1. Put on your safety goggles and gloves.
2. Tap the -amylase bottle so that the powder collects at the bottom of the container.
3. Carefully remove both the lid and the tip of the bottle and set aside.
4. Use the 25 mL graduated cylinder to measure and add 12.0 mL of distilled water to the bottle.
5. Return the tip and lid to the bottle and shake vigorously until the contents are mixed.
6. Create a hot water bath using the burner fuel, burner stand, and 250 mL glass beaker.
7. While the water is heating, use the permanent marker to label three test tubes as "starch" and another three test tubes as "amylase."
8. Place the six test tubes into empty wells of the 24-well plate, using the well plate as a test tube holder.
9. Add 4 drops of 1% starch solution to each of the starch test tubes.
10. Add 4 drops of -amylase solution to each of the amylase test tubes.
11. Extinguish the burner flame once the water achieves a rolling boil.
12. Carefully place one starch test tube and one amylase test tube into the water bath. See Figure 8.

Hot water bath treatment.

1. Allow the tubes to remain in the water bath for 5 minutes.
2. During the 5 minute period, use the thermometer to measure the temperature of the water and record in Data Table 2.
3. After 5 minutes, carefully pour the contents of the -amylase test tube into the starch test tube and return the starch test tube to the water bath.
4. Allow the enzyme and starch solution to react for 1 minute.
5. After 1 minute, use a clean pipet to add 1 drop of IKI indicator to the test tube and immediately observe the color of the solution.
6. Record your observations of the initial solution color in Data Table 2.
7. Compare the color of the solution in the test tube with the solutions in Figure 9 to determine the presence of starch.

IKI results for testing for starch. Gray, brown, or black solutions indicate the presence of starch. Clear amber solutions indicate the absence of starch.

1. Record the presence or absence of starch in Data Table 2.
2. Remove the starch test tube from the water bath and place it upright in an empty well on the left side of the 24-well plate.
3. Allow the hot water bath to cool below 40C.
4. Carefully remove the 250 mL beaker from the burner stand and discard the water.
5. Create a 37C water bath in the 250 mL beaker.

Note: You may use either warm water from a faucet or the burner to create the water bath. It is only important that the water is 37C.

1. Repeat steps 12-20 using the 37C water bath.
2. Remove the starch test tube from the water bath and place it upright in an empty well in the middle row of the 24-well plate.
3. Discard the water from the beaker.
4. Create an ice bath in the 250 mL beaker by filling it half full of crushed ice and cold tap water.
5. Repeat steps 12-20 using the ice bath.
6. Remove the starch test tube from the water bath and place it upright in an empty well on the right side of the 24-well plate.
7. Allow the three test tubes to remain in the well plate at room temperature for 10 minutes.
8. After 10 minutes, observe the solutions in each tube and record the final solution color in Data Table 2.
9. Compare the color of the solution in the test tube with the solutions in Figure 9 to determine the presence of starch.
10. Record the presence or absence of starch in Data Table 2.
11. Remove the test tubes and clean the well plate for use in Part 2.

Part 2: Effects of pH

1. Write your name date on new sheet of paper.
2. Place the clean 24-well plate on the sheet of paper containing your name and date.
3. Label the sheet 1-4 below the bottom four wells.
4. Use the permanent marker to label four clean pipets "pH 3.5 buffer," "pH 5.0 buffer," "pH 6.8 buffer," and "pH 11.5 buffer".
5. Remove the dropper tips from each of buffer solution bottles and draw the solutions into the correctly labeled pipets.
6. Place each pipet bulb-side down in empty wells of the 24-well plate, using the well-plate as a pipet holder. See Figure 10.

pH set up.

1. Add the following solutions in the listed order to the labeled wells:
   • Well 1: add 6 drops of pH 3.5 buffer, 2 drops of -amylase solution, and then 2 drops of 1% starch solution
   • Well 2: add 6 drops of pH 5.0 buffer, 2 drops of -amylase solution, and then 2 drops of 1% starch solution
   • Well 3: add 6 drops of pH 6.8 buffer, 2 drops of -amylase solution, and then 2 drops of 1% starch solution
   • Well 4: add 6 drops of pH 11.5 buffer, 2 drops of -amylase solution, and then 2 drops of 1% starch solution
2. Allow each of the reactions to occur for 1 minute.
3. After 1 minute, add 1 drop of IKI indicator to the solutions in each well.
4. Observe the color of the solutions in each well and record in Data Table 3.
5. Compare the colors of the solutions in the wells with the solutions in Figure 9 to determine the presence of starch.
6. Record the presence or absence of starch in each well in Data Table 3.
7. Take a photo of the well plate displaying the solutions in each well and your name and date on the sheet of paper.
8. Upload the image into Photo 2.

Cleanup:

• Properly dispose of the solutions in the well plate and the used chemical pipets.
• Clean all lab equipment with soap and water and allow to thoroughly dry.
• Return cleaned and dried items to the lab kit for future use.

Table 2

Teprature Initial solution color Initial solution color Final Solution color Starch present-final (Y/N)

HOT 91C Yellow - Green Y Green Y

37C 37 C Yellow N Yellow N

ICE 3 C Orange - Yellow N Yellow N

Table 3

pH Color Starch present (Y/N)

3.5 Orange Y

5.0 Orange Y

6.8 Yellow N

11.5 Black Y

Exercise 2 - Questions

Question 1

1. Describe the relationship between temperature and the enzymatic activity of -amylase.

a. Of the three temperatures tested, which is the optimal temperature for enzyme activity?

b. Did any of the temperatures cause the enzyme to denature?

Explain your answer by referencing your results in Data Table 2.

• Question 2

2. What is the pH range for -amylase activity? Did any of the pH levels cause the enzyme to denature? Explain your answer by referencing your results in Data Table 3 and Photo 2.

Question 3

3. Do your results for the effects of both temperature and pH on -amylase activity support digestion of starch occurring in the mouth and intestines but not the stomach of humans? Explain your results by referencing your data and the temperature and pH of the mouth, stomach, and small intestines.