Experiment 1: Enzymes in FoodAmylase is used by humans to facilitate digestion. Specifically, it is an enzymewhich breaks down starch molecules into sugar molecules. This is why peoplesometimes observe a sweet taste after sucking on a starch-containing food foran extended period of time. Amylase is found naturally in human saliva andthe pancreas. However, it is also present in some of the common plant foodsconsumed by humans.This experiment tests for the presence of amylase in food by using IodinePotassium Iodide, IKI. IKI is a color indicator used to detect starch. Thisindicator turns dark purple or black in color when in the presence of starch.Therefore, if the IKI solution turns to a dark purple or black color during theexperiment, one can determine that amylase is not present (becausepresence of amylase would break down the starch molecules, and the IKIwould not change color).Materials(1) 2 oz. Bottle (Empty)(1) 100 mL GraduatedCylinder30 mL Iodine-PotassiumIodide, IKIPermanent MarkerRuler2 Spray Lids30 mL Starch (liquid)*Cutting Board*2 Food Products (e.g., gingerroot, apple, potato, etc.)*Kitchen Knife*Paper Towel*Saliva Sample*Tap Water*You Must ProvideProcedure:1.Remove the cap from the starch solution. Attach the spray lid to thestarch solution.2.Rinse out the empty two ounce bottle with tap water. Use the 100 mLgraduated cylinder to measure and pour 30 mL of IKI into the empty twoounce bottle. Attach the remaining spray lid to the bottle.3.Set up a positive control for this experiment by spraying a paper towelwith the starch solution. Allow the starch to dry for approximately onehour (this time interval may vary by location).4.In the mean time, set up a negative control for this experiment. Use yourknowledge of the scientific method and experimental controls toestablish this component (hint: what should happen when IKI solutioncontacts something that does not contain starch?) Identify your negativecontrol in Table 1.Note: Be sure to space the positive and negative controls apart fromeach other to prevent cross-contamination.5.When the starch solution has dried, test your positive and negativecontrols. This step establishes a baseline color scale for you to evaluatethe starch concentration of the food products you will test in Steps 7 11. Record your results in Table 1.6.Select two food items from your kitchen cabinet or refrigerator.7.Obtain a kitchen knife and a cutting board. Carefullycut your selectedfood items to create a fresh surface.Figure 3: Sample set-up.8.Gently rub the fresh/exposed area of the food items on the dry, starchsprayed paper towel back and forth 10 – 15 times. Label where eachspecimen was rubbed on the paper towel with a permanent marker(Figure 3).9.Provide a saliva sample by spitting in a separate bowl and rubbing thepaper towel in the saliva. Be sure not to spit on the paper towel directlyas you may unintentionally cross-contaminate your samples. Repeatthis step until you are able to adequately moisten the paper towel.10.Wait five minutes.11.Hold the IKI spray bottle 25 – 30 cm away from the paper towel, andmist with the IKI solution.12.The reaction will be complete after approximately 60 seconds. Observewhere color develops, and consider what these results indicate. Recordyour results in Table 1.Table 1: Substance vs. Starch PresenceSubstanceResulting ColorPresence ofStarch?Positive Control: StarchNegative Control: Student Must SelectFood Product:Food Product:Saliva:experiment 2: Effect of Temperature on Enzyme ActivityFigure 4: Catalase catalyzesthe decomposition ofhydrogen peroxide to waterand oxygen.Yeast cells contain catalase, an enzyme which helps convert hydrogenperoxide to water and oxygen. This enzyme is very significant as hydrogenperoxide can be toxic to cells if allowed to accumulate. The effect of catalasecan be seen when yeast is combined with hydrogen peroxide (Catalase: 2H2O2 ? 2 H2O + O2).In this lab you will examine the effects of temperature on enzyme (catalase)activity based on the amount of oxygen produced. Note, be sure to remainobservant for effervescence when analyzing your results.Materials(2) 250 mL Beakers3 Balloons30 mL 3% HydrogenPeroxide, H2O2Measuring SpoonPermanent MarkerRuler3 Test Tubes (Glass)Test Tube RackThermometerYeast Packet*Hot Water Bath*Stopwatch20 cm String*You Must ProvideProcedure1.Use a permanent marker to label test tubes 1, 2, and 3. Place them inthe test tube rack.2.Fill each tube with 10 mL hydrogen peroxide. Then, keep one of the testtubes in the test tube rack, but transfer the two additional test tubes totwo separate 250 mL beakers.3.Find one of the balloons, and the piece of string. Wrap the string aroundthe uninflated balloon and measure the length of the string with theruler. Record the measurement in Table 2.4.Create a hot water bath by performing the following steps:a.Determine if you will use a stovetop or microwave to heat thewater. Use the 100 mL graduated cylinder to measure and pourapproximately 200 mL of water into a small pot or microwave-safebowl (you will have to measure this volume in two separateallocations).b.If using a stovetop, obtain a small pot and proceed to Step 4c. Ifusing a microwave, obtain a microwave-safe bowl and proceed toStep 4e.c.If using a stove, place a small pot on the stove and turn the stoveon to a medium heat setting.d.Carefully monitor the water in the pot until it comes to a soft boil(approximately 100 Â°C). Use the thermometer provided in your labkit to verify the water temperature. Turn the stove off when thewater begins to boil. Immediately proceed to Step 5.CAUTION: Be sure to turn the stove off after creating the hotwater bath. Monitor the heating water at all times, and neverhandle a hot pan without appropriate pot holders.e.If using a microwave, place the microwave-safe bowl in themicrowave and heat the water in 30 second increments until thetemperature of the water is approximately 100 Â°C. Use thethermometer provided in your lab kit to verify the watertemperature. Wait approximately one minute before proceeding toStep 5.5.Place Tube 1 in the refrigerator. Leave Tube 2 at room temperature, andplace Tube 3 in the hot water bath.Important Note: The water should be at approximately 85 Â°C when youplace Tube 3 in it. Verify the temperature with the thermometer toensure the water is not too hot! Temperatures which exceedapproximately 85 Â°C may denature the hydrogen peroxide.6.Record the temperatures of each condition in Table 2. Be sure toprovide the thermometer with sufficient time in between eachenvironment to avoid obscuring the temperature readings.7.Let the tubes sit for 15 minutes.8.During the 15 minutes prepare the balloons with yeast by adding Â¼ tsp.of yeast each balloon. Make sure all the yeast gets settled to the bulb ofthe balloon and not caught in the neck. Be sure not spill yeast whilehandling the balloons.9.Carefully stretch the neck of the balloon to help ensure it does not ripwhen stretched over the opening of the test tube.10.Attach the neck of a balloon you prepared in step 8 to the top of Tube 2(the room temperature test tube) making sure to not let the yeast spillinto the test tube yet. Once the balloon is securely attached to the testtube lift the balloon and allow the yeast to enter the test tube. Tap thebulb of the balloon to ensure all the yeast falls into the tube.11.As quickly and carefully as possible remove the Tube 1 (cold) from therefrigerator and repeat steps 9 – 10 with Tube 1 using a balloon youprepared in step 8.12.As quickly and carefully as possible remove Tube 3 (hot) from the hotwater bath and repeat steps 9 – 10 with Tube 3 using a balloon youprepared in step 8.13.Swirl each tube to mix, and wait 30 seconds.14.Wrap the string around the center of each balloon to measure thecircumference. Measure the length of string with a ruler. Record yourmeasurements in Table 2.Table 2: Balloon Circumference vs. TemperatureTube1 – (Cold)2 – (RT)Temperature(Â°C)BalloonCircumference(Uninflated; cm)BalloonCircumference(Final; cm)3 – (Hot)To conduct your laboratory exercises, use the Laboratory Manual located underCourse Content. Read the introduction and the directions for each exercise/experimentcarefully before completing the exercises/experiments and answering the questions.Save your Lab 4 Answer Sheet in the following format: LastName_Lab4 (e.g.,Smith_Lab4).You should submit your document as a Word (.doc or .docx) or Rich Text Format(.rtf) file for best compatibility.Pre-Lab Questions1. How could you test to see if an enzyme was completely saturated during an experiment?2. List three conditions that would alter the activity of an enzyme. Be specific with yourexplanation.3. Take a look around your house and identify household products that work by means of anenzyme. Name the products, and indicate how you know they work with an enzyme.Experiment 1: Enzymes In FoodData Tables and Post-Lab AssessmentTable 1: Substance vs. Starch PresenceSubstanceResulting ColorPresence of Starch?Positive Control: StarchNegative Control: Student MustSelectFood Product:Food Product:Saliva:Post-Lab Questions1. What were your controls for this experiment? What did they demonstrate? Why wassaliva included in this experiment?2. What is the function of amylase? What does amylase do to starch?3. Which of the foods that you tested contained amylase? Which did not? Whatexperimental evidence supports your claim?4. Saliva does not contain amylase until babies are two months old. How could this affectan infantâs digestive requirements?5. There is another digestive enzyme (other than salivary amylase) that is secreted by thesalivary glands. Research to determine what this enzyme is called. What substrate does itact on? Where in the body does it become activated, and why?6. Digestive enzymes in the gut include proteases, which digest proteins. Why donât theseenzymes digest the stomach and small intestine, which are partially composed of protein?Experiment 2: Effect of Temperature on Enzyme ActivityData Tables and Post-Lab AssessmentTable 2: Balloon Circumference vs. TemperatureTubeTemperature (Â°C)BalloonCircumference(Uninflated; cm)1 – (Cold)2 – (RT)3 – (Hot)Post-Lab Questions1. What reaction is being catalyzed in this experiment?2. What is the enzyme in this experiment? What is the substrate?Balloon Circumference(Inflated; cm)3. What is the independent variable in this experiment? What is the dependent variable?4. How does the temperature affect enzyme function? Use evidence from your data to supportyour answer.5. Draw a graph of balloon diameter vs. temperature. What is the correlation?6. Is there a negative control in this experiment? If yes, identify the control. If no, suggest howyou could revise the experiment to include a negative control.7. In general, how would an increase in substrate alter enzyme activity? Draw a graph toillustrate this relationship.8. Design an experiment to determine the optimal temperature for enzyme function, completewith controls. Where would you find the enzymes for this experiment? What substrate wouldyou use?
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