TitleEnzyme kinematics as a method to establish optimal pH and temperature levels for alpha amylase reactions.
...nzyme work optimally and at levels thereafter, the enzyme may decrease in its effectiveness. In an experiment involving the enzyme alpha amylase, the optimal levels of temperature and pH were calculated as the absorbance of individual I_2_KI solutions were measured at different time intervals in order to find reaction rates at different temperatures and pH levels (Vliet, 1993). Based on the fact that an increase in heat would make substrate molecules move faster and there by increase their chance to attach to enzymes, and the assumption that a higher acidity level (lower pH) will cause molecules to react as well, I hypothesized that experiments done at the highest temperature (70 degrees F) and the lowest pH level (4.0) would produce optimal reaction rates. Materials and Methods In this enzyme kinematics experiment I was able obtain a calculation of what temperature and pH levels provide the best environment for enzyme reactions. The temperature and pH experiments were done separately and independent of each other. Materials I used included a spectrophotometer (a device used to measure absorbance, the amount of light absorbed by a substance), and a heating bath used to set the reactant solution to different temperatures. My procedures utilized solutions of the alpha amylase enzyme, starch that acted as a substrate, and an iodine indicator, I_2_KI. In preparation for the experiment, I allowed the spectrophotometer to warm up for several minutes prior to its usage. The heating bath also had to be given ample time to reach the desired temperature for each different test. For the temperature experiments, a reaction flask was created by mixing 35 ml of starch solution provided by the lab instructor with 35 ml of distilled water. This reaction flask was prepared for each experiment and heated to various temperatures, 15, 30, 45, 55, 60, and 70 degrees Celsius. In the pH experiments, 35 ml of starch was mixed with buffer solutions of varying acidities, pH levels of 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5 respectively. Next, a blank was prepared in order to set the spectrophotometer to a zero absorbance level. This was done by adding 5 ml of distilled water to 0.1 ml of I_2_KI indicator, also provided by the instructor, to a blank cuvette. Additional cuvettes were prepared with 0.1 ml I_2_KI indicator for each time reading in the experiment. In order to begin the experiment, a reading at time 0 was measured by taking 5 ml of solution from the reaction flask (starch and water for temperature experiments; starch and buffer for pH experiments) and mixing it with the I_2_KI indicator. I placed the cuvette into the spectrophotometer, then read and recorded the absorbance measurement into the proper table. The lab instructor then placed 1 ml of alpha amylase solution into the reaction flask. The mixture was quickly swirled and the reaction began. Timing began immediately after the enzyme was placed into the solution For temperature experiments, additional absorbance measurements were taken each minute for the first 8 minutes, again at 10 minutes and finally at 20 minutes. In pH experiments, additional absorbance measurements were taken every other minute, ending at 20 minutes. At each of the aforementioned time intervals, five ml of solution was taken out of the reaction flask and placed into a cuvette containing I_2_KI indicator. I used a pipette in order to extract solution from the reaction flask and transfer it to the cuvettes. All resulting absorbance readings were recorded in their appropriate places in the time vs. absorbance charts. This experiment was performed using each of the temperatures and pH levels mentioned above so that an optimal pH and temperature for enzyme reactions could be established. In analyzing the data acquired from this experiment, the rates at which reactions occur for the alpha amylase enzyme at different temperature and pH levels was calculated. I did this by utilizing the lab manual's method. The equation Reaction Rate= (1/2 Delta Absorbance)/Time that Delta Absorbance Took was used to calculate Reaction Rates for the various reaction rate. In order to find the time that delta Absorbance took, I plotted an Absorbance v Time (min) graph for temperature and pH. Best fit lines of the points obtained from each temperature and pH experiment were drawn. delta Absorbance was used to find the corresponding time value for each respective temperature and pH level. Once reaction rates were determined, a final analysis of the data was obtained by plotting graphs of reaction rates vs temperature and reaction rates vs pH level. From these graphs, I was able to determine the optimal pH and temperature for alpha amylase enzyme reactions. Results After conducting the enzyme kinematics experiment to determine optimal temperature and pH for the alpha amylase enzyme, I found that a pH of 4.5 and a temperature of 70 degrees C are the optimal conditions for reactions involving this enzyme. Table 1 and Table 2, absorbance readings over time of reactions at different temperatures and pH levels, show the data necessa...