VARIABLES
MATERIALS
APPARATUS
Thermostatic water bath Spectrophotometer
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METHOD
- Set up eight water baths at 15°C, 25°C, 35°C, 45°C, 55°C, 65°C, 75°C, and 85°C and in 500 mL. (If you cannot have eight water baths set up, set up four at 35°C, 55°C, 75°C. After the first reaction is stopped have set thewater baths to 45°C, 65°C, 85°C)
- Add 5 mL of the starch solution to five test tubes. Place each of the test tubes in separate water baths for 5 minutes. Allow the temperature of each of the starch solutions to come to equilibrium with that of the water bath.
- Add 5 mL amylase solution to another five test tubes. Place each of the test tubes in separate water baths for 5 minutes. Allow the temperature of each of the amylase solutions to come to equilibrium with that of the water bath.
- Mix the solution with clean glass rod. Continue to monitor the temperature.
- Stop the reaction after exactly 10 min by adding 5 mL of HCl.
- Add 0.5 mL of the above mixture to 5 mL iodine solution to develop color. Shake and mix. The solution should turn deep blue if there is any residual, unconverted starch present in the solution. The solution is brown-red for partially degraded starch, while it is clear for totally degraded starch.
- Measure the absorbance with a spectrophotometer at 620 nm.
RESULTS
Quantitative data:
Qualitative data:
Processed data:
CONCLUSION
The purpose of this experiment was to determine the effect of different temperatures on the reaction of amylase. The rate of absorbance and enzyme activity which was calculated using the spectrophotometer are what determine the effect of the different temperatures. Iodine was used to determine whether the starch was still present in the mixture. The hypothesis was that if the amylase was tested in different temperatures (15°C, 25°C, 35°C, 45°C, 55°C, 65°C, 75°C, and 85°C), then it will react best at 35°C because that is the nearest to 37°C which is same to human body temperature and the optimum temperature at which that enzyme works. The hypothesis was accepted.
The major findings of this experiment show that as temperature increases, the rate of reaction of the enzyme amylase also increases; a positive correlation until it reach 37°C and decreases beyond the temperature.The rate of amylase enzyme activity started very low at the temperature of 15°C. That was expected because in that cold temperature the enzyme wouldn’t activate. The rate increase and reach it peak at the temperature of 35°C. This was hypothesized to be the temperature with the highest rate of amylase enzyme activity because it is the optimum temperature for enzymes performance. When it begins to digest food in our body its environment is supposed to match that. The rate of amylase enzyme activity decrease as the temperature decreases until 85°C. It make sense because at this high temperature the enzyme would already become denatured. It can be concluded that the closer the temperature to the human body temperature(37°C) the higher the rate of amylase enzyme activity.
EVALUATION
Overall, the experiment yielded reliable results. The results were as expected and according to the hypothesis made. Although there is no definite trend between the time at which the enzyme completely hydrolyzes the starch and the temperature, the experiment still shows that amylase works best at temperature closest to human body temperature, 37°C . Both cold and hot conditions as well as extreme temperature are shown to make enzyme activity slower or even denature the enzyme.
Some aspects of the experiment may have yielded some inaccurate data. For example, the time interval at which the mixture of starch and enzyme solution were added together may not have been very accurate and not consistent and sometimes too long at some temperature. This also happened during the addition of hydochloric acid to stop the reaction between starch and amylase enzyme. The large and inconsistent time difference between each interval may have generalized the data too much. This, thus, may be responsible for the nearly identical results which are not really accurate.
In addition, 0.5 mL of the mixture of amylase, starch and iodine solution which was put into the cuvette was not diluted before entering the spectrophotometer. The mixture was very concentrated which can be seen obviously from their dark colors. Although the wavelength used was 620 nm which is quite high but it not enough to penetrate the molecules of the high concetrated mixture. This, thus, may be responsible for the identical duplicate results in rate of absorbance reading at some temperatures like 55°C, 65°C, and 75°C which have same rate of absorbance. This is not accurate because with 10°C difference, the rate of absorbance should be different not the same.
On top of that, the experiment was only carried out once. This might produce inaccurate because we only depended on only one trials. The results may be exposed to many threats of errors. The data is insufficient because mean of the data cannot be calculated due to no repetition. Finally, the imprecise amount of stirring that applied to the mixture of starch and amylase solution in different temperatures may have yielded unreliable data. For example, because the solutions were very sensitive during reactions, a harder stir at the solution may have speeded up the reaction.
SUGGESTED IMPROVEMENTS
Numerous ways can be implemented in order to improve the results of the experiment. Firstly, the time intervals for mixing the starch and amylase solution as well as the addition of hydrochloric acid into the mixture should be standardized and short for an instant 10 seconds. The period of 10 seconds in between each interval is suitable and short enough in order to find out more accurate results about the rate of amylase enzyme activity.
Before entering the cuvette into the spectrophotometer, the mixture of starch, amylase and iodine solution should be diluted first with distilled water. The justification will be easier for the penetration of wavelength so that more accurate results with more diversity will be produced. Hence, we can see clearer distinctions of the rate of absorbance and amylase enzyme activity in every temperature.
Moreover, the experiment must undergo repetition at least 3 times. With more trials, mean of the data can be calculated with higher accuracy and consistency. Therefore, the results will be more reliable because we use the average value of the data instead of depending on only a single trial. Lastly, we can set a constant number for inserting and stirring the solution using the glass rod. This would add more uniformity on the reading of the rate of absorbance and amylase enzyme activity in every temperature.
REFERENCES
A, A. (2001). Enzymes in action. In A. A, Biology for the IB Diploma (p. 15). Oxon: Oxford University Press.
Amylase. (2001-2012, April 28). Retrieved July 10, 2012, from Lab Tests Online: http://labtestsonline.org/understanding/analytes/amylase/tab/test
Blauch, N. D. (2009). Spectrophotometer Basic Principles. Retrieved July 1, 2012, from Virtual Chemistry: http://www.chm.davidson.edu/vce/spectrophotometry/Spectrophotometry.html