Katie Huber and Pat Carney
Staci Riehl and Dawn Jerbeck
Becky Mugaini and Amy Bein
We plan to create a Web Site Page.
Have you ever wondered why you are not suppose to add fresh pineapple to Jell-O and expect it to set? The answer is, because of the enzyme bromelain which is found in fresh pineapple.
An enzyme serves as a catalyst, changing the rate of a chemical reaction without itself being changed (Campbell, Mitchell, Reece, 801). Without enzymes many metabolic reaction would occur too slowly to sustain life. (763) It was for this reason, to take a closer look at this particular topic.
We hope to explore how enzymes are effected by temperature and pH, and illustrate how amylases in saliva breaks down starch and changes it to sugar.
Five labeled test tubes included the following ingredients:
Five drops of Gram's iodine solution was added to each of the test tubes. The test tubes were then heated to room temperature by allowing them to settle for about 5-10 minutes, or until they changed color. The test tubes were next heated gradually over a Bunsen burner. Benedict's reagent was added to each test tube (10 drops each). Then the test tubes were warmed over the Bunsen burner until the color changed.
In test tube #1 with the distilled water, iodine caused a weak reaction and no real color change occurred. Test tube #2 with the starch solution had a moderate reaction with the iodine in it and it turned a dark blue color. Test tube #3 with the maltose solution and test tube #4 with the saliva had weak reactions and no visible color change. Test tube #5 with the saliva and starch had a moderate reaction and turned dark blue.
The test tubes were then warmed over the Bunsen burner and the Benedict's solution was added. Test tubes #1, #2, and #4 changed to a light blue with the solution but no other reaction occurred. Test tube #3 had the strongest reaction in that the color went from light blue to green to yellow to orange. Test tube #5 also had a strong reaction and the color changed from light blue to green to yellow.
The following items will be used in this project: Ten test tubes and lids, any flavor Jell-O (R), pineapple, refrigerator, microwave, blender, incubator, small strainer, one hard boiled egg white, knife, cutting board, water, several 250 m beakers, and two large bowls.
To begin this experiment 10 test tubes will be marked with wax pencils in the following fashion: Test tubes 1-4 in one color and test tubes 5-10 in another color.
Jell-O (R) gelatin was prepared according to the directions on the box and approximately 10 ml poured into four test tubes to set overnight. The next day, 1/3 of a fresh pineapple was cut into small chunk size pieces with a corresponding amount of water. Both items were placed in a blender and mixed.
After mixing, the pineapple was strained into a 250 m beaker. The juice was separated into equal amounts using another 250 m beaker. One of the beakers containing juice was microwaved for several minutes until the juice reached its boiling point, the other beaker remained at room temperature.
The hard boiled egg white was cut into 1/2 cm cubes. One egg white cube was placed in each of the remaining six test tubes (test tube stand used). 10 m of fresh pineapple juice was placed in test tubes #1, #3 and #5. 10 m of cooked pineapple juice was placed in test tubes #2, #4 and #6. The test tubes were covered. The test tubes were stored as follows: # 1 and #2 at room temperature in a designated area and #3 and #4 in the refrigerator, and #5 and #6 in the incubator set at 37 degrees Celsius.
On day three the test tubes were retrieved and changes were recorded.
Materials and Methods: 144 grams of red cabbage were weighed, placed in a blender with 144mL of dH20 and blended until liquefied. After being strained, the cabbage juice was poured into a 250 mL beaker.
Seven test tubes were numbered and each one was filled with 5 mL of a different substance. A vortex was used to thoroughly mix the solutions. Color changes and reactions were noted. After mixing, pH paper was used to test the pH of each solution, 5 mL of 3% H202 was then added to each of the test tubes and any changes and/or reactions were recorded.
Alone w/H202 Tube Contents Color pH Reactions? Color 1 dH20 Clear 5 /Weak Clear 2 cabbage juice Dark Purple 7 /Strong Lt. Purple 3 vinegar Clear 3 /Weak Clear 4 ammonia solution (1:10 dilution) Clear 11 /Weak Clear 5 dH20+ cabbage juice Purple 7 /Strong Lt. Purple 6 vinegar+ cabbage juice Red 3 /Moderate Lt. Red 7 ammonia solution + cabbage juice Green 11 /Moderate Lt. Green
Note that the cabbage juice and the cabbage juice mixed with dH20 both have a pH level of 7, even though the cabbage juice was also added to the other substances. The more basic solutions generated more of a reaction than the acidic solutions.
Enzymes are large proteins that speed up chemical reactions. Enzymes involved directly in this project are ptylain, bromelain, and catalase. "Enzymes are very selective in the reactions they catalyze. Their selectivity determines which chemical processes occur in a cell at any particular time."
PICTURE # 1 GOES HERE
The activity of an enzyme is effected by its environment, and for every enzyme there are conditions under which it is most effective. Temperature for instance, affects molecular motion, and an enzyme's optimal temperature produces the highest rate of contact between reactant molecules and the enzyme's active site. Enzymes have a higher activity rate in regards to temperature. This is commonly referred to as enzyme kinetics. (Glanze, 1992) Enzymes can become denatured when exposed to high temperatures for a period of time. The temperature and time required to denature will change from enzyme to enzyme.
The pH also influences the enzyme activity. For some enzymes the optimal pH is near neutrality, in the pH range of 6-8. In regards to pH, a basic substance will react more strongly than an acid. Yet not as much as a neutral substance.
The enzyme in saliva, ptylain, breaks down starches into sugar, maltose. Starch is not a single molecule but a mixture of amylose and amylopectin. The identifier for starch is iodine. The identifier for sugar is Benedict's reagent. These identifiers were used to show that saliva can break down starches in sugar.
In Part A, the effect of salivary amylase were tested, The hypothesis arrived at was "Amylase breaks starch down into sugar." This was proven by the reaction that took place in test tubes #3 and #5 when the Benedict's solution was added. In test tube #3, the color changes indicated a high amount of sugar, which seems reasonable considering that it contained maltose which is a sugar. This same reaction took place in test tube #5. However, what happened to the starch in test tube #5? Was it reacting also? We are led to believe that the enzyme in the saliva broke the starch down into sugar because no reaction occurred in test tube #2 or #4 that only contained the starch and saliva respectively.
Part B, the effect of temperature on enzyme activity of Bromelain were tested. The question was asked, "What is the effect of temperature on the action of Bromelain?" Our hypothesis was, "The cooler the temperature the slower the Bromelain reacts." This was seen in the reaction of the various test tubes. In the test tubes that were refrigerated, the Bromelain even after a week, had reacted very little. The cooked juice had no reaction but the fresh juice had a slight reaction. The room temperature test tube that contained fresh juice reacted more and the egg was starting to break up. However, the cooked juice had no reaction even though the temperature was warmer. The test tube that had the fresh juice and was in the incubator dissolved the egg after a week had passed. The cooked juice still had a chunk of egg left in it. This proves the hypothesis that the cooler the temperature, the slower the Bromelain reacts. The temperature hinders the activity. However, we could even explore this more and state that the result of too much heat on the enzyme could eventually denature it. This is proven by the results of the cooked juice versus the fresh juice. The cooked juice had no to very little reactions regardless of the temperature. This assures us that the fresh juice still had the Bromelain intact, but the cooked juice had the Bromelain denatured due to the heating and destroying of it. We could explore this more by varying temperature to see the results and trying to find if there is an optimum temperature when the Bromelain would react the most.
Part C, the effect of pH on catalase activity were tested. The hypothesis could either that "Catalase enzyme in purple cabbage works best at neutral pH," "Catalase enzyme activity works best at acidic pH," or "Catalase enzyme works best at basic pH." After testing, it was seen that the reactions were strongest at a pH of 7. This leads us to believe that the hypothesis that catalase activity works best at neutral pH is proven and the other two are disproved. We could test this farther by using different materials that contain the catalase enzyme and seeing if the color of the juice and the activity correlate: and then testing the pH and see if the hypothesis is further proven.
Amylase Research Society of Japan. 1995. Enzyme chemistry and molecular biology of amylases and related enzymes. Boca Raton: CRC Press.
Campbell, Neil A., Lawrence G. Mitchell, Jane B. Reese. 1994. Biology Concepts and Connections. Benjamin/Cummings Publishing Company inc. 39, 42, 76-77, 104 pp.
Fogarty, William M., 1983. Microbial enzymes and biotechnology. London; New York: Applied Sciences and Publishers. 382 pp.
Glanze, Anderson and Anderson, eds. 1992. The Mosby Medical Encyclopedia. New York:
Microsoft (R) Encarta. 1994. Structure and Function of an Enzyme.
Wong, Dominic W.S., 1995. Food Enzymes: Structure and Mechanism. Ohio Links Central Catalog. XVI. 390 pp.