By: Pat Ebbert

Amylase is an enzyme found in the body that breaks down starch into simple sugars.

Amylase is found in the saliva and in the pancreas of humans, breaking down the starch from the foods we consume into simpler sugars, which are later converted into glucose needed for energy.

Alpha-Amylase is the structure of amylase found in human saliva and pancreas.

It breaks down long strands of carbohydrates into simpler sugars such as maltose or glucose.

This form of amylase is considered superior because it can act on any portion of the substrate , unlike Beta-Amylase, making reactions occur much faster.

To the right is a diagram showing the structure of the Alpha-Amylase enzyme.

The green dot represents a chloride ion, the pale yellow a calcium ion. Both are used to help the enzyme bind to the substrate.

Iodine has the property of binding to starch to create a dark blue color.

If starch has been broken down into simpler sugars, the iodine cannot bind and therefore leaves an area of no color visible.

If amylase has broken down surrounding starch into simpler sugars, this area’s diameter can be measured to give a relative value of how much starch has been broken down.An iodine atom

Three variables are used in this experiment to test their effect on amylase activity. All variables are liquids that could be found in the oral cavity, because that is where salivary amylase is most prominent.

Listerine is used to see if mouthwash effects amylase just as it kills bacteria.

Diet Pepsi is used to see if carbonation has any effect on the amylase.

At last, Mylanta antacid drink is used to see if a change in pH has an effect on amylase activity.

The purpose of this experiment is the determine whether or not the presence of various liquids that may be found in the mouth has any effect on amylase activity.

Null Hypothesis: It is hypothesized that the presence of Mylanta, Diet Pepsi, or Listerine will have NO significant effect on the activity of enzyme alpha-amylase when compared to the controls.

Alternate Hypothesis: It is hypothesized that the presence of Mylanta, Diet Pepsi, or Listerine WILL have a significant effect on the activity of enzyme alpha-amylase when compared to the controls.

2% stock solution of 1,4-α-D-glucan glucanohydrolase (alpha-amylase) in distilled water

3% stock solution of iodine in distilled water

5ml macro-pipette 50-200µl micro-pipette Micro-pipette tips 16 starch-agar plates

8 test tubes (5ml) 45ml mixing tube Squirt bottle Sharpie marker Drinking straws Diet Pepsi Mylanta antacid

drink Listerine mouthwash

1. Create eight different test tube solutions to be used in experiment (explained on following slide).

2. Fill five wells in each of 16 starch agar plates with 50µl of amylase for eight different data sets (10 trials of each set, 80 total wells).

3. Two data sets are positive and negative control sets, three are 10% variable solutions, and three are 50% variable solutions (all of these come from the test tubes).

4. After one hour, spray iodine solution onto each plate and measure zone of starch-breakdown in millimeters.

Neg. Control

Pos. Control

10% Var.* 50% Var.*

Amylase solution

0ml 1ml 1ml 1ml

Distilled water

5ml 4ml 3.5ml 1.5ml

Variable 0ml 0ml 0.5ml 2.5ml

Total 5ml 5ml 5ml 5ml

This table represents what will be present in the eight test tubes used in the experiment. After the solutions are prepared, 50µl of each tube are pipetted into ten different starch-agar plate wells.

*The variable columns are repeated three times (Mylanta, Diet Pepsi, Listerine)