Total Dissolved Solids
Lactase Action
Intestinal gas occurs normally in humans. It is composed primarily of carbon dioxide, nitrogen and oxygen gases. Gas is produced when indigestible sugars are metabolized by certain bacteria in the intestines. Nearly one out of four individuals lack the genetic ability to digest these sugars.
Lactase is an enzyme responsible for breaking bonds in lactose, a disaccharide sugar. When lactose cannot be metabolized within the cells of a person, bacteria digest lactose in the intestines. This bacterial digestion produces carbon dioxide gas as a by-product. When this gas builds up in the intestines, flatulence is the result. Recently, researchers have discovered a way to mass produce the enzyme lactase. Lactase converts lactose into glucose and galactose, both easily digestible monosaccharides. Using biotechnological processes, they have formulated the product Lactaid as a way to lower the production and expulsion of rectal gas. The enzyme lactase is used to break the bond in lactose before it is ingested.
In this lab, you will test the function of lactase. One way to test its activity is to determine if the enzymes are capable of converting the disaccharide into glucose and galactose. It is easy to test for glucose. You can use a special test paper originally made for diabetics to help detect blood sugar in urine. If glucose is present in a liquid, the test paper turns a different color.
An alternative way to test the activity of lactase is to determine whether the sugar can support life. Presumably, yeast are unable digest lactose. They can, however, digest glucose and use it as an energy source. Yeast can burn sugar anaerobically during fermentation, according to the equation:
C6Hl2O6 → 2 CH3CH2OH + 2 CO2 + energy
Yeast release CO2 when they metabolize sugar. By monitoring a pressure change caused by the production of CO2, we can use yeast to indicate the activity of lactase.
OBJECTIVES
IN THIS EXPERIMENT, YOU WILL
* Test the action of lactase.
* Use glucose test paper to monitor the presence of glucose.
* Determine if yeast can metabolize glucose, lactose, or galactose.
[pic]
Figure 1
MATERIALS
|COMPUTER |600 ML BEAKER (FOR WATER BATH) |
|VERNIER COMPUTER INTERFACE |DROPPER OR BERAL PIPET |
|LOGGER PRO |FORCEPS |
|VERNIER GAS PRESSURE SENSOR |FOUR 18 ( 150 MM TEST TUBES |
|1-HOLE RUBBER STOPPER ASSEMBLY |HOT AND COLD WATER |
|LACTASE (DROPLET FORM) |STOPWATCH |
|5% GALACTOSE SOLUTION |TES-TAPE OR OTHER GLUCOSE TEST PAPER |
|5% GLUCOSE SOLUTION |TEST-TUBE RACK |
|5% LACTOSE SOLUTION |THERMOMETER |
|10 ML GRADUATED CYLINDER |YEAST SUSPENSION |
PROCEDURE
TESTING FOR THE PRODUCTION OF GLUCOSE
1. You will determine if lactase can produce glucose from the conversion of lactose.
2. Obtain two test tubes and label them test tube 1 and test tube 2.
|Table 1 |
|Test Tube |Sugar Solution (2.5 mL) |Lactase |
|1 |lactose |2 drops |
|2 |lactose |none |
3. Obtain the lactose sugar solution. Add 2.5 mL of the sugar solution to both test tubes, as shown in Table 1.
4. Prepare a water bath for the sugar solutions. A water bath is simply a large beaker of water at a constant and controlled temperature. To prepare the water bath, obtain some warm and cool water from your teacher. Combine the warm and cool water in the 600 mL beaker until it reaches 35°C. The beaker should be filled with about 400 mL of water.
If you need to add more hot or cold water to maintain a constant temperature, first remove about as much water as you will be adding, or the beaker may overflow. Use a basting bulb or a Beral pipette to remove excess water.
5. Measure the glucose concentration. To do this:
a. If the test paper is supplied in a continuous strip, tear off a small piece (0.5 cm) of glucose test paper. Otherwise, obtain one test strip.
b. Using a dropper pipette, withdraw a drop or two of sugar solution from test tube 1.
c. Place one drop of sugar solution onto the glucose test paper.
d. Follow the instructions on the glucose test paper package to develop the test paper. This usually requires a 30 or 60 second wait before you compare the color of the tape to the supplied color chart.
e. Record the approximate concentration of glucose in Table 3 in the “Time 0” column.
f. Discard any sugar solution remaining in the dropper. Rinse the dropper by taking up clean water and expelling it into a waste beaker.
6. Repeat Step 5 for the second test tube.
7. Add lactase to the test tube 1:
a. Place 2 drops of lactase into test tube 1.
b. Gently mix the contents of the tube.
8. Set both tubes in the water bath. Start the stopwatch. Be sure to keep the temperature of the water bath close to 35°C.
9. Incubate the test tubes for 10 minutes, taking a glucose test once every minute for 10 minutes. Repeat Step 5 and record the concentrations of glucose in Table 3 once every minute.
Testing for the Ability of Yeast to Ferment Sugars
10. Connect the Gas Pressure Sensor to the computer interface. Prepare the computer for data collection by opening the file “24B Lactase Act (Press)” from the Biology with Computers folder of Logger Pro.
11. Connect the plastic tubing to the valve on the Gas Pressure Sensor.
12. Check the water bath so it remains at a constant temperature range between 33°C and 37°C.
13. You will perform one of the four tests outlined in Table 2 and obtain the results of the other tests from your classmates. Ask your instructor which test you will be performing and record the test number in Table 4.
|Table 2 |
|Tube |Sugar |Volume (mL) |Enzyme |
|1 | Lactose |2.5 |Lactase |
|2 | Lactose |2.5 |none |
|3 | Glucose |2.5 |none |
|4 | Galactose |2.5 |none |
|5 | None (water only) |2.5 |none |
14. Prepare the sugar solution:
* If you are assigned tests 1 or 2, you have already made the solution. Use the same test tube as in Table 1.
* If you are assigned tests 3, 4, or 5, obtain the appropriate solution. Place 2.5 mL of the solution into the test tube, as in Table 2.
[pic]
Figure 2
15. Set the test tube in the water bath.
16. Obtain the yeast suspension. Gently swirl the yeast suspension to mix the yeast that settles to the bottom. Put 2.5 mL of yeast into the test tube and mix the solution.
17. In the test tube, place enough vegetable oil to completely cover the surface of the yeast/sugar mixture as shown in Figure 3. Be careful to not get oil on the inside wall of the test tube. Set the test tube in the water bath.
18. Insert the single-holed rubber-stopper into the test tube. Note: Firmly twist the stopper for an airtight fit. Secure the test tube with a utility clamp and ring-stand as shown in Figure 1.
19. Incubate the test tube for 10 minutes in the water bath. Be sure to keep the temperature of the water bath constant. If you need to add more hot or cold water, first remove about as much water as you will be adding, or the beaker may overflow. Use a basting bulb to remove excess water.
[pic]
Figure 3
Note: Be sure that most of the test tube is completely covered by the water in the water bath. The temperature of the air in the tube must be constant for this experiment to work well.
20. When incubation has finished, connect the free-end of the plastic tubing to the connector in the rubber stopper as shown in Figure 3.
21. Begin collecting pressure data by clicking [pic]. Maintain the temperature of the water bath during the course of the experiment.
22. Data collection will end after 15 minutes. Monitor the pressure readings displayed on the computer screen. If the pressure exceeds 130 kilopascals, the pressure inside the tube will be too great and the rubber stopper is likely to pop off. Disconnect the plastic tubing from the Gas Pressure Sensor if the pressure exceeds 130 kilopascals.
23. When data collection has finished, disconnect the plastic tubing connector from the rubber stopper. Remove the rubber stopper from the test tube and discard the contents in a waste beaker.
PROCESSING THE DATA
YOU WILL NOW DETERMINE THE RATE OF FERMENTATION. THE RATE OF FERMENTATION CAN BE MEASURED BY EXAMINING THE SLOPE OF THE PRESSURE VS. TIME PLOT FOR EACH TEST.
1. Find the rate of fermentation:
a. Move the mouse pointer to the point where the pressure values begin to increase. Hold down the mouse button. Drag the pointer to the end of the data and release the mouse button.
b. Click the Linear Fit button, [pic], to perform a linear regression. A floating box will appear with the formula for a best fit line.
c. Record the slope of the line, m, as the rate of fermentation in Table 4.
d. Close the linear regression floating box.
e. Share your data with other classmates by recording the test tube number and the rate of fermentation on the board.
2. Print the graph of pressure vs. time if directed to do so by your instructor.
3. Average the rate values for each of the four tests performed by the class and record them in Table 5.
4. On Page 2 of the experiment file, make a graph of rate of fermentation vs. sugar/enzyme combination.
DATA
|TABLE 3: GLUCOSE CONCENTRATIONS |
|TIME (MINUTES) |LACTOSE + LACTASE |LACTOSE ONLY |
|0 | | |
|1 | | |
|2 | | |
|3 | | |
|4 | | |
|5 | | |
|6 | | |
|7 | | |
|8 | | |
|9 | | |
|10 | | |
|Table 4: Your results |
|Test |Rate (kPa/min) |
| | |
|Table 5 |
|Test |Type of Sugar / Enzyme |Fermentation Rate |
| | |(kPa/min) |
|1 | Lactose + Lactase | |
|2 | Lactose only | |
|3 | Glucose | |
|4 | Galactose | |
|5 | None (water only) | |
QUESTIONS
1. FROM THE RESULTS OF THIS EXPERIMENT, HOW DOES LACTASE FUNCTION? WHAT IS YOUR EVIDENCE?
2. Considering the results of this experiment, can yeast utilize all of the sugars equally well? Explain.
3. Hypothesize why some sugars are not utilized by yeast while other sugars are metabolized.
4. How did the results of testing lactase’s activity using glucose test paper compare with the results of using yeast as an indicator of activity? What is your evidence?
5. Which test tube served as a control in this experiment? What did you conclude from the control? How did this affect the interpretation of data in this experiment?
Extensions
1. DESIGN AN EXPERIMENT TO TEST THE ACTIVITY OF BEANO ON THE SUGAR MELIBIOSE. DOES BEANO HAVE ANY EFFECT ON THE SUGAR LACTOSE? FROM THE RESULTS OF THIS EXPERIMENT, HOW DOES BEANO FUNCTION? WHAT IS YOUR EVIDENCE?
2. Design an experiment to test whether Beano has any effect on the sugar lactose.
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