Elise Curtis
Professor Loughman
Bio-105L
30 October 2006
The Effects of Environmental Temperature and pH on the Activity of Porcine Pancreatic Amylase
Introduction
Porcine pancreatic amylase is an enzyme found in a human’s saliva and small intestine and is used for the breakdown of starch by hydrolysis. As soon as starchy food is consumed, it begins to breakdown in the mouth because of the amylase in the saliva. The complete breakdown of the starch is done in the small intestine which will break it down into disaccharides called maltose. The maltose will then be broken down by a separate enzyme into maltase by means of hydrolysis. Here the process of glycolysis begins. Glycolysis is the process that extracts energy from carbohydrates which are consumed.
Amylase is considered an enzyme. Because of it being an enzyme, the environmental temperature and pH of the amylase have a great effect on the way it will function. This change in environmental temperature or pH will change the secondary and tertiary structures of the enzyme which will completely change the enzymatic activity. Once the shape of the enzyme changes it begins to denature. When an enzyme denatures, the substrate cannot join with the active site, which will stop the enzyme from functioning.
The enzymatic action of amylase will increase as the environmental temperature gets warmer. Even a change in temperature as small as one or two degrees Celsius can change the action drastically.
This experiment will show whether or not amylase activity will work best at higher temperatures; somewhat higher than the human body. Most proteins usually start to denature at 40 to 50 degrees Celsius. Amylase, although, does not begin to denature until about 65 degrees Celsius or higher. This experiment will give us a good idea as to how much tolerance amylase has for temperature changes in the environment.
We will also experiment on the level of activity in previously frozen amylase. The amylase will be frozen only one time and will not be thawed until the time it is to be used. It can be kept in the freezer for up to twelve months and still retain good quality.
Extreme pH changes can also completely stop the activity in the enzyme. Amylase does not have much tolerance for drastic changes in pH. Thus, the pH must be kept at a fairly even level so that its enzymatic structure will not be altered. If the pH gets too low, the enzyme will gain hydrogen ions and will change the enzyme’s shape. If the pH gets too high, the enzyme will lose hydrogen ions and will also quickly lose its shape. The amylase enzyme works best in a neutral pH environment, which on the scale, is about six or seven. We will find the best pH environment for enzymatic activity in this experiment.
These three experiments will be examining amylase activity while making changes in its pH and temperature environments, and the nature of the enzyme after being frozen and boiled. Starch will be used to check for the presence of the porcine pancreatic amylase over certain predetermined time periods.




Experiment #1:
Investigating the Effect of Environmental Temperature on the Activity of Porcine Pancreatic Amylase
As I run the amylase/starch reactions at increasingly higher temperatures, I predict that the amylase will work better. As the environment grows warmer, the amylase will become more active and become more effective. Although, if the environment reaches temperatures nearing the boiling point, it will become too hot and will stop the enzyme from working. I predict that the best environment for the amylase will be somewhat higher than body temperature.
Materials and Methods
Amylase experiment #1 was done to see if environmental temperature affected the quality of the enzyme. First we gathered all of the materials that were necessary to perform the experiment. We needed five clean test tubes, a test tube rack, a beaker of 1% starch solution (pH 7.0), a test tube of porcine amylase solution--- which was previously prepared by mixing porcine pancreatic amylase powder with 0.9% NaCI concentration to create an amylase concentration of 0.0375mg/mL, one 1-mL pipette, one 5-mL pipette with pipette pump, five transfer pipettes, two spot plates, a bottle of iodine solution, a timer, one beaker half way filled with ice and a small amount of water, two thermometers, and three water baths each with a different temperature.
In this experiment, we first labeled the test tubes as T1, T2, T3, T4, and T5. We then added 5-mL of 1% starch solution with a pH of 7.0 to each of them. After each tube was filled, T1 was placed in the ice water at -8 degrees Celsius, T2 was placed in room temperature water with a temperature of 32 degrees Celsius, T3 was placed in warm water at 40 degrees Celsius, T4 was placed in hot water at 62 degrees Celsius, and T5 was placed in extremely hot water at 85 degrees Celsius. Each test tube was then left in the designated place for ten minutes so that the starch solution could adapt to its surrounding temperature. After ten minutes, we then used a 1-mL pipette to add 0.3-mL of the amylase mixture to each of the tubes. The contents were then thoroughly mixed together with a transfer pipette, and then the timer was set. After two minutes, we put two drops of the starch/amylase mixture from each of the five tubes into a separate well on the spot plate, and then added two drops of iodine to each well and recorded the outcome. After every two minutes had passed, these same steps were repeated four more times, until ten minutes altogether had passed. We then performed the same routine but waited for five minutes between tests. At fifteen minutes altogether, we ran the same test again and took three more tests using the same procedure every five minutes until thirty minutes altogether had passed. With each of these tests the outcomes were recorded.
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TABLE I: Effect of Temperature on Amylase Activity

Effect of Temperature on Amylase Activity











Time (min)
0oC
22oC
38oC
55oC
82oC
0
+
+
+
+
+
2
+
+
+
-
+
4
+
+
-

+
6
+
+


+
8
+
-


+
10
+



+
15
+



+
20
+



+
25
+




30
+




35
+/-





||
||
Graph I: Effect of Temperature on Amylase Activity
external image octet-stream.png graph 1.xls
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graph.jpg
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Experiment #2:
Investigating the Effect of Environmental pH on the Activity of Porcine Pancreatic Amylase
As I run the amylase/starch reactions at increasingly higher pHs, I predict that the amylase will not work well. A significant change in pH causes the enzyme to denature and will cause it to stop working. An enzyme’s environmental pH has to be fairly stable in order for it to work properly.
Materials and Methods
Amylase experiment #2 was done to see if pH levels affected the enzyme. In this experiment, we collected five test tubes and labeled them T1, T2, T3, T4, and T5. We also needed five separate 1% starch solutions--- each with a different pH level, a test tube rack, a transfer pipette, one 5-mL pipette with pipette pump, a bottle of iodine solution, and a spot plate. Each of the test tubes received starch solution all with different pH levels. T1 received pH3 solution, T2 received pH5 solution, T3 received pH7 solution, T4 received pH9 solution, and T5 received pH11 solution. Then we added 0.3-mL of the amylase mixture to each one of the pH solutions in the test tubes. As soon as the amylase was added to the starch solutions, we started the timer. After two minutes, we took about three drops of the starch/amylase solution from each of the tubes and put them on a spot plate into separate wells. Then we added about one or two drops of the iodine to each one. We repeated this procedure four more times at two minute intervals, until ten minutes all together had passed. We then performed the test using five minute intervals and tested the starch/amylase solution again in the same way. We repeated this four more times every five minutes until thirty minutes all together had passed. We recorded the outcome after each one of these tests.
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TABLE II: Effect of pH on Amylase Activity
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Effect of pH on Amylase Activity






pH
pH
pH
pH
pH
Time (min)
3
5
7
9
11

+
+
+
+
+
2
+
+
+
+
+
4
+
+
+
+
+
6
+
+
+
-
+
8
+
+
+

+
10
+
+
-

+
15
+
-


+
20
+



+
25
+



+
30
+



+

Graph II: Effect of pH on Amylase Activity
graphpH.jpg
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Experiment #3:
Investigating the Effect of Freezing and of Boiling on the Activity of Porcine Pancreatic Amylase
If I freeze some amylase solution, thaw it, and try to digest starch with at it at room temperature, I predict the previously-frozen amylase will not work quite as well as amylase that has not been previously frozen because it will affect the substrates ability to bind to its active site in the enzyme.
If I boil some amylase solution, cool it, and try to digest starch with at room temperature, I predict the previously-boiled amylase will work much worse than amylase that has not been previously boiled. Boiling the amylase causes the enzyme to denature and completely stops it from functioning; therefore it will have no effect.


Materials and Methods
Amylase experiment #3 was done to see if boiling or freezing the amylase would have an effect on the enzyme. For this experiment, we collected three clean test tubes, porcine pancreatic amylase solution, a test tube containing frozen amylase solution, two 600-mL beakers each containing 200-mL of room temperature water, a hot plate, three transfer pipettes, two spot plates, a bottle of iodine solution, and a test tube rack. First we took the frozen amylase solution out of the freezer and put it in one of the 600-mL beakers in room temperature water. It was let to sit in the water until it fully melted down to room temperature. We labeled this test tube “frozen amylase.” Then we put about 1-mL of amylase solution in a test tube and boiled it in the other 600-mL beaker for two minutes. We labeled this test tube “boiled amylase” and then placed it in the other 600-mL beaker with the room temperature water to cool it. We allowed it to sit in the water for at least ten minutes until it cooled down to room temperature. Then we put 5-mL of starch solution (pH 7.0) in three test tubes and labeled them “boiled amylase,” “frozen amylase,” and “regular amylase.” We used a 1-mL pipette to add 0.3-mL of the boiled, frozen, and regular amylase solutions to their properly labeled test tubes. The solutions were then thoroughly mixed together with a transfer pipette and the timer was set. After two minutes, we did the first test. We took out about three drops of starch/amylase solution from all three of the test tubes and put them on a spot plate into three separate wells. Then about two drops of iodine solution were added to each one. This same routine was repeated four more times at two minute intervals until ten minutes all together had passed. We then waited for five minutes in between tests and took another sample at the fifteen minute mark. This same routine was done three more times at five minute intervals until thirty minutes all together had passed. All of these tests were recorded prior to each one.
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TABLE III:
Effect of Boiling and Freezing on Amylase Activity
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TABLE III: Effect of Boiling and Freezing on Amylase Activity





Time (min)
Boiled
Frozen

+
+
2
+
+
4
+
+
6
+
+
8
+
+
10
+
-
15
+

20
+

25
+

30
+

Graph III: Effect of boiling and Freezing on Amylase Activity

graphbf.jpg
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Discussion
In experiment #1 the porcine pancreatic amylase has shown to work best from anywhere between 40 and 65 degrees Celsius. These temperatures are very warm environments, as the prediction stated. Porcine pancreatic amylase works at its full potential at warmer temperatures because it causes the molecules to move faster. This increase of motion creates greater activity which will cause more enzyme and substrate properties to be linked together in the amylase and it will break down the starch much faster. You can see the difference in the graph as the temperature rises, until it gets too hot and the action completely stops. You can see that at 85 degrees Celsius the enzyme has denatured and it is no longer working.
In experiment #2 you can see that the amylase functions best from pH5 to pH9. Its highest activity level occurred in pH7 and pH9 environments. This is the pH level of the environment in human saliva and the small intestine, where the amylase enzyme is present. The amylase has shown no activity in pH3 and pH11 environments. Because of the higher amount of hydrogen and hydroxide ions, the enzyme cannot bind to the active site and the enzymatic activity stops.
In experiment #3 we see the effects of boiling and freezing porcine pancreatic amylase. In the boiled sample it shows that the enzyme was denatured and can no longer function properly. This experiment shows that the prediction is correct. The frozen sample still dissolved the starch, but it took four minutes longer compared to the regular amylase. This experiment shows that the prediction is correct that freezing amylase will not have a great affect on it.
The effect that high temperatures have on an enzyme can be seen in the preparation of butter beans. Butter beans contain an acetone based glycoside called linamarin. Sometimes large amounts of linamarin can be found in these beans. To eat the beans uncooked could be very harmful if the amount of linamarin is too high. Cooking the butter beans changes the shape and causes the enzyme to denature, which then makes them safe to consume.
These experiments could be done again to find a more specific temperature level where the amylase would work at its fullest potential. We could run these experiments in more depth and test the amylase in temperatures between 40 and 65 degrees Celsius to find the absolute highest level of activity. We could also run the pH experiment again and test the amylase at pH7 and pH9 but run the test for a longer period of time to find the best possible pH level for amylase activity.











Works Cited
Arnott, Corinne A. “Hazards of Enzyme Action.” University of Paisley. Copyright
2001. 2 Nov. 2006. <http://www-biol.paisley.ac.uk>
DeAngelo, Eric. Fundamentals of Biology: Laboratory Manual-Biology 105.
Lehigh Carbon Community College. (5~1 – 5~6)
Loughman, John. “The Effects of Environmental Temperature and pH on the Activity of
Porcine Pancreatic Amylase.” 7 Aug. 2006. 1 Nov. 2006. <http://bio105l.wikispaces.com>
Wikimedia Foundation, Inc. Wikipedia: The Free Encyclopedia. 31 Oct. 2006.
1 Nov. 2006. <http://www.en.wikipedia.org>