the reaction it will displace the air trapped in the test tube, this will be forced through the delivery tube into the measuring cylinder. The air will rise to the top as the measuring cylinder is full of water and its volume can be measured. Take readings of gas produced every 30 seconds for four minutes.
Repeat the experiment twice for reliability and verification of results. Conduct similar experiments with water bath temperatures of 10°C, 30°C, 40°C, 50°C, 60°C and 70°C. In each case make sure the temperature of the substrate has reached the temperature of the water bath before adding the potato.
Other methods which can be used are counting the number of bubbles produced, this would not be accurate as the bubble sizes are not the same and the volume is not measured. Measuring the mass of the gas lost is a better method as readings on the scale will be accurate, however this would require equipment which is not available.
To make the test fair all other factors affecting the rate of reaction must be kept constant. This includes surface area of the potato and the concentration of the hydrogen peroxide. The experiment will be kept a fair test by:
· Using the same length of potato, to keep the surface area constant,
· Using the same volume of hydrogen peroxide,
· Washing the test tube out with water and drying it, this will prevent concentration changes in hydrogen peroxide,
· Using the same potato, as different potatoes will have different levels of catalase present,
· Using the same concentration of hydrogen peroxide,
· Using the same cork borer to cut the potato, to keep the surface area constant.
The experiment will be safe by:
· Always wearing safety goggles, as hydrogen peroxide an damage your eyes,
· Not spilling the hydrogen peroxide as it is an irritant, and bleaches,
· Taking care when cutting the potato,
· Taking care when handling hot water.
Results
The results obtained are as follows:
There was not enough time to conduct two repetitions, however, one repetition was conducted. The rest of the method was followed as planned.
1st Set of Results
2nd Set of Results
Averages
Rate of reaction
Analysis
The amount of gas produced is proportional to the rate of reaction because if the rate of reaction doubles then twice as many reactions are occurring per second so the amount a gas produced is doubled. The rate of reaction can be found by dividing the gas produced by the time. This has been done in the results.
From the graph of temperature against rate of reaction we can see the highest rate of reaction, of 0.013 /s, occurs at 40°C. The rate is slow, 0.00125 /s, at 10°C it then rises with temperature until it reaches its maximum at 40°C. This happens because at low temperatures the hydrogen peroxide has less energy a moves more slowly. It will collide with the catalase less often, meaning the frequency of collisions is low. They are less likely to have the right activation energy so there are less collisions resulting in reactions. This will mean the rate of reaction will be low. At higher temperatures the hydrogen peroxide has enough energy to reach its activation energy and it is also colliding more often, so the rate of reaction will be higher.
After 40°C the rate of reaction falls , this is because the catalase begins to denature. When enzymes denature the attractions between amino acids in the enzyme break and the enzyme begins to return to its original shape. The shape of the active site also changes so it cannot break down the hydrogen peroxide.
The rise in rate of reaction between 10°C and 40°C complies with the Q10 coefficient, as a 10°C rise causes the rate of reaction to approximately double. This can be shown by dividing the higher rate of reaction by the lower one. For example dividing the rate of reaction at 20°C by the rate of reaction at 10°C should give a figure close to 2.
These figures are all close to 2, they will not be exact because Q10 is only an approximate and the results are not perfect.
From the table of results showing the average volume of gas produced every 30 seconds we can see that at 50°C the enzymes denature within 2 minutes, as gas is no longer produced. This happens because the enzyme takes time to heat up, while it is still reacting with the substrate. Once it is heated to the temperature of the hydrogen peroxide not all the enzymes are denatured. At 60°C the potato is heated faster and it takes 90 seconds for the enzymes to denature, at 70°C the potato is heated even faster and it takes 30 seconds to denature.
The results agree with my hypothesis because I have predicted that the rate of reaction will rise between 10°C and 40°C, and the rate will fall after 40°C. The graph obtained for the results is also similar to the one predicted, and the results seem to follow as predicted.
From conducting the experiment and gathering data I can conclude that the rate of reaction between catalase and hydrogen peroxide rises as the temperature of the mixture rises. This happens until 40°C, after which the rate of reaction falls because the catalase begins to denature. When enzyme denature attractions between the amino acids break so the enzymes loses its shape. The active site will no longer have its unique shape and the enzyme will be unable to react with its substrate. This is because only the right active site shape can break down hydrogen peroxide, according to the ‘lock and key’ hypothesis, which suggests the substrate, like a key, will only have one lock, enzyme, it fits into. The reaction follows Q10 until 40°C, because a 10°C rise will give enough energy to the substrate to increase the number of collisions and give more molecules the right activation energy to react when the collide. This will double the rate of reaction.
Two anomalous results occurred during the experiment.
1. During the repeat reading of the experiment at 10°C the reading at 90 seconds is higher than that of 120 seconds. This does not affect the analysis as the reading was ignored when taking averages.
2. During the 50°C experiment the first time the volume of gas produced stopped increasing between 120 and 180 seconds. Gas was then released, it may have been trapped in the delivery tube. When drawing the best fit line this was taken into account, so it shouldn’t affect the analysis.
Evaluation
The experiment was conducted successfully, the results obtained indicates a clear pattern which can be used to draw and support a valid conclusion. The experiment could not be conducted as planned because there was not enough time to repeat each experiment twice. However, one repetition was conducted which did make the results more reliable.
The results are reliable because the experiment was a fair test. This was done by keeping all variables constant. The concentration of hydrogen peroxide was not changed, however, it did vary as it naturally decomposed into water and oxygen. It also decomposed more during the higher temperature experiments because the hydrogen peroxide had more energy. This was a slight change and could not vary the volume of the gas produced significantly. The same volume oh hydrogen peroxide was used. The surface area of the potato was kept constant by using the same size cork borer and cutting it to the same size.
The results are accurate because a narrow measuring cylinder was used, so the volume measured is more accurate. Hot and cold water were mixed to achieve accurate temperatures.
An anomalous results occurred during the repeat reading of the 10°C experiment, the reading at 90 seconds is higher than that at 120 seconds. This reading was ignored when taking the average so It does not affect the analysis. It occurred because the measurement was misread, it may have been 0.1 cm3, instead of 0.2 cm3. Another anomalous result occurred during the first taking of the 50°C experiment, gas was released at 210 seconds when the experiment seemed to have stopped. The gas may have been trapped in the delivery tube and should have been released earlier in the experiment. This was taken into account when drawing the best fit line on the graph, so does not affect the analysis.
The method used was good enough to achieve reliable readings , but it can be improved by measuring the mass of the gas lost, this would be more accurate as digital readings would be taken. Using a smaller frequency and a larger range of temperatures would give more evidence for the conclusion. However would require more time and equipment.
Other improvements are, using thinner measuring cylinders, to measure out the hydrogen peroxide and the gas produced. Using a thermocouple thermometer to accurately measure the temperature. The hydrogen peroxide took time to heat up as the test tube is glass and is insulated. Using a better material would save time and would have allowed the plan to be completed. Using electronic equipment to take readings a exactly 30 seconds, would eliminate human error.
Further work that would extend the investigation and give more evidence to the conclusion would be, to use different concentrations of hydrogen peroxide and lengths of potato, to see how these affect the rate of reaction. Using other substances with catalase, like liver, to see how enzyme concentration affects the rate.