Enzyme Hydrolysis of Starch

In this exercise, students actually observe an enzyme-catalyzed reaction and the effects on its rate of pH, temperature, and a disinfectant. The value of the exercise depends on your purpose in introducing enzymes. I prefer to do it after class discussion of proteins and the relationship between their structures and their functions, both of which can be disrupted (denaturation) by many treatments. In some texts, enzymes are only discussed in the context of hydrolysis/digestion of carbohydrates. In years when we do this lab before covering proteins, we can only talk about disruption of enzyme activity in a simplistic sense, i.e. enzymes work best in their natural environments and can be "killed" by harsh treatments.

Starch hydrolysis is detected by altered reaction with iodine. The starch we use is what is sold as "soluble starch". The source of amylase can be either saliva or a commercial preparation. To prepare an amylase solution from saliva, students dilute 1 mL of saliva with 9 mL of water, then adding 60 mL of 0.5% NaCl. Some students are inevitably squeamish about saliva, but that seems to be less a problem when I tell them that adding the salt destroyed the sliminess and it isn't saliva any more, just a solution of proteins. Alternatively, to avoid the squeamishness, and for more consistency of results, I have prepared a solution of 15 mg powdered amylase (Sigma A3176 or equivalent) per 100 mL of water.

In all tests below, the same approach was used: equal volumes of enzyme and starch are incubated for 5 minutes in separate test tubes to equilibrate temperatures. After mixing, three-drop samples are taken immediately and at one minute intervals for 10 minutes and mixed immediately in a color test plate with a couple drops of the iodine solution described in another exercise. As hydrolysis proceeds, the color formed with iodine changes from blue-black to dark red to pale orange. Students are instructed to clean their test tubes carefully between tests to prevent cross-contamination.

Effect of pH: To 1 mL of buffer (pH 4, 7, or 10) in each of three test tubes, students add 1 mL of 2% unbuffered starch. Three separate tubes contain 2 mL of enzyme solution. After preincubating at 37 degrees for 10 minutes, pairs of tubes are mixed and samples are tested for color with iodine. Generally, there is a fairly rapid disappearance of starch at pH 7 and none at pH 4 and 10.

Effect of temperature: Students put 2 mL of enzyme solution in three tubes, and 2 mL of buffered 1% starch (pH 7) in three other tubes. Pairs of tubes are preincubated for 10 minutes at either 0, 37, or 70 degrees, then mixed and processed as above, keeping them at the test temperature between samples. The same disappearance of starch is seen at 37 degrees as at pH 7 above (since the conditions should be identical), and little or no reaction occurs in the cold or hot water. (We do this test second because the manipulations are a little more challenging.)

Effect of disinfectant: Students add one drop of full-strength Lysol disinfectant to 2 mL of enzyme solution and preincubate at 37 degrees for 10 minutes along with 2 mL of buffered starch in a separate tube. Then the tubes are mixed and processed as above. Generally, there is no detectable disappearance of starch in this test. Lysol at this strength seems to interfere with color stability, causing the blue color to last only briefly, so students need to record observations immediately after mixing. (Diluted Lysol solutions are less effective at inhibiting the enzyme.)

The buffers we use are as follows:

  • pH 4: 0.2 M citric acid adjusted to pH 4 with NaOH
  • pH 7: 0.2 M monopotassium dihydrogen phosphate adjusted to pH 7 with KOH
  • pH 10: 0.2 M sodium bicarbonate adjusted to pH 10 with NaOH

After reporting their observed colors in tables, students are asked to compare their results with the observations that in the absence of enzyme, starch hydrolyzes faster in acid, and faster at higher temperatures, and to explain similarities or discrepancies in terms of what they know about enzymes, and to predict whether all enzymes in nature would work best at pH 7 and 37 degrees. Finally, they are asked why heat, cold, and disinfectants prevent microbial growth.

 

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Revised 9/14/09