Establishing that an Enzyme is Present

1. Crumble a piece of cracker (about 1 x 1 cm) into a test tube. Add warm water (about 37șC) to a depth of about 5 cm.

2. Shake and pour into a funnel with filter paper.

3. Collect the fluid that runs through (the filtrate) in a second test tube to a depth of about 1 cm, and into a third tube to a depth of about 2 cm.

4. Test the filtrate in tube #2 for the presence of starch, using the iodine reagent. Add several drops of the solution to the filtrate. A blue-black color is a positive test for starch.

5. Test the filtrate in tube #3 for sugar by adding Benedict's or Fehling's solution, then heating until a green, orange, or yellow color appears. If there is no color change after heating for several minutes, the test is negative meaning no sugar is present.

6. If the test for sugar is positive, try another brand of cracker! We do not want sugar in the cracker. It will interfere with determining whether or not amylase is able to convert the cracker's starch into sugar.

7. To produce salivary amylase, someone will have to provide saliva! To do this, a student will chew on a rubber band or a piece of paraffin wax. DO NOT CHEW GUM! Why not?

8. After a few milliliters of saliva have been collected in a test tube, test the saliva for sugar. If the test is positive for sugar, another person will have to provide the saliva.

9. The student who tests negative for sugar should next chew a piece of cracker, roughly 3 x 3 cm. After thoroughly chewing the cracker for several minutes, the wad of cracker and residual saliva should be deposited into a funnel lined with filter paper.

10. Add about 5 ml of warm water (37șC) to the material in the filter paper, collecting about 3 ml of filtrate in a new test tube.

11. Test the filtrate for sugar. What does a negative test mean? What does a positive test mean?

Variable Conditions for Enzyme Activity

1. Using a rubber band or paraffin wax, again collect saliva from the student who provided the saliva in Step 9. The amount of saliva to be collected must fill each of 7 test tubes to a depth of about 2 cm.

2. Using pH paper, measure the pH of the saliva.

3. In test tube #1 containing saliva, add a few drops of starch solution and place the tube in a beaker containing water at 37șC for 10 minutes. Remove the tube and test for sugar.

4. Place test tube #2 with its saliva in a beaker containing boiling water. Add a few drops of starch solution and leave the tube in the boiling water for 10 minutes. Remove the tube and test for sugar.

5. Place tube #3 with its saliva in a beaker containing crushed ice and water. Add a few drops of starch and leave the tube in the ice water for 10 minutes. Remove the tube and test for sugar.

6. In test tube #4 with its saliva, add an equal amount of hydrochloric acid solution, pH 6. Mix the contents by gentle “banging” of the tube bottom against the palm of the hand. Place the tube in a water bath at 37șC and add a few drops of starch solution. Allow the tube to stand for 10 minutes, then remove and test for sugar.

7. In test tube #5 with its saliva, add an equal volume of hydrochloric acid solution, pH 3. Mix by hitting the tube bottom against the palm of the hand. Place the tube in a water bath at 37șC and add several drops of starch solution. Allow the tube to stand in the water for 10 minutes after which the tube is removed and the solution is tested for the presence of sugar.

8. To test tube #6 with its saliva, add an equal volume of sodium hydroxide solution (NaOH) of pH 8 and mix. Add several drops of starch solution, then place in a water bath at 37șC for 10 minutes after which the tube is removed and the solution tested for the presence of sugar.

9. To test tube #7 with its saliva, add an equal volume of sodium hydroxide, pH 11 and mix. Place the tube in a water bath at 37șC and add several drops of starch solution. Allow the tube to stand for 10 minutes, after which the tube is removed and tested for the presence of sugar.

Data Analysis

1. Based on your data, what are the best conditions for amylase activity in terms of temperature and pH? Do any of these combinations apply to the human mouth?

2. Was there any enzymatic activity when the test tube was placed in boiling water?

3. Since amylase is a protein molecule, its activity dependents on maintaining a certain physical structure that is affected by temperature. Did boiling water affect the activity of the enzyme? Did it increase, decrease, or stay the same compared with room temperature (23șC) or body temperature (37șC)? Would any of these changes be related to the effect of temperature on the rate of a reaction (rate of molecular collisions)?