The Effects on Enzymes
In this lab exercise, the study of enzyme catalase, we viewed the breakdown of hydrogen peroxide into water and oxygen. The purpose was to isolate catalase from starch and measure the rate of activity under different conditions. Changes in temperature and pH along with Substrate Concentration and Enzyme Concentration were the conditions tested in the experiment. Our class performed this experiment by breaking up into three mega-groups and the data presented in this report will reflect the data of each group. Throughout the experiment, each group encountered difficulties, which threw off the exact data that was collected. However, by working hard and forming realistic hypothesis, we were able to find the correct data.
The chemical reaction that is being studied is the hydrolysis of starch and the enzyme that is being studied is amylase. Amylase is found in the saliva. This experiment is looking at the various effects on the rate of the enzymatic hydrolysis of starch. Enzymes are biological catalysts that carry out thousands of chemical reactions that occur in living cells. Generally large proteins, enzymes are made up of several hundred amino acids. When an enzyme can no longer function at all, it is said to be denatured. There are several factors that contribute to the denaturing of the enzyme that also determine the enzyme's shape. These factors are very closely regulated in both living organisms and in laboratory environments. The temperature of the enzyme reaction or enzyme itself, as well as the enzyme’s pH, can affect the rate of activity in the function. The other factors include the Substrate Concentration and Enzyme Concentration. In this lab, we tested the enzyme reaction when adding more enzymes, the substrate, temperature, and the pH. Materials and Methods
Before the lab took place, we were given a spot/well plate, starch, amylase, iodine and water. Due to time, the class was broken up into groups to help expedite the lab. We started out by placing one drop of iodine into three separate wells of the spot/well plate. We then used a drop of starch into the first well and noted the color change. For the second well, we used a drop of amylase to mix with the iodine. For the third well, a drop of water was placed into the well and mixed with the iodine. Each well had a different color change, and each color change was recorded. The steps to complete Experiment 1, testing the effect of enzyme concentration on reaction time, was first by placing 5 ml of amylase into five test tubes. The dilution process was performed by placing 5mL of 2% Amylase into tube 1, which already contains 5mL of water, and noting the color of the mixture. Then 5mL of tube 1’s contents were placed into tube 2. This was repeated for tubes 3,4, and 5 along with the observation of color changes as each tube was diluted. However due to some problems along the way the lab results indicated that “there was no change.” With the help of our lab instructors Sam and Jing, they guided us along the way to help show the correct results which were: Tube 1 had a 1% concentration of Amylase, tube 2 had a 0.5% concentration of Amylase, tube 3 had a 0.25% concentration of Amylase, tube 4 had a 0.125% concentration of Amylase, and finally tube 5 had a 0.0625% concentration of Amylase. We then gathered more test tubes and added 1 mL of starch to each mix-test tube. Later we added one drop of iodine to each well in the spot plate and as soon as we dropped 1ml of starch, we began timing and recorded the color change. We continued the placement of one drop of the starch-amylase mixture into each well of the spot plate throughout the remainder of the experiment and each well was timed and recorded the same way. For experiment 2, we found the effect of substrate concentration on Amylase Activity. A spot plate was prepared by placing two drops of iodine into each well. 5mL of Amylase was placed into 5 separate test tubes. Then 5mL of starch was placed into test tube 1 and the dilution process, presented in experiment 1 was performed. The results from the starch concentration were: Tube 1 contained 0.5% starch concentration, whereas tube 2 contained 0.25% concentration, test tube 3 contained a 0.125% concentration, test tube 4 contained 0.0625% concentration, and test tube 5 contained a 0.03125% concentration. We then gathered additional test tubes and created mixture tubes, the same way constructed in experiment one. Once we placed one drop of mixture into the first iodine well, we recorded the color changes and any additional notes. We then placed one drop from mixture tube 1 into each well. We stopped the timer when the color of the mixture in the well changed from light to dark or vice versa. Then we created 4 additional mixture tubes each containing 5mL of 2% amylase and either 0.25% starch solution, 0.125% starch solution, 0.0625% starch solution, or 0.03125% starch solution. For experiment 3, the effect of temperature on fungal amylase activity, we began by dropping one drop of iodine in each well. We added 3mL of Amylase to a test tube and placed the tube into 80 degrees Celsius for 2 minutes. After 2 minutes we removed the test tube from the ice and added .5mL of 1% starch to the tube and immediately began a timer. We then prepared another spot plate with 1 drop of iodine in each well. We placed another test tube with 3mL of Amylase into a hot water bath set at 36 degrees Celsius. We removed the tube after two minutes and placed .5mL of 1% starch into the tube and immediately began the timer. I was not able to perform this experiment due to time, however we tested the temperature at other degrees such as: 22 degrees Celsius, and 4 degrees Celsius. For experiment 4, the effect of pH of the amylase activity, we were not able to perform this procedure step-by-step. However, from discussing the experiment in class and taking a hypothetical guess, I believe that the closer the pH is to 5, then the rate of reaction will be higher We were given five pre-determined buffers with the pH concentrations 3.0, 5.0, 7.0, 8.0, 10.0. We placed 5 drops of starch solution into 5 test tubes and the added 10 drops of the buffer into test tube 1. Test tube 1’s starch pH was 3.0, test tube 2 was 5.0, test tube 3 was 7.0, test tube 4 was 8.0, and test tube five was 10.0 pH. We performed the same procedure as we did with experiments one and two, where you take the mixture and add to another test tube and so forth. We noted the color changes and any other additional notes that we saw. We began timing as soon as the starch met the well, and timed until the reaction was over. We used this same process for the mix test tubes 2,3,4, and 5. Results
Experiment 1: Enzymes present
1| 2| 3| 4| 5|
0 seconds| 2 seconds| 5 seconds| 15 seconds| 4 minutes|
This chart shows the shows the length of time for the starch and concentration to react in seconds. If you increase the amount of enzymes, then the rate of reaction increases.
Experiment 2: Substrate
0.5%| 0.25%| 0.125%| 0.0625%| 0.03125%|
2seconds| 3 seconds| 5 seconds| 3 seconds| 4 seconds|
This chart describes the relationship between the amount of substrate that is present in a reaction versus the reaction time. If the substrate is lower, then the reaction rate will be lower.
Experiment 3: Temperature
80 ̊C| 36 ̊C| 22 ̊C| 4 ̊C|
2 minutes| 4 minutes| 4 minutes| 4 minutes|
This chart shows the reaction rate when the enzyme is exposed to certain temperatures. If the enzyme is exposed to higher temperature then the rate of reaction will be faster.
Experiment 4: pH
This chart is showing the time of the reaction under each pH. If the pH is close to 5, the biological pH of the fungus from which our amylase is derived, then the rate of reaction will be higher.
Throughout this lab, many procedures were issued and it took a lot of trial and error for us to complete this lab successfully. However throughout problem solving and hard work, we were able to use realistic hypothesis, charts, and tables to conclude that the data throughout the lab is correct. The results matched up with most of experiments, except for experiment 1, where we found no change until the help of our lab instructors. By stating a hypothesis at the beginning of an experiment, it allows you to set a goal on what to expect and then you begin to build around it. Although we did not have enough time to complete each experiment, we formed mega-groups to allow us to be able to obtain the right data for each experiment.
Jane B. Reece; Lisa A. Urry; Michael L. Cain; Steven A. Wasserman; Peter V. Minorsky; Robert B. Jackson. 2011. Campbell: Biology, Ninth Edition. Benjamin Cummings.