Amylase Lab

1
The Limits of Amylase
2-1-14

Abstract

This report explains the purpose of this experiment in a way that conveys information to the reader about Amylase’s ability to withstand acidic or basic pH. To do this, two test tubes were both filled with 5mL of a 5% amylase solution. The first one was filled with an acid, while the other was filled with a base. After dropping liquid Iodine and Benedict’s solution into each one, the tube with a basic pH tested positive for glucose. The acidic solution tested (mostly) negative for glucose, although there were trace amounts at the bottom. Overall, my conclusion is that only acidic solutions inhibit Amylase’s ability to digest starch.

Background

The goal of the experiment is to test whether or not Amylase can withstand abnormal changes in pH without denaturing itself. Salivary Amylase is an enzyme that acts upon any polysaccharide that enters the mouth (mainly starch). However, it has a few other clinical uses. It is used for the diagnosis of acute pancreatitis, an inflammation of the pancreas, and other medical conditions regarding Amylase levels in the body. Amylase’s first discovery in 1831 led to new experiments about this enzyme. By separating pancreatic Amylase from Trypsin, they were able to realize that it acted upon starch, breaking it down into a simple sugar, or monosaccharide. In the diagram (featured below), the active site of this enzyme contain three major acidic groups.
These contain a calcium ion (the large gray sphere)
, the chloride ion (the large green sphere), and the chain of five sugar units (in yellow & orange). Previously mentioned, this enzyme will act upon starch and break it down into simple sugars. 2
Hypothesis

If we expose Amylase to a variety of acidic and basic solutions to test whether it will still digest starch or not, then I think that it will denature both ways, because Amylase is normally intolerable to any pH above 8 or below 6.

Materials and Procedures

The following will be useful in this experiment:
20mL of a 5% Amylase solution
20mL of a 1% starch solution
8 test tubes
At least 5mL of HCl
At least 5mL of NaOH
Hot plate
Beaker full of water (for incubation)
Liquid Iodine Solution
Benedict’s Solution

Follow this procedure:

1. In your first 4 test tubes, add the following solutions accordingly to each tube: -Test tube 1: 5mL of distilled water -Test tube 2: 5mL of the 5% Amylase solution -Test tube 3: 5mL of Amylase solution + 2-3 drops of HCl -Test tube 4: 5mL of Amylase solution + 2-3 drops of NaOH

2. Add 5mL of the starch solution into each tube.

3. Incubate each tube in the beaker of water for 30 minutes. The temperature should be 37°C (98.6°F).

4. After incubation, split half of all contents in each tube into 4 new tubes.

5. Insert 2-3 drops of the Iodine solution into the first set of beakers (the original 4). Then, insert 2-3 drops of the Benedicts solution into the second set of test tubes.

6. Record any color changes.

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Results

The first test tube (containing distilled water) was light brown when mixed with iodine, and blue when mixed with Benedict’s solution. The second test tube (5% Amylase solution) was light brown when mixed with iodine. It became orange when Benedict’s was added to the mix. The third test tube turned dark brown when the iodine was added. The other beaker with test tube 3’s contents remained blue when Benedict’s solution was added, although traces of Orange were found. The last test tube appeared Light Brown with the iodine mixed in (3 drops are always used), and appeared Orange when combined with the Benedict’s solution.

Discussion

Many of the test tubes ended up looking like I expected them to, except the fourth beaker. I had not expected that Amylase, under the influence of a very basic solution, could still act as a catalyst for starch. With a pH of 9 or higher, Amylase would normally denature itself. This could mean one of two things: Amylase is capable of functioning normally under a very high pH, or it could’ve been human error that led to this surprising result. To minimize chances of this happening again, assuming that this was human error only, would be to maximize the pH in the fourth beaker to 14 instead of 12, should a new lab arise, focusing on Amylase’s ability to withstand a high pH without getting denatured.

Conclusion

Overall, most of my results agreed with my notes and background knowledge about Amylase, apart from the difference in test tube 4. Like I predicted, the Amylase solution (tube 2) tested positive for simple sugars, the acidic solution denatured the enzyme, and the distilled water did absolutely nothing.

Sources

Dugdale, D. (2013, October 31). Amylase - blood. Retrieved from http://www.nlm.nih.gov/medlineplus/ency/article/003464.htm
(n.a.) Alpha- amylases. (2006, February 18). Retrieved from http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/download/Alpha-amylase.pdf
(n.a.) Alpha amylase. (2010, January 29). Retrieved from http://science.marshall.edu/murraye/alpha_amylase.htm