Procedures:
(Since the time was not enough to do the whole experiment for each group, only one temperature of the mixture was done by different groups.)
- 20g of green beans was measured by using the electronic balance.
- The beans was put in a mortar and grinded by a pestle to obtain a mixture of beans’ residue and amylase extract.
- The mixture was evenly poured into two Perspex tubes.
- The perspex tubes were put in the centrifuge for centrifugation; amylase extract was obtained from the upper layer of the product and placed in a test tube.
- Meanwhile, a drop of iodine solution was added to a spot tile.
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3cm3 of starch solution and 2cm3 of acetate buffer were measured and mixed together in a beaker.
- 5 drops of the mixture and 2 drops of the amylase extract were added to the spot tile with the iodine solution.
- Time for the disappearance of dark-blue colour in the mixture was recorded.
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6cm3 of starch solution and 4cm3 of acetate buffer were added to two boiling tubes respectively. The tubes were labeled with “amylase” and “no amylase” to indicate whether the tube contained amylase.
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At the same time, 4cm3 of amylase extract was prepared in another test tube with the help of a measuring cylinder.
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The two boiling tubes and the test tube with 4cm3 amylase extract were placed in the water bath with 60℃ for 5 minutes. Two test tubes with tap water were placed in the same water bath for 5 minutes.
- A drop of iodine solution was added into each of the boiling tubes.
- The hot water in the test tubes was poured in a beaker. Then the two boiling tubes were put into the hot water.
- The amylase extract in the test tube was put into the test tube with “amylase”. The stopwatch was started.
- The stopwatch was stopped when the dark-blue colour in the boiling tube containing amylase disappeared.
Results:
In part I
In part II, each group has to do the experiment with a certain temperature (0℃, 25℃, 40℃, 60℃ and 100℃). Our group had done the experiment with 60℃. The experimental results of us and the other groups are shown below.
Experimental result taken by our group: Time taken for the disappearance of dark-blue color in the solution is 48 seconds and the rate of action is 0.0208 (cor. to 3 sig. fig.)
The experimental results taken by colleagues:
From the data, firstly we know the amylase used in the experiment could function properly as it could convert starch into maltose.
Next, it was found that only the mixtures in 40℃ by other group and 60℃ by our group could take the time reading. For the mixtures in other temperature, the time taken was very large or not recorded as the lessons were over before they could start recording.
Discussions:
It is the first Biology report done in F.6. As we did not have much experience to handle the experiment and missed some procedures, a lot of errors are resulted in the experiment done by both our group and other classmates and thus the data collected by us is very inaccurate compared to that learned from the biological theory.
In the experiment, some assumptions were made. First, the starch in the solution would not decompose to form glucose during the experiment. Next, all the solutions in water bath should attain to the temperature same as the bath. Also, the pH value of all the solutions should be the same. Finally, the substances provided by the laboratory were correct and not be contaminated.
Let’s talk about the errors done in the experiment. In our group, there was no obvious error in the 1st part, but were in the 2nd part of the experiment.
1st error: The temperature in the 60℃ water bath was not exactly 60℃, but was a little bit lower. In fact, the actual temperature of the water bath shown by the thermometer, which was immersed in the bath for a long time, was about 58℃.
Problem: It could not be the error in this experiment as it was one of the assumptions. However, the result of our group was totally different with that taken from other group. Thus I think that the slight decrease of temperature could be the great cause. Not all the amylases were denatured and some of them could still have the normal function, and also have a great activity because of the high temperature, so the starch could decompose in a short time.
Improvement: The temperature of the water bath should be adjusted to become 60℃ so to ensure temperature did not contribute to the mistake in the experiment.
2nd error: The methods of doing the experiment by different groups were not the same. For instance, some groups mixed the mixture of starch solution and acetate buffet and the amylase extract in the water bath while some took out the solution and mixture out of the water bath before mixing.
Problem: The main purpose of the experiment is to test how temperature affects enzyme activities. As different methods were employed, the problem of heat loss or gain to the surrounding existed. For the solutions treated in 0℃, 25℃ and 40℃, their enzyme activities are not the same. If there is something that could cause heat gain or loss of the enzyme such as putting the extract outside the water bath for a while before mixing it with the starch solution, the enzyme activities will not represent to the temperature of them in the water baths.
Improvement: All our classmates should do the experiment with the same method and the solutions should be mixed and allowed to stay in the water baths so that the temperature of the solutions would not change during the experiment.
3rd error: Different amount and proportion of solution were used by different group. For example, some groups mixed the amylase extract and starch solution in the ration 1:3, but some others mixed the solution in the ratio 2:3.
Problem: It is one of the most serious problems in the experiment. As the amount of enzyme acting on the substrate could affect the rate of change of substrate, the amount of amylase extract used should be equal and have the same proportion in using the extract and starch solution in each group. Otherwise, the test is not fair and the data collected from the test could not only show the effect of temperature to enzymes activities but also the effect of the amount of amylase to the rate of change of substrate.
Improvement: All the groups should use the same amount and proportion of solution so that temperature is the only factor affecting the results.
There were one more group in class to do the experiment with 60℃, except our group. However, the data collected by the two groups are totally different. The time taken for the decomposition is 48s by our group while that is infinity by another group. It is clear that problems arise in the procedures of the experiment done by us because all enzymes should be denatured in 60℃ and thus the time taken should be infinity instead of 48s. After deduction, I think there are mainly two reasons to explain the abnormality.
The first reason is the actual temperature of the water bath, which has been discussed above.
Another reason is that the amylase extract used by our group was double to the other group. It might be the main cause why our data was different from it. Since we used a larger amount of the amylase extract and the temperature of the water bath was not very high actually (60℃), the heat given to the extract might not be enough and thus it did not attain to the temperature of 60℃ in 5 minutes time. In this way, some of the amylase might not be denatured and still carry the function. Moreover and the most importantly, as the volume of amylase extract used was much greater, the amount of amylase used was greater, too. This could cause a higher probability of the existence of normal amylase in the experiment. From our group, the rate of decomposition of starch is the highest among the data from different groups.
If the problems are solved, the data would be much more accurate.
Conclusions:
The results of the experiment find out that only the solution mixtures in 40℃ and 60℃. There are lots of mistakes in taking the experiment. For example, different methods were used in different groups and the overuse of amylase extract. That is why the data collected is not accurate enough with the biological theory.
To have improvement, the instruction of the experiment should be read carefully for better preparation to the experiment next time.
Reference:
Biological Science 3rd edition by CAMBRIDGE
Wikipedia (http://en.wikipedia.org/wiki/Main_Page)