The same concentration and size of drop of iodine solution will be used each time
Buffer solution will be used to eliminate small variations in pH The end point will be judged against the original iodine solution
I will take 3 test tubes each containing 2 cm3 of 1 % starch solution made up in pH 7.2 buffer solution to prevent small pH changes affecting my results and place in a water bath at 25°C. 1 will then take 3 test tubes containing 2cm3 of the selected amylase solution and place it in a water bath for temperature treatment (between 40°C and 70°C) I will then return the treated amylase to the 25°C water bath to equilibrate at this temperature. When the amylase has cooled to 25°C I will mix pour each tube into a separate tube of starch. As soon as I do this I will start the stop watch and each minute I will take a small sample with a pipette and test it by mixing with a drop of iodine solution on a white tile. I will continue to do this until there is no change in the colour of iodine on the tile.
I intend to test each amylase at four different temperatures and three different times (5, 10, 15 min:). I will repeat each reading as many times as the time available for the experiment allows.
As a control experiment I intend to test the enzymes without any heat treatment so that I can compare this with the effects of different temperatures.
Risk Assessment
Amylase Enzymes:
These enzymes are bought in powdered form. All enzymes have biological activity and need to be treated with care.
In powdered form:
Handle carefully USE EYE PROTECTION. Avoid inhaling powder
As 2% solutions:
USE EYE PROTECTION
Wash any spillage from skin with water as soon as possible Low risk.
lodine in potassium iodide:
Can be toxic and irritant USE EYE PROTECTION Wash any spillage from skin with water ' Use only small drops
Rinse dropping tiles carefully under running water. Use only in clearly labelled dropping bottles
Starch solution:
Very low risk
Keep in clearly labelled container
Pilot experiment
In order to determine suitable concentrations that will give me a measurable result within the time I have available, different concentrations of starch and enzyme were tried. I was aiming to adjust the concentrations to give me a reaction time of 1-2 minutes which I felt would allow me to take accurate readings but also allow me to carry out a number of repeats in a reasonable time scale
Results of pilot tests
Using 2 cm3 1 % starch solution
Using 2cm3 0.5% starch solution
I also did a single trial to determine the range of temperature treatment that would give me sensible results. I initially thought that treating the enzyme for 10 minutes at 70°C would be sufficient to cause full denaturation. In my trial I found that at this temperature the enzymes were only slightly affected so I extended the temperature range to 90°C.
As a result of these trials I decided to carry out my experiment at 25°C using 0.5% starch solution over a range of 40-90°C. It was evident that the fungal amylase was more active than the bacterial amylase at these concentrations but I decided to keep to the same concentration for each rather than introduce another variable. I am planning to calculate the relative effect of temperature on each which should take into account the difference in the rates of each one.
Method
I measured 2cm3 of 1 % bacterial amylase solution with a small pipette and placed this in a clean test tube. In a second test tube I measured 2cm3 of 0.5% starch solution dissolved in buffer solution of pH 7.2 and then placed it in a water bath at 25°C.
I prepared a second water bath at 40°C and placed the bacterial amylase in this for exactly 5 minutes. I checked the temperature of the amylase with a thermometer to ensure that it was maintained at the correct temperature for 5 minutes.
Whilst this was being treated I prepared a spotting tile by placing equal drops of iodine solution in each depression and marking them in pencil with 30 s intervals.
As soon as the 5 minutes was completed I removed the amylase from the water bath and quickly cooled it to 25°C. When both the amylase and the starch were equilibrated at this temperature I mixed them together a started the stopwatch. At 30 s interval I withdrew a small sample of this mixture from the tube and mixed it with one of the iodine drops on the spotting tile. I continued this until I found that the colour of the iodine remained exactly the same as one of the untreated drops. At this point 1 recorded the time taken.
I repeated this experiment for 10 minutes and 15 minutes at each of the temperatures tested (40,50,60,70,80 and 90°C). I then began again and repeated exactly the same procedure with the fungal amylase solution. I found that the end point was not always clear so I recorded three different iodine colours to help me analyse the data later. These were, a definite blue/black colour, a dark brownish red and a light reddish brown which matched the original iodine drops.
As a control experiment three tests were made in an identical manner with fungal and bacterial amylase except no temperature treatment was given to the enzymes before testing.
RESULTS
Bacterial amylase
Fungal amylase
Results of control experiments
Bacterial amylase
Fungal amylase
To enable me to compare the effects of temperature treatment on each enzyme I calculated the overall rate of reaction for each using the formula:
I then expressed the rate of each reaction as a percentage of the rate of the untreated enzyme. The results are shown in the following summary table:
Conclusions
Main trends and patterns
The results show that there is a significant difference in the response of bacterial and fungal amylase to different temperature treatments.
The raw data shows that bacterial amylase is significantly less active in these conditions compared with fungal amylase.
Fungal amylase retains 100% of its activity when treated at temperatures up to 60°C. Above this temperature fungal amylase activity rapidly decreases to less than 5% of its original rate but still retains some limited activity even at when treated at 90°C. Length of time of treatment appeared to have little effect on this pattern since there were only small variations at 70°C and at 90°C.
In contrast bacterial amylase activity was inhibited by treatment at 40°C for 600s and 900s. It could be said therefore that the bacterial amylase was more sensitive to temperature but although activity was inhibited the bacterial enzyme was affected much less by treatments in the range 50-90°C. It can be seen from the graph that the bacterial enzyme retained over 50% of its activity up to 80°C. It also appeared that length of time of treatment was also more important to the activity of bacterial amylase as there was an obvious relationship showing that the longer the treatment the slower the reaction at three of the temperatures tested.
The results for bacterial amylase also showed a much greater degree of variability. There were obvious anomalies at 60 and 80°C for the 900s treatment although it was interesting to note that all of the rates rose unexpectedly between 50 a n d 60°C. This may be a n important trend or experimental error which would need further investigation. It is possible that because of the much slower basic rate of the bacterial amylase that experimental errors could have a much more significant effect on the final results when converted percentage differences.
Explanation of results
Enzymes are globular proteins made up of folded polypeptide chains. The shape of the final protein is vital to the activity of an enzyme because it forms an active site into which a substrate molecule must fit to allow the enzyme to work. In large proteins this 3-D shape is maintained by holding the folds of the polypeptide in position by using cross links between them. These cross links are mainly disulphide bridges and hydrogen bonding between amino acids. Increases in temperature make it more likely that these cross links become broken and as the polypeptide unfolds it is more and more likely that the change in shape will affect the active site and stop the enzyme working.
Whilst the active site of enzymes acting on the same substrate must be very similar it is possible that variations in the protein structure of different enzymes can differ meaning that different types and numbers of cross links can be used to make the same shape. It is possible therefore that the effect of increasing temperature can be different.
In this case if the results for bacterial amylase were shown to be correct it would be possible to speculate that as temperature increased some cross links were broken which slowed the rate of reaction but other stronger bonds remained to keep its activity over a wide temperature range. The effect of increasing the time of treatment of bacterial amylase especially 'at 60, 70, and 80°C showed an increased inhibition with longer treatment. This could support the idea that the remaining links were stronger but prolonged treatment at these higher temperatures broke them down too.
In the case of fungal amylase the cross links could resist change up to 60°C but at this point many similar links are broken and activity falls quickly. It would be possible to investigate this idea by detailed analysis of the protein structure of each enzyme.
Experimental limitations
The most important limitation to this experiment is the difficulty in making an accurate judgement of the final end point. The colour of the iodine changes slowly in some cases. In the slower reactions this was particularly difficult. Whilst some effort was made to distinguish between reactions where the dark brown colour remained after 630 s and those where the drops remained blue/black by counting the rate of reaction as zero where there was no sign of change this remained an inaccurate method.
The other major limitation involved temperature treatment. Especially at higher temperatures it was difficult to determine the exact time of temperature treatment. The solution itself was checked but inevitably it took longer for the enzyme to reach these higher temperatures so the exact time of treatment did vary.
Whilst exactly the same concentration of each enzyme was used the powdered enzymes themselves were rather crude preparations and it is likely that they contain different amounts of active ingredients. This may have accounted for the big difference between the activity of the two enzymes and makes direct comparison more difficult.
The overall reliability of the investigation was also limited by time constraints. To cover the full range of temperatures and times of treatment meant that 36 separate experiments were required. This meant unfortunately there was not time to carry out repeat tests. It is evident from the variability of the results especially for bacterial amylase that this was an important limitation.
Overall it appeared that this investigation did provide some evidence to support my hypothesis that bacterial amylase would work better over a wider temperature range than fungal amylase.
Clearly repeating these tests at least three times would be the most important type of further work which would be needed. If the trends shown in the first set of data are consistent it would also be important to investigate smaller increases in temperature between 50 and 70°C.
If time was available it would also be profitable to investigate a wider range of fungal and bacterial enzymes possibly from different
sources.
Comparing the denaturation rate of fungal and bacterial amylase
MODERATOR'S COMMENTARY
Planning
(a)The hypothesis is stated in a concise manner and in clearly testable form. The use of biological knowledge, principles and concepts is concise with no irrelevant detail showing that material has been carefully selected. It was evident from inspecting the work of other students in this group that this was an independent analysis of the problem. The criteria for 8 marks were met to a good standard in this section.
(b) The planned procedure is clearly the result of individual planning and there is good evidence of the students' involvement from the pilot study employed. The candidate gives further evidence of their original thinking in deciding to treat the enzymes at different temperatures but test each the effects at a standard temperature. The plan details clearly how important variables are to be controlled and the planned number and type of measurements is likely to be sufficient to produce reliable results. This exceeds the criteria for 6 marks and just qualifies for a maximum of 8.
(c) There is a good attempt to consider the risks associated with the compounds used and suggestions for their control. Again this is well above 6 marks and meets most of the criteria for 8 marks.
Overall this met all the criteria to a high standard and justified the award of a maximum 8 marks.
Implementing
The observing teacher reported that the experiment was carried out with exceptional care and all apparatus was handled with a high level of skill well above average for an AS student. There are sufficient demands in terms of accurate volume measurements, maintaining water baths and organising numerous samples to justify the award of maximum marks.
All safety precautions were meticulously followed without the need for prompting and the candidate applied their risk assessment carefully. She showed excellent organisational ability which was again well above average for an AS student and therefore once again the award of maximum marks could be justified.
There are extensive results which are recorded methodically in suitable tables with SI units. She has avoided the common confusion with minutes and seconds. Obviously there is a serious limitation in that there are no repeats, which the candidate discusses later. In hindsight the candidate could have limited the temperature range and allowed time for at least one set of repeats but there are important trends shown from 40°C upwards. To carry out one or two repeats of this investigation would certainly far exceed the suggested time allocated to these investigations.
Overall most of the criteria have been met to the highest standards but this limitation would preclude a maximum but certainly 7 marks would be appropriate.
Analysing evidence and drawing conclusions
(a)There is good evidence of careful tabulation with consistent application of SI units. The manipulation of the data and the design of the graph show careful thought has been used to consider rates of reaction and a useful method of allowing a direct comparison between the two enzymes. This is the work of an able candidate but very high marks would also be accessible in this section for a clear calculation of rates of reaction and their graphical presentation. The graph has been carefully chosen to illustrate main trends and patterns and to facilitate comparison between the two enzymes. There is sufficient numerical manipulation and careful selection of methods of presentation to justify maximum marks.
(b) The conclusions start with a clear description of the main trends and patterns which is well separated from any theoretical speculation. Most of the important trends are clearly and concisely identified as are obvious anomalies. This exceeds the criteria for 6 marks and would qualify for 8 marks.
(c)The explanation of these experimental results is concise and avoids simply regurgitating long descriptions of enzyme theory. It would be improved by a more detailed discussion of the possible reasons for the differences in the effect of time of treatment in addition to the actual temperature. In this respect it is a little limited but it was felt that it would be harsh not to allow a maximum 8 marks.
Overall this is an excellent section which in some ways exceeds the standard expected for AS, a maximum 8 marks were awarded.
Evaluating evidence and procedures.
There is good evidence from the conclusions and the separate limitations section that the candidate is well aware of the major limitations and anomalies in this investigation: The cautious nature of the final conclusion and the analysis of the shortcomings of the experimental technique show an awareness of the tentative nature of the results obtained. Whilst this is rather short it does identify all the important points and is close to a maximum 8 marks
The candidate recognises that the variability of the bacterial amylase results in particular needs further investigation and the suggestions for further study in carrying out more repeats, investigating the important temperature range in more detail and looking at a wider range of enzymes from bacteria and fungi are well chosen. and clearly closely related to the hypothesis under investigation. Once again this would benefit from a little more discussion in depth but it is refreshing to see important points selected and commented on with some conciseness rather than being hidden in long accounts of sometimes irrelevant background theory.
This again is a good concise summary which meets the criteria for 8 marks well.
8 marks were awarded for analysing evidence and drawing conclusions.
TOTAL
Planning = 8
Implementing = 7
Analysing = 8
Evaluating = 8
Overall total: 31/32
Context of this investigation
Following theoretical work on enzymes, the group of students performed simple laboratory experiments on amylases and proteases recapping their Key Stage 4 work on peroxidase.
They were provided with samples of different enzymes and asked to investigate some basic properties in detail. Several students investigated temperature effects but initial planning and pilot tests were individual and this resulted in very different approaches to the problems of heat treatment, assessing end points and numerical analysis of data which were clearly reflected in the final reports.
In awarding the highest mark the centre was therefore confident that this was the individual work of a very able student. The candidate spent approximately three hours on the practical work.
