Enzymes and Temperature

Zaquia Austin Enzymes and Temperature Laboratory #6 3/13/2013 Purpose (Objectives): The purpose of this experiment was for students to be able to understand the enzyme-facilitated reaction and explain how enzyme activity can be affected by environment conditions. Abstract: This experiment mainly delt with enzymes and the reaction that enzymes have with different solutions in various temperatures. Three different exercises were done. The first exercise had to do with enzymes and temperature. During this exercise three different test tubes were used for three different temperates. The first test tube which involved 0 ?C water bath had a total conversion of starch to sugar in the first 15 minutes, and the second test tube which involved 37? C water bath had a total conversion of starch to sugar after the first five minutes. While the third test tube took a little longer. The second exercise had to do with enzymes and substrate concentration. This exercise took longer for the all of the starch to be removed from the test tubes, and it involved 37? C water bath for all five test tubes. The last exercise had to do with enzymes and pH. This exercise involved four test tubes all put into a water bath of 37? C.The fourth test tube had a total conversion of starch to sugar in the first five minutes while the other three took a little longer to convert. Experiment & Observation: First the water baths and the 1% Alpha-amylase was prepared. Three different water baths were prepared. One being set at 0? C, the next being set at 37? C and the last one being set at 100? C. Next a 96-well microplate was marked with times and numbers based on the test tubes number. Then using a pipet one drop of iodine was placed into each well (1,2,3) up to total of 30 minutes. Then the test tubes were marked 1cm and 6cm from the bottom.Afterward, 1cm of alpha-amylase was pipetted into each test tube. Then, test tube 1 was incubated at 0? C, test tube 2 was incubated at 37? C, and test tube 3 w as incubated at 100? C for five minutes. After that 1% starch solution was added to the 6cm mark. Next it was put back into its assigned water bath for another 5 minutes. Then two drops of solution was put into its corresponding number on the microplate in the 5 minute row. The color was immediately recorded. The steps were repeated for a interval of 30 minutes or until each well had an amber color in it.Test tube 3 did not change within the 30 minute interval so it was placed into the 37? C water bath for another 30 minutes. Then one drop of solution and one drop of iodine was added into the well. There was still no color change, so another 1cm of alpha-amylase was added to it and was incubated for another 30 minutes, the color became amber. Table 1. Effect of Temperature on Amylase Enzyme Conversion of Starch to Sugar |Time/Minutes |Test Tube 1 / 0? C |Test Tube 2 / 37? C |Test Tube 3 / 100?C | |5 |+ |- |++ | |10 |+ | |++ | |15 |- | |++ | |20 | | |++ | |25 | | |++ | |30 | | |++ | | | | | | |60 | | |++ | | | | | | |90 | | |- | The color of iodine that indicated that starch was still present in the test tube was the Black/ Blue-Black color. The color that indicated that the starch was gone was the Amber color. Amylase breaks up the starch which makes it disappear, it does not react with iodine anymore. The variable in this experiment can be an independent variable. The change in the experiment is the starch to sugar. The temperature that is optimal is 37? C.The temperature that facilitated first was the 37? C, then it was 0? C. No 37? C was chosen as the mid-range temperature because that degrees in Fahrenheit is 98. 6? F. It was important to immediately observe the color because after a while it changes colors again. If you would have just added the iodine in test tubes that would have ruined the entire experiment because there wouldn't be anything to test if it didn't work the first time. Once the test tube was incubated at 37? c nothing happen. The starch d id not disappear, maybe because there wasn't enough amylase. After 1cm of alpha-amylase was added and incubated at 37? C the starch eventually disappeared.This probably didn't happen the first time because the more amylase there is the better chance of it breaking up the starch. [pic] My initial hypothesis was that the more starch solution there is the longer it will take for it to break down amylase. My hypothesis was actually supported, there was more starch then amylase, when there needed to be more amylase then starch. I learned that amylase breaks up starch, and that its an digestive enzyme. Exercise 2: First a water bath was set at 37? C. Then the microplate was label this time five test tubes were used. One drop of iodine was placed into each well (1,2,3,4,5) up to 30 minutes. Then 1/2cm was marked from the bottom of the test tube. Next for each tube an additional cm was added.For test tube one 2cm above the bottom, test tube two 3cm above the bottom, test tube three 4cm abov e the bottom, test tube four 5cm above the bottom, test tube five 6cm above the bottom. Then 1/2 alpha-amylase was added to the 1/2 cm mark on the test tube. Afterwards the test tubes were placed into the water bath for five minutes. Next 1% starch solution was added to the next cm mark on the test tubes. The test tubes were put back into the water bath for another five minutes. Then two drops of the solution was added to each of the corresponding wells. This was done for all five test tubes. Immediately record the colors. The steps were completed for an interval of 30 minutes.The tubes that had not changed color within the 30 minute interval was put back into the water bath for another 30 minutes. Two drops of solution and one drop of iodine was put into the well and the amber color was recorded. Table 2: The Effect of Concentration on Amylase Enzyme Conversion of Starch to Sugar |Time/Minutes |Tube 1 |Tube 2 |Tube 3 |Tube 4 |Tube 5 | |Concentration of Amylase: |0. 5cm/2cm |0. 5cm/ 3cm |0. 5cm/4cm |0. 5cm/5cm |0. 5cm/6cm | |Per test tube |25% |17% |12. % |10% |8% | |5 |++ |++ |++ |++ |++ | |10 |++ |++ |++ |++ |++ | |15 |++ |++ |++ |++ |++ | |20 |++ |++ |++ |++ |++ | |25 |++ |- |++ |- |++ | |30 |++ | |++ | |++ | | | | |++ | |++ | |60 |++ | |- | |- | The variable in this exercise the substrate concentrations. Test tube 3 This experiment could be improved if each test tube had its own water bath.My hypothesis was that test tube five would convert to sugar first. The reason I hypothesized this was because test tube five had the most starch. In this exercise I learned that enzymes can be used over and over again to facilitate the conversion of substances before they are denatured. Some practical applications can be Food and Beverages D. Another way this experiment could be done is by using different temperatures of water baths. Exercise 3: First a water bath was set at 37? C. The microplate was labeled, this time only four test tubes were used. Each test tube was m arked 1cm, 2cm, and 4cm from the bottom. Next one drop of iodine was added to the weel (1,2,3,4) and u to a 30 minute interval.Then a different pH buffer was added to each test tube at the 1cm mark. For test tube one pH 3. 5 buffer was added, for test tube two pH 5 buffer was added, for test tube three pH 6. 8 was added, and for test tube four pH 11. 5 was added. Afterwards, 2cm of alpha-amylase was added. Then the test tubes was placed into the water bath to be incubated. After five minutes starch solution was added to the remaining 4cm mark, then placed back into the water bath. After five minutes two drops of solution was put into each corresponding well. Immediately record color. The steps were completed for an interval of 30 minutes. The fourth test tube showed color immediately, but the other three test tubes did not.Table 3: The Effect of pH on Amylase Enzyme Conversion of Starch to Sugar |Time/ Minute |Test Tube 1 |Test Tube 2 |Test Tube 3 |Test Tube 4 | |pH |3. 5 |5. 0 |6. 8 |11. 5 | |5 |++ |++ |++ |- | |10 |++ |++ |++ | | |15 |++ |++ |++ | | |20 |++ ++ |++ | | |25 |++ |++ |++ | | |30 |++ |+ |+ | | | | | | | | |60 |+ |+ |+ | | The variable in this exercise is the pH. Only the fourth test tube converted starch to sugar, I think this happend because it had an higher amount of buffer. Yes the first three test tube did not exhibit a change in color.. There pH was much lower then the last one. I hypothesized that the test tube with the pH buffer would convert to sugar first. My hypothesis was refute. Conclusion: Temperature, and the times of incubation.You can get different substrates if you change the incubation times. In this laboratory I learned how to recognize enzyme-facilitate reactions, and how to tell when starch is converted into sugar. Some practical applications could be yeast, detergent, leather and bioethanol. Discussion/Error Analysis/Conclusion: The first part of this laboratory was based upon testing the alpha-amylase enzyme activity on sta rch under three temperature environments, 0? C, 37? C, and 100? C. The next part was to demonstrate the effects of substrate concentration on enzyme reactivity. The last exercise was based upon testing how alpha-amylase functions at four different pH levels (3. ,5,6. 8, and 11. 5). A few errors that occurred was one, the changing of the water bath temperatures. If the bath stayed at a constant temperature then it probably would have made a difference to some of the tubes. Another laboratory error could have been that there were only a select few of pipets. If there were pipets for each exercise that could have made a difference even though the pipets were cleaned after each experiment, it still would have made a different if it was a clean dry pipet. Another laboratory error could have been the incubating times. These errors could be minimized in the future if there were a few arrangements before hand.