Lesson 10 - DEMONSTRATION OR STUDENT ACTIVITY: Enzyme activity
Student Performance Objectives1. Describe an experiment to demonstrate the effects of lipase, bile and temperature on litmus cream. 2. Describe an experiment to demonstrate the effects of amylase, temperature and pH on starch.Lesson OutlineA. Fat digestion in cream by pancreatic lipase: utilize 8 tubes. 1. First 4 Tubes are at room temperature. The contents of each tube are the following: a. Tube 1 - Litmus cream, pancreatic lipase and bile. b. Tube 2 - Litmus cream and lipase. c. Tube 3 - Litmus cream and bile. d. Tube 4 - Litmus cream and distilled water. 2. Second 4 Tubes are the same as the first 4 but placed in ice with all ingredients added except thelipase - the lipase is only added after the tubes get cold. 3. Note from the degree of change of color of the litmus cream from blue to reddish that the reactionoccurs at both room temperature and at ice temperatures, but faster at room temperature (what a surprise. Of course you could have predicted this, but it is one thing to "know" something and another thing to actually demonstrate it. So this is not an experiment - it is an exercise. 4. Note the use of controls: the reaction does not proceed without the enzyme, pancreatic lipase.B. Starch digestion by porcine amylase: utilize 15 tubes: 5 to run the experiment, then 5 to testfor sugar presence, and 5 to test for starch presence. 1. First 5 tubes are used to set up and to run the experiment and then to extract samples for furthertesting for the presence of sugar and starch. Experimental tubes contain: a. Tube 1: Starch solution, pH 7 buffer, amylase solution - perfect conditions for enzyme activity. b. Tube 2: Starch solution, pH 9 buffer, amylase solution - high pH control. c. Tube 3: Starch solution, pH 3 buffer, amylase solution - low pH control. d. Tube 4: Starch solution, pH 7 buffer, boiled amylase solution - enzyme control #1 (denaturedenzyme). e. Tube 5: Starch solution, pH 7 buffer, distilled water - enzyme control #2 (no enzyme). 2. Tube contents are prepared and the experiment runs for one hour in a 37º C water bath, thistemperature simulating the temperature of the human mouth. 3. After one hour of incubation, two samples are taken from each tube. If you are using Clinitest reagent tablets to test for sugar, a "sample' is just 5 drops. Then add 10 drops of distilled water, and drop in the tablet and let it boil in a test-tube rack for a few seconds before noting the color. a. Tubes 6- 10: the first sample deom each tube (5 drops from each tube, using a different dropper for each tube) is placed in tubes 6- 10 and tested for sugar (utilizing Fehling's solution or Clinitest reagent tablets). Color change to shades of green and then brick red, indicates breakdown of starch to sugar, and is ideally observed in tube 1 only. Other tubes (controls) show only a negative reaction (blue color) for the reasons noted in B, part 1, a-d, above. b. Tubes 11-15: the second sample from each tube is placed in tubes 11-15 and tested for starch(utilizing Lugol's iodine solution). Color change to a blue-black color indicates starch is still present. Digestion either did not occur at all, or, possibly, only slightly. 4. Note the sensitivity of enzymes to pH, temperature, and the presence or absence of a substrate. 5. Note the need for care in preparing for an experiment - attention to detail in preparation for an experiment (or even an exercise like this in which you can easily predict the result).