Effects Of Temperature, Ph, And Gelatain On Bromelain Essay, Research Paper

Effects of Temperature, pH, and Gelatin on BromelainIntroduction

Bromelain is an organic catalyst that speeds up the rate of chemical reactions. Bromelain is used in this experiment to catalyze the break down of proteins and is called a proteolytic enzyme. Enzymes enable reactions to occur at a lower temperature and energy levels. This allows many reactions to occur inside living organisms that would otherwise have been impossible to take place. This enzyme is found in fruits, leaves, and stems of many species of pineapple. Pineapple is a tropical fruit native to northern South America. It is referred to as a multiple fruit because it consists of enlarged ovaries of many flowers more or less grown together into one mass. Ananas comosus is the type of pineapple used in this experiment. The bromelain is found in the pineapple attached itself to the substrate and broke it apart. The time required to complete the disassociation of the substrate was very important to the results of the lab, therefore time keeping must be precise in the experimentation. The effects of bromelain was compared under different temperatures and pH levels, the effects of bromelain were also observed in gelatin, there were three experiments performed and three hypothesis were formed. Temperature experiment: the higher the temperature the faster the reaction will occur to a point. The temperature will become to hot around 60 degrees Celsius and the enzyme will under go denaturation. Proteins are structurally complex and are very diverse. They have a high molecular weight and consist of amino acids liked by peptide bonds. Proteins are divided into three levels of structure: primary, secondary, and tertiary. Although some proteins have a fourth level which is the quaternary. Gelatin Experiment: The fresh pineapple juice will not solidify in gelatin. The hot juice and the control will solidify in the gelatin mixture.pH Experiment: The hypothesis for this experiment is that only a few numbers of pH will cause a reaction within a narrow range. pH levels at the extremes will cause the enzyme to under go denaturation. (Unfolding of the enzyme, loosing its ability to ac as an enzyme)Materials and Methods In the first experiment, effect of temperature on rate of enzyme activity, two test tubes were acquired for each temperature difference: 20 degrees C, 30 degrees C, 40 degrees C, 50 degrees C, and 70 degrees C. Each test tube was then cleaned with soap and water and rinsed out. The first five test tubes were then filled with five ml of fresh pineapple juice from the dispenser and labeled. The second five test tubes were then filled with five ml of a pH 3.5 buffer. The two test tubes labeled 70 degrees C and 70 degree C buffer and then placed in a test tube rack in an electronic water bath. Next, 20, 30, 40, and 50 degrees C water baths were made and the other four sets of test tubes were put into them. The bath temperatures were maintained throughout the experiment. All the test tubes sat in their respective water bath for five minutes to equilibrate. Next, filmstrips were placed in each of the test tubes and time began to be recorded. Every ten minutes the film was vigorously agitated for five seconds in the tubes in the 70 degrees C bath. In the 20 degrees C bath every five minutes the film was vigorously agitated for five seconds and checked for clearing. In the 30, 40, and 50 degrees C baths filmstrips were vigorously agitated every one minute and checked for clearing. When each of the test tubes cleared off their respective filmstrip then the time was recorded on the table as to how long it took. The fastest reaction happened in the 50 degrees C tube with a time of three minutes. Then the 40 degrees C tube was next to react at five minutes. The 30 degrees C tube was third to react at a time of 11 minutes. After twenty the test tube in the 20 degrees C reacted. The tube in the 70 degrees C never reacted. After the experiment was complete, the contents of the test tubes and strips of film were dumped into a bucket. The test tubes were then washed and dried out.

In the second experiment, effect of bromelain on gelation of gelatin, four clean test tubes were attained. The first test tube was labeled fresh juice and filled with one ml of fresh pineapple juice using a Pasteur pipette. The second test tube was labeled hot juice and then filled with one ml of the pineapple juice heated to 70 degrees C using a Pasteur pipette. The third test tube was labeled canned juice and then filled with one ml of canned pineapple juice using a Pasteur pipette. A fourth test tube was labeled control and filled with the pipette to one ml with water and dispensed into the test tube. Next, one ml of JELL-O was added to all four test tubes using a Pasteur pipette and the contents of the all four test tubes were mixed by placing a piece of wax paper over each tube and shaking it. Then all four test tubes were placed in a beaker of ice for five minutes. After five minutes in the ice, all four test tubes were removed and tilted slightly. The consistency of the gelatin in each test tube was observed and recorded in Table 2. The unused ice was then returned into the cooler. The test tubes were then emptied and washed out. The results of this experiment were that three test tubes turned to gel and one was still a liquid. The only one that didn t gel was the test tube which contained fresh pineapple juice.In the third experiment, effect of pH on rate of enzyme activity, ten clean test tubes were attained and labeled one through ten. Then they were filled with the following:Tube 1: 5ml of pineapple juice and 2ml of 1M HCltube 2: 5ml of water and 2ml of 1M HCL Tube 3: 5ml of pineapple juice Tube 4: 5ml of water and 2ml of pH 3.5 buffer Tube 5: 5ml of pineapple juice and 2ml of pH 5 buffer Tube 6: 5ml of water and 2ml of pH 5 buffer Tube 7: 5ml of pineapple juice and 2ml of pH 7 buffer Tube 8: 5ml of water and 2ml of pH 7 buffer Tume 9: 5ml of pineapple juice and 2ml of 1M NaOH Tube 10: 5ml of water and 2ml of 1M NaOH Each test tube was quickly inverted with a piece of wax paper covering the oppening to mix the solution. The solutions were then tested for pH levels using pH paper. These levels were recorded on the data chart. The ten test tubes were placed in 45 degrees celsius water bath. It important that the temperature remain constant. After five minutes, allowing the temperature inside the test tube to reach 45 degrees, a film strip was placed into each test tube. Every minute the strip were shaken vigorously for five seconds and cheched for clearing. This was done unyill the strips had reacted. Tubes 1,3,5,7, and 9 were the experimental and tubes 2,4,6,8, and 10 were the corresponding control tubes. As soon as the experimental tubes reacted the control group were considered done. The observations were recorded on the table 3. results In the first experiment the tubes containing juice reacted in 20 minutes at 20 degrees celsius, in 11 minutes at 30 degrees celsius, in 5 minutes at 40 degrees celsius, in 3 minutes at 50 degrees celsius, and did not react at 70 degrees celcius. The buffers at each temperature did not react. In the second experiment the heated juice in the gelatin and Discussion The first experiment supported the first hypothesis that bromelain activity will increase as temperature increases. Also the fact that there is a temperature limit, which causes the bromelain activity to not work, was learned. The temperature limit was 70 degrees C in our experiment. As the temperature was raised in ten degrees C increments from 20 degrees C to 50 degrees C the reaction time of the juice substance steadily decreased. The reaction rate of the juice test tubes increased because of the increase in kinetic energy due to the increase in temperature. As the temperature increased, enzymes and substrates moved faster and collided more often, causing a quicker reaction rate. The buffer test tubes showed no reaction because they lacked and enzyme to catalyze the breakdown. When the temperature was raised from 50 degrees C to 70 degrees C there was no reaction in the juice substance, signifying that somewhere around 50 degrees C is the optimum temperature for bromelain activity to take place. This appears to be supported because the highest reaction rate was at 50 degrees C and the lower temperatures had slower reaction rates. See graph Reaction Rate vs. Time The juice test tube in the 70 degrees C water bath showed no reaction because the water was too hot. Denaturation occurred because the high temperature caused the proteins to undergo the unfolding of the peptide chains, which make up an enzyme. The unfolding of the peptide chains caused the enzyme to become disordered resulting in no reaction. Pineapple pickers wear protective gloves when harvesting pineapples to prevent coming in contact with the bromelain enzymes, which catalyze the breakdown of proteins into their amino acid building blocks through a hydrolysis reaction. This would irritate the skin and could cause toughening and possibly decay of the skin. The second experiment supported the second hypothesis that heated juice and gelatin, canned juice and gelatin, and pure gelatin would solidify while fresh juice and gelatin would not solidify.

The heated juice and gelatin solution solidified because the bromelain enzyme of the pineapple juice had been denaturated in the other forms: heated juice, canned juice. The bromelain couldn t function as an enzyme because the high temperature had neutralized it. Therefore, gelation occurred when the test tube was placed in the ice because the gelatin was not catalyzed. The canned juice and gelatin solution solidified because the canned juice had been exposed to high temperatures during pasteurization and the bromelain had been denatured. Therefore, if the bromelain had been denatured it couldn t catalyze the gelatin and the JELL-O could solidify. The solution of pure gelatin solidified because there was no enzyme in the solution. If there is not an enzyme, catalysis can t take place and gelation will occur. Adding canned pineapple to JELL-O would be better than adding fresh pineapple because the bromelain in canned pineapple has been denatured through pasteurization and would allow the JELL-O to solidify where as fresh pineapple couldn t be used because the bromelain would catalyze the gelatin and the JELL-O couldn t solidify. If the pH is an extreme high or low it will cause no reaction. While the more neutral the pH is, the higher the rate of activity will be. Proteins are active in only a narrow range of pH on either side of seven. In a very acidic or basic solution the molecular shape of an enzyme is permanently changed and the enzyme is inactive. The optimum pH for maximum bromelain activity would be a pH of five because this was the fastest reaction time of any pH in the experiment. See Graph of pH vs. Reaction Time. The bromelain enzyme doesn t digest proteins in the stomach because it has a pH of 2 and the stomach pH is 1.5. Bromelain activity is optimal at a pH of 5, therefore the enzyme would quickly be denatured in the stomachs acidity, changing the molecular structure of the bromelain.