The Effects of Environmental Temperature and pH on the Activity of Porcine Pancreatic Amylase.
By:
John Loughman
Bio 105L
8/7/06



Introduction
Amylase is a digestive enzyme found in saliva and in pancreatic secretions to the small intestine. The function of amylase is to catalyze the hydrolysis of carbohydrates, such as the polysaccharide, starch, to the disaccharide maltose. Maltose is subsequently hydrolyzed to glucose by another digestive enzyme, maltase. These glucose molecules are the starting point of glycolysis.
Glycolsis is the first process organisms employ to harvest energy, (ATP), from ingested carbohydrates. In cellular respiration, glycolosis is the initial step in energy production as it converts glucose to two pyruvate molecules. These pyruvate molecules are further processed in the Krebs cycle and then through oxidative phosphorylation to eventually generate a combined ~ 38 ATP’s per glucose molecule processed(1, 2).
Amylase is an enzyme. Since enzymes are proteins, their secondary and tertiary structures are affected by temperature and pH. The enzymatic activity is closely associated with the structure of the enzyme, so any change in the secondary or tertiary structure leads to a change in enzyme activity. (3)
Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature rises. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results (4).
This study will investigate if amylase activity is best at temperatures near 37C, body temperature. Many proteins are denatured by temperatures around 40­C - 50C, but some are still active at 70C - ­80C (5). The amylases are quite robust thermally, with most reaching their thermal optima in the range of 50- 60C and losing significant catalytic activity >65C (6). This experiment will shed light on the temperature tolerance of porcine pancreatic amylase.
The effect, or lack thereof, of freezing the amylase solution prior to use will be observed. Prepared aliquots of concentrated stock solutions, or even premixed ready-to-use solutions, may be stored frozen for six or even twelve months, but only in a freezer that does not have an automatic defrost cycle. The only freeze- thaw to which these should be exposed is the initial freeze and final thaw. This is not due to damage to the amylase structure, but to precipitation of the amylase on the sides of the container (6).
Extreme pH environments also have been shown to diminish and even eliminate amylase activity (7). Enzyme amino acid side chains contain groups such as -COOH and -NH2 that readily gain or lose H+ ions. As the pH is lowered, an enzyme will tend to gain H+ ions, and eventually enough side chains will be affected so that the enzyme's shape is disrupted. Likewise, as the pH is raised, the enzyme will lose H+ ions and eventually lose its active shape. Many enzymes have an optimum in the neutral pH range and are denatured at either extremely high or low pH (6, 7). This experiment will determine the optimum pH range for porcine pancreatic amylase. Neutral pH is viewed as optimal for amylase activity (8).
The following experiments will monitor porcine pancreatic amylase activity while varying pH, temperature, and the nature of the stock amylase solution, (whether it has been frozen or boiled). The activity of the porcine pancreatic amylase will be determined by the presence or absence of starch in the samples over time.



Experiment #1:
Investigating the Effect of Environmental Temperature on the Activity of Porcine Pancreatic Amylase

Porcine pancreatic amylase is affected by environmental temperature. I predict that the amylase activity will work best at near body temperature, 38oC. As the temperature moves from this point I predict that the amylase activity will decrease and eventually stop.
Materials and Methods
The following stock solutions were prepared in advance;
1% Starch Solution pH 7
0.0375 mg/ml Porcine Pancreatic Amylase Solution (amylase powder in 0.9% NaCl )
Iodine Solution
5 ml of 1% starch solution were pipetted into each of 5 test tubes. Four tubes were immersed for >10 minutes into different temperature bathes. One tube was held at room temperature. These bathes were 0C, 38C, 55C and 83C. Room temperature was 22C.
0.3 ml of porcine pancreatic amylase solution was then pipetted into each tube. The pipette was used to mix the sample and then left in the tube. A timer was started and 3 drops of the starch / amylase mixture were pipetted into a spot plate cell every 2 minutes until 10 minutes had elapsed, then the sample was pipetted into a spot plate cell every 5 minutes. As soon as the sample was in the spot plate 2 drops of iodine indicator was dropped in to the starch amylase mixture. Iodine reacts with starch to change from yellow to deep blue /black in the presence of starch. A lightening of the blue/ black to a brown color will occur as less starch is present. Results were reported as (+) for presence of starch in the sample or (–) for the absence of starch in the sample.
(See Table I), N/A – No Activity, (See Graph I)
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TABLE I: Effect of Temperature on Amylase Activity

Effect of Temperature on Amylase Activity











Time (min)
0oC
22oC
38oC
55oC
83oC

+
+
+
-
+
2
+
+
+

+
4
+
+
-

+
6
+
+


+
8
+
-


+
10
+



+
15
+



+
20
+




25
+




30
+/-




35
+/-





||
||
Graph I: Effect of Temperature on Amylase Activity

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graph.jpg
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||

||

||

||

||
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Experiment #2:
Investigating the Effect of Environmental pH on the Activity of Porcine Pancreatic Amylase

Porcine pancreatic amylase is affected by environmental pH. I predict that the amylase activity will work best at a pH 6-8. As the pH moves from this point I predict that the amylase activity will decrease and eventually stop.
Materials and Methods
The following stock solutions were prepared in advance;
1% Starch Solution pH 3
1% Starch Solution pH 5
1% Starch Solution pH 7
1% Starch Solution pH 9
1% Starch Solution pH 11
0.0375 mg/ml Porcine Pancreatic Amylase Solution (amylase powder in 0.9% NaCl )
Iodine Solution
5 ml of 1% starch solution were pipetted into each of 5 test tubes. Each tube contained a 1% starch solution at a different pH. All tubes were held at room temperature. Room temperature was 22C.
0.3 ml of porcine pancreatic amylase solution was then pipetted into each tube. The pipette was used to mix the sample and then left in the tube. A timer was started and 3 drops of the starch / amylase mixture were pipetted into 8 spot plate cells for every sample tube. As soon as the timer reached 2 minutes 2 drops of iodine solution were added to a spot plate cell for each sample. Iodine reacts with starch to change from yellow to deep blue /black in the presence of starch. A lightening of the blue/ black to a brown color will occur as less starch is present. Results were reported as (+) for presence of starch in the sample or (–) for the absence of starch in the sample.
(See Table II), N/A – No Activity, (See Graph II)
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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 Boiling on the Activity of Porcine Pancreatic Amylase
Porcine pancreatic amylase is affected by boiling. I predict that the amylase activity will not occur in the boiled sample, and that the amylase activity will be unchanged in the frozen sample.

Materials and Methods
The following stock solutions were prepared in advance;
1% Starch Solution pH 7
0.0375 mg/ml Porcine Pancreatic Amylase Solution (amylase powder in 0.9% NaCl )
Previously Frozen - 0.0375 mg/ml Porcine Pancreatic Amylase Solution
Iodine Solution
A sample of frozen porcine pancreatic amylase was taken out of storage and held in a 22C water bath for 1.5 hours until melted and at room temperature.
A sample of porcine pancreatic amylase was suspended in a boiling water bath for three minutes. The sample was then placed in a room temperature water bath for 1.5 hours until cooled to room temperature.
5 ml of 1% starch solution were pipetted into each of 2 test tubes. Both tubes were held at room temperature. Room temperature was 22C.
0.3 ml of the previously frozen porcine pancreatic amylase solution was then pipetted into a test tube containing 5 ml of 1% starch solution. 0.3 ml of the previously boiled porcine pancreatic amylase solution was then pipetted into a test tube containing 5 ml of 1% starch solution. A timer was started and 3 drops of the starch / amylase mixture were pipetted into a spot plate cell every 2 minutes until 10 minutes had elapsed, then the sample was pipetted into a spot plate cell every 5 minutes. As soon as the sample was in the spot plate 2 drops of iodine indicator was dropped in to the starch amylase mixture. Iodine reacts with starch to change from yellow to deep blue /black in the presence of starch. A lightening of the blue/ black to a brown color will occur as less starch is present. Results were reported as (+) for presence of starch in the sample or (–) for the absence of starch in the sample. (See Table III), N/A = No Activity, (See Graph III)
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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
The results of the experiments showed the marked effect of temperature and pH on porcine pancreatic amylase activity. In experiment #1, the optimum temperature for porcine pancreatic amylase activity was 55C. This was higher than body temperature, the predicted 38C. The cause for this increased activity is that generally, reaction rates increase with temperature due to increased molecular motion. The increased motion increases the opportunity for substrate enzyme contact. This is evident from the increased porcine pancreatic amylase activity from 22C to 38C to 55C samples. The opposite is occurring in the 0C sample. The system is so cold that the system lacks the kinetic activation energy to initiate the reaction. The 55C temperature must also be below the temperature where porcine pancreatic amylase will denature. Denaturing changes the protein structure and thereby its ability to function properly. This is visible in the 83C sample where no starch reduction, (no amylase activity), occurred.

In experiment #2 the results indicate that between pH 5 to pH 7 and up to pH 9 the porcine pancreatic amylase activity continues to increase. This may show that the porcine pancreatic amylase activity is best at pH ranges of the saliva and the small intestine. The two extreme pH values of pH 3 and pH 11 showed no porcine pancreatic amylase activity. This may be due to the predicted competition at the enzyme active sites with increased hydrogen ion or hydroxide ion concentrations. Also the attraction of H or OH ions to the protein functional groups can alter the shape of the enzyme and thereby limit its activity.

In experiment #3 the porcine pancreatic amylase activity was zero in the boiled sample. This confirms the prediction that boiling the porcine pancreatic amylase will denature it and prevent it from functioning. The frozen sample results were comparable to our initial pH 7 room temperature sample. The slight difference of two minutes for reaction completion may be due to the sample cooling a little from 22C before the test. This demonstrated that freezing the amylase before use did not significantly affect its performance.
The experimental results are relevant to food preparation in that a dish made of protein is sensitive to pH and temperature. For example, a piece of fish can be cooked (denatured) by raising its temperature until the flesh flakes. At this point the fish protein has irreversibly changed structure. This change, or denaturing, was also observed in the high temperature and boiled amylase samples. An alternate way to cook fish would be by the ceviche method. This method entails soaking, or marinating the fish in lime and or lemon juice for from 8 hours to overnight depending on the size of the fish. This technique also cooks the fish by denaturing the proteins with acid. This effect was also exhibited in the low pH sample where although the temperature remained constant, the activity of the amylase protein was destroyed. In both cases the protein is denatured and permanently altered.
The experiments raised a couple of questions which would require additional testing. The test can be rerun with the temperatures between 55C and 83C to determine a more accurate temperature that porcine pancreatic amylase will denature. Also, additional tests can be run at pH 9 to pH 11 to determine the optimum pH, as well as the point where denaturing occurs.

Literature Cited

1) Campbell, Neil A., Jane B. Reese, Martha R. Taylor, Eric J. Simon. Biology 105 Taken From; Biology: Concepts & Connections (Fifth Edition) Benjamin Cumming, San Francisco, CA, 2006.

2) DeAngelo, Eric, Lehigh Carbon Community College, Lecture notes from July 31, 2006, Chapter 6 Cell Energy Production, Biology 105 Taken From; Biology: Concepts & Connections (Fifth Edition) Benjamin Cumming, San Francisco, CA, 2006.

3) Pugh, Michael E., Anderson, Wayne P., Experiment 12, Factors that Influence the Activity of an Enzyme, (Rev. 3/2004),*Adapted from Experiment 74, “Factors Affecting Enzymatic Activity” in: Holum, John R., Olmstead, Sandra L., Laboratory Manual for Fundamentals of General, Organic and Biological Chemistry, 5th Ed. New York: Wiley, 1994.

4) Manual of Clinical Enzyme Measurements, Introduction to Enzymes Worthington Biochemical Corporation, 1972, 730 Vassar Ave., Lakewood, NJ, 08701

5) Nichols Beth A. D., Cholewiak Linda B., A Quantitative Enzyme Study
Using Simple Equipment,
Departments of Molecular Biology and Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey 08544, 1991

6) Erickson, Hal, "Usage Recommendations for α-Amylases: Maximizing
Enzyme Activity while Minimizing Enzyme-Artifact Binding Residues" in The Book and Paper Group Annual, Volume 11 (Washington, DC: The Book and Paper Group of the American Institute for Conservation of Historic and Artistic Works, 1992), pp. 24-33.

7) Eckert, Roger, Randall, David, Animal Physiology: Mechanisms and Adaptations, W.H. Freeman Company, New York, 1983 pgs.55-60

8) Worthington Biochemical Corporation, Alpha Amylase (pancreatic porcine amylase) Product Bulletin, 730 Vassar Ave., Lakewood, NJ, 08701, 2006