At high temperatures the heat energy increases and causes the atoms that make up the enzyme molecules to vibrate. If these vibrations become too violent this eventually leads the enzyme structure to change from its precise three-dimensional shape because the chemical bonds inside it have broken down. This change in shape renders it useless to perform its function, as its specially structured form, essential for enzyme activity, is altered. In other words at higher temperatures bonds are broken which mean that the lipase enzyme key can no longer fit in the milk substrate lock (diagram of lock and key hypothesis is show below as diagram A). This process is called denaturing. Preliminary experiments showed that after 40°C the rate of reaction began to decrease rapidly, I think that this is due to the enzymes being denatured by the heat and therefore being useless. In my opinion after 40°C denaturing destroys so many enzymes that the increased kinetic energy cannot compensate and the reaction slows. This denaturing is the reason why I did not predict a temperature above 40°C.
I predict 40°C to be my optimum temperature because I feel that at this temperature there is sufficient heat to supply kinetic energy to molecules yet there is not such excessive heat that vibrations are caused in the enzyme’s bonds causing them to denature. At 40°C the enzymes are not necessarily at their most stable and there may be a small amount of denaturing.
I predict that 30°C and 50°C will be fairly fast reaction which are slightly behind the optimum since there is only a 10°C difference from the predicted optimum. I predict that there will be no reaction within fifteen minutes for 70°C since I feel that the majority of the lipase enzymes will be denatured due to the excessive heat making the chemical bonds vibrate so violently that they break, causing the enzyme to denature. I think that 60°C will be quite a slow reaction as the majority of the enzymes will be denatured. I feel that 20°C will be a slow reaction since there will be less heat to supply kinetic energy to the molecules thus resulting in fewer molecular collisions and therefore less successful enzyme-substrate complexes. I feel that 40°C will be the optimum because I feel that this is the temperature where there is a balance between denaturing and the need for kinetic energy.
My prediction for a graph of temperature against rate of reaction (shown below as Graph A) is therefore that there is an optimum at 40°C, that goes down to the lower rates at 20°C and 70°C.
Apparatus
12 Test tubes
Waterbath
Bunsen Burner
Timer
Undiluted full cream milk
3% bile salt solution
Phenolphthalein indicator solution
0.1M Sodium Carbonate solution
Lipase solution
Ice
Diagram of Apparatus
Method
- Take two test tubes labelled A and B
- In tube A add: 5ml of undiluted full cream milk
1ml of 3% bile salt solution
2ml of 0.1M sodium carbonate solution
- To tube B add 2ml of lipase solution.
- Set up a water bath to obtain the desired temperature. Ice can be used to obtain the cold temperatures and a Bunsen Burner should be used for warmer temperatures.
- Place both tubes in the water bath
- After two minutes mix tubes A and B, immediately replace the tube holding the mixture in the appropriate water bath and start the timer.
- Using a test tube of milk as a control check when the mixture changes to the same colour as the milk. Stop the timer and record the time taken for the reaction to occur. (if there is no change within fifteen minutes stop the experiment)
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Repeat the experiment until you have three readings for each of the temperatures 20°C, 30°C, 40°C, 50°C, 60°C and 70°C.
Explanation of Method
In this experiment the independent variable which will be changed during the experiment is temperature. The dependent variable being measured is the time taken for the enzyme lipase to breakdown all of the milk. The other control factors to stay constant are the pH of the solutions in all tubes, all of the volumes and concentrations of the solutions should be constant and all of the guidelines in the method should be followed carefully. These other factors must stay the same in order for the experiment to be fair.
Preliminary experiments showed that measuring cylinders were not accurate enough for measuring the volumes of the solutions in the experiment. I rectified this problem in my final method by using 1cm3 and 5cm3 syringes as measuring equipment.
To keep the experiment a fair test I have taken some precautionary steps for example the milk has been taken from the same carton and has not been mixed with any other substances. The tests shall be carried out 3 times for each temperature with new (clean) apparatus for each individual test. A test tube of milk will be used as a control check for when the final mixture is being timed for breakdown rate.
Preliminary experiments also led me to choose phenolphthalein as my indicator since it gave a pink starting colour (when the solutions were first mixed) that was distinctly different from the white finishing colour of the milk (this enabled the finishing point of the reaction to be more easily detected than when using other indicators). The fact the phenolphthalein was pink in alkali (when the reaction started) and colourless in acid (when the reaction finished) meant it was the best indicator I had at my disposal. The change in pH due to the reaction made phenolphthalein an ideal indicator.
The bile salt and sodium carbonate quantities were also manipulated during preliminary experiments to give an adequate pH for the lipase enzymes to work at and to also provide a suitable pH for the phenolphthalein indicator (i.e. a colour change when the reaction is completed).
The volumes of full cream milk (eventually 5ml) and lipase solution (2ml) were also found by preliminary experiments and secondary sources, these eventual volumes were chosen since they did not make the experiment move too fast or too slow.
Before mixing I decided to put the two tubes in the appropriate waterbath for two minutes before mixing. The idea behind this was to equilibrate the two tubes, so that they are at the same, appropriate temperature when the reaction actually starts. This enables more accurate results to be achieved since the molecules are at the temperature from the very beginning of the reaction and do not need time to change to the different temperature (from room temperature).
Safety
Safety goggles must be worn throughout this experiment. This is especially important when using the Bunsen burner as there is a possibility that hot water (and other solutions) may ‘spit’ into the eyes. Tongs should also be used when handling the hot waterbaths and any other hot materials. Care should be taken while handling the thermometer as it contains Mercury. Long hair should be tied back and ties should be tucked in to avoid any contact with flames or harmful substances. The Bile salt solution and sodium carbonate solution should also be handled with care.
Conclusion
The final results show 30°C to be the optimum temperature with a rate of reaction of 30. This is followed by my predicted optimum temperature which was 40°C with a rate of reaction of 25. The difference from 20°C to 30°C is rather drastic as it changes from 12 to 30. After 40°C the temperature begins to drop steadily until at 60°C the rate is roughly the same as at 20°C. 70°C had no reaction within the fifteen minutes. My predicted optimum temperature was wrong, however the results I have obtained do roughly follow my prediction.
With the exception of the incorrect optimum (the results show the optimum to be at 30°C rather than at the predicted 40°C) the graph of results is very close to the prediction curve shown at the end of my prediction. This infers that up to the optimum the reaction is speeded up by heat (up to 30°C) the heat supplies kinetic energy to the molecules, which increases the chances of molecular collision. This means that the lipase and milk molecules ‘meet’ more often and thus the reaction is speeded up. At the optimum (30°C) there is more heat, and therefore more kinetic energy, than at the lower temperatures so there is a higher chance of the lipase and milk molecules colliding than at the lower temperatures. Any collisions are also more likely to be successful since the molecules meet at higher speeds, (with more kinetic energy) and they therefore are more likely to have the activation energy required to start the reaction. There is not however such excessive heat that the atoms in the enzyme vibrate so violently causing the chemical bonds to break and the enzyme to denature. The denaturing of the enzyme means that the active centre of the enzymes is irreversibly altered and the substrate can no longer interact with the denatured enzymes (the lipase enzyme is no longer the right shape for the milk molecule substrate). At 30°C and 40°C there would be little or no denaturing. This does not however mean that the optimum is when the enzyme is most stable.
After the optimum there is a fall in the reaction (shown by the graph), this is as the heat increases enzymes start to denature and the increased kinetic energy cannot compensate for this. The higher the temperature the more enzymes denature and this results in the rate falling after the optimum, this trend is illustrated by the best fit line on the graph.
The results show that 30°C has a sufficient amount of kinetic energy to speed up the particles in the mixture and not too much energy so that the enzymes are denatured. My general prediction that there would be a rise up to the optimum and then a fall was correct. I did predict the wrong optimum, however I did correctly predict that there would be no reaction within the fifteen minutes for 70°C. My prediction that 20°C and 60°C would be slow was also proved correct. I could not however accurately test my idea that if the reaction was increased by 10°C the time rate for the reaction would halve since I could only compare the results for 20°C and 30°C before denaturing started to radically affect the results. I do however feel that the majority of my predictions were correct and the results supported rather than undermined them.
Evaluation
I think that the method I used was sufficient to obtain a firm conclusion and I believe that the results I have are accurate. I also feel that the procedure used was generally quite successful.
Although there are no obvious anomalies there are however indications of discrepancies. The range for the three readings for 40°C have a range of 10 seconds and 60°C also have a range of 10 seconds. These scattered results show that there are slight anomalies within the readings for each temperature. These results are only every 10°C so they are not extremely accurate, the anomalies within the temperature repeats also show that there are inaccuracies within the results. In addition to this the graph of rate of reaction against temperature is not the same smooth curve as shown in my prediction and in many secondary sources. These results do however follow roughly the same trend as the prediction and secondary sources show, the graph of results does also show similarities to the prediction curve shown in the prediction.
I think that the method was suitable but I realise that it would have been improved if better equipment had been used. One of the flaws in this method was that it was difficult to maintain the temperature of the waterbaths during the experiments, this problem could be solved by using thermostatic waterbaths to ensure proper control of the temperature. In addition to this it was difficult to ascertain exactly when the milk molecules had been broken down and the reaction was finished; judging the end of the reaction at different shades of white could have allowed inaccuracies into the experiment. This could have been avoided by using special machinery that could have detected the pH changes (and therefore the end of the reaction) much more accurately. Better equipment and machinery could also improve other aspects of the experiment as the time could be more precise than the nearest second. These flaws in the method may explain the variation within the results and any other possible anomalies.
The evidence obtained by this experiment is quite reliable and there was a fairly wide range of results available, with three repeats for each of the six temperatures. We have to take into account that there were anomalies since there was a lot of variation within the repeat results and this indicates that the experiment may have had flaws. I think that you can base a conclusion upon these results but for the results to be justified I feel that you would have to repeat the experiment in controlled condition making sure all flaws have been accounted for. By repeating the experiment over a wider range of temperatures and more than three times for each temperatures and by implementing my improvements I think that you would achieve much more reliable results.
Use of different types of milk with different amounts of fat could be an idea for further work. Also the idea of the effect of temperature on enzyme activity could be broadened to other reactions such as the reaction of amylase and starch. Also the effect of other variables such as pH, concentration and volume on this reaction could be tested. By using machinery the precision and accuracy could be improved and results that could support a firm conclusion could actually be found.
Bibliography
Background Information and secondary sources used:
Biological Studies by Henry Wilkinson, published by Nelson, page number 46.
Encarta ’97.
GCSE Double Science Biology Revision Guide by Richard Parsons, published by Coordination Group Publications, page numbers 20-22.
Key Science for GCSE Biology by David Applin, published by Stanley Thornes, page numbers 227-230.