The Effect of Temperature on Amylase Activity

Abstract:
This study of the effect of temperature on the enzyme amylase was performed to determine the correlation between the two. The experiment was developed to test the enzymes reaction rate of amylase digesting starch at several different temperatures and see how the rate changed. The rate of reaction was found to increase as the temperature of the environment was raised. As the temperature was raised from incrementally to 4�°C, 22�°C, 37�°C and finally 80�°C the rate of reaction followed this trend and also increased. However as predicted in the hypothesis of this experiment when the temperature was raised too high the enzyme would denature. In this experiment the value which the enzyme denatured at was 80�°C. To summarize, the temperature of the environment was increased the reaction rate increased until the enzyme denatured.

Introduction:
Amylase is an enzyme found in the human body. Enzymes are structural proteins which can catalyze a reaction. They cause an increase in the rate of the reaction, while not being consumed in the reaction (Campbell, Neil, J. Reece, and L. Mitchell, 2002). Enzyme activity is affected by several factors such as temperature, pH, and sometimes even stress. Psychological stress can increase reaction rate in humans because more amylase will be released (Takai 1). However, the factor which we are testing in this experiment is temperature. Enzyme activity is changed by variation in temperature. As temperature rises the rate of chemical reactions increases because temperature increases the rate of motion of molecules. This leads to more interactions between an enzyme and its substrate (Morgan 109). In this experiment the substrate was starch, which is a polysaccharide of glucose. However, if the temperature is too high, enzymes can be denatured and they can no longer bind to a substrate and catalyze reactions. The hypothesis was therefore concluded that the amylase activity would increase as the temperature was risen, until a certain high temperature at which the amylase would denature and be non-functional.

Materials and Methods:
Five ml of 1% starch solution were placed into four separate test tubes. Test tube one was then placed into an 80�°C water bath. Test tube two was then placed into a 37�°C water bath. Test tube three was placed in a test tube rack at room temperature, approximately 22�°C. Lastly, test tube four was placed into a beaker of crushed ice, approximately 4�°C.

Subsequently, one ml of 1% amylase was placed into another four separate test tubes. Test tubes one through four were placed in the respective temperatures listed above for each test tube. The contents of the two sets of tubes were not mixed. The eight test tubes were allowed to sit in their individual environments for ten minutes.

A test plate was obtained, and several rows of the test plate were filled with one or two drops of I2KI (iodine-potassium-iodide). The test tubes were left in their respective environments as they are being tested. The tubes in the 80�°C water bath were mixed. The time at which they were mixed was marked as zero. A pipette was used to add one or two drops of the mixture to a drop of I2KI. Every thirty seconds another drop of the solution was placed into another well containing fresh I2KI. This process was Thorough continued until a blue color was no longer produced and the I2KI solution remained a yellow-amber (indicating all the starch was digested) (Morgan 110). If the process exceeded ten minutes the experiment was stopped with that particular reaction mixture. This procedure was repeated with the test tube sets in the 37�°C, 22�°C, and 4�°C environments.

Results:
Temp (�°C) (Tube #)Time of Starch Disappearance (in minutes)
4�° (4)7.5
22�° (3)3.5
37�° (2)3
80�° (1)>10
This table shows the results of temperature on the reaction rate of amylase expressed by the time of starch disappearance.

The results above support the hypothesis of the experiment (the reaction rate would increase as temperature was increased, until a certain point at which the amylase would denature and would stop working ) as the amount of time for the amylase to digest all the starch at 4�°C was 7.5 minutes. As the temperature was increased to 22�°C the time decreased to 3.5 minutes, showing the reaction was occurring at a faster rate. When the temperature was raised to 37�°C the rate of reaction increased yet again completing its reaction in 3 minutes. However, when the temperature was raised to 80�°C the enzyme apparently denatured.

This graph shows the time it took for the starch to be digested by the amylase as the temperature is increased.

Discussion:
Looking at the data collected, it appears as though the hypothesis which was developed at the beginning of the experiment was in fact correct. The hypothesis was that the reaction rate would increase as temperature was increased, until a certain point at which the amylase would denature and would stop working. The reason for this conclusion is that the rate of reaction exceeded the ten minute limit, meaning that the amylase was not breaking down the starch.

Although it appears that the data supports our hypothesis it would be a good idea to repeat the experiment several times to establish a trend, and prove that this set of data was not a coincidence. Also it would be interesting to actually find the point of denaturization, this would be achieved by performing the experiment again with environmental conditions between 22Ã?°C and 37Ã?°C, as it appeared as though the denaturization of the enzyme occurred during this interval. However we were able to establish a general range at which amylase denatures, not all enzymes denature at this range. Some enzymes which have just recently been discovered can thrive in temperatures of 100Ã?°C. This newly discovered enzyme could possibly give some information on the early development on life on the earth, as the climate of the primordial earth was much warmer than it is now (Cowen 11). This enzyme was discovered in an organism that thrived in 100Ã?°C temperatures near volcanic vents on the sea floor. However amylase has a much lower denaturization temperature than this newly discovered enzyme; it is important not to make the wide assumption that “all” enzymes denature at the same range as amylase.

In conclusion this experiment further confirmed that enzymes do increase/decrease their rate of reaction due to change in temperature of the environment. Each enzyme has its own individual prime functional temperature, therefore if they are removed from these environments they will not function as well or possibly denature if it the temperature is too high. Although, if an enzyme’s optimal temperature is maintained then the enzymes will catalyze reactions at optimum rates.

Resources
Campbell, Neil, J. Reece, and L. Mitchell. Biology 5th Edition. Menlo Park, CA.
Benjamin/Cummings, 1999.
Morgan, Judith. General Biology Laboratory Bio III. Menlo Park, CA. Pearson Custom
Publishing, 2000.
Takai, Noriasu. “Effects of Psychological Stress On the Salivary Cortisol and Amylase
Levels in Healthy Young Adults.” Archives of Oral Biology 49.12 (2004): 963.
Cowen, Robert. “Study of Life’s Origins May Improve Everyday Life Today.” Christian
Science Monitor 87.84 (1995):11.

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