Words: 1982
Abstract Enzymes are proteins that lower the activation energy needed for chemical reactions. The two main environmental factors that can affect the enzyme’s activity are temperature and pH, and each enzyme works best at a particular temperature and pH. The purpose of this enzyme kinetic experiment was to observe the effect of temperature and pH on the reaction of barley alpha-amylase enzyme with starch substrate and establish the optimum temperature and pH for this reaction. The optimum temperature and pH for the reaction of alpha-amylase and starch was predicted to be a temperature of 50??C and a pH of 5.
The optimum temperature and pH for the reaction was determined by monitoring the reaction rate of alpha-amylase at different temperatures and pH’s by means of using a spectrophotometer to measure the disappearance (in absorbance) of the substrate starch. As a result, the absorbance of the substrate starch decreased at different rates for each temperature and pH as time continued to increase. The results showed that the reaction rate with the enzyme is highest when it reaches a temperature of 50??C and has a pH of 5. Therefore, a farmer should grow barley seeds in soil that has a temperature of 50??C and a pH of 5.
Introduction The purpose of this enzyme kinetic experiment was to observe the effect of temperature and pH on the reaction of barley alpha-amylase enzyme with starch substrate and establish the optimum temperature and pH for this reaction. Enzymes are essential to all living organisms. An enzyme is a globular protein that acts as a catalyst, a chemical agent that speeds up reactions without being consumed by the reaction. They lower the activation energy needed for chemical reactions, allowing the reactant molecules to absorb enough energy to reach the transition state (Vliet 2008).
When an enzyme binds to a substrate, it forms an enzyme-substrate complex. The substrate-enzyme complex allows the enzyme to react with the substrate in order to form the product (Vliet 2008). The enzyme is neither changed nor consumed during this process. Without the presence of enzymes, many of the chemical reactions in living cells would occur too slowly (Johnson and Raven 1998). Specifically, the alpha-amylase enzyme is a type of enzyme found in barley plant seeds (Vliet 2008). Alpha-amylase catalyzes the breakdown of starch into glucose.
Starch is a polysaccharide made up of glucose molecules (Johnson and Raven 1998). Starch is the substrate of alpha-amylase enzyme. Because glucose is a major cellular fuel, starch represents stored energy. According to Johnson and Raven (1998), the sugar can later be withdrawn from this carbohydrate “bank” by hydrolysis. Organisms have enzymes (such as alpha-amylase) that can hydrolyze plant starch (Vliet 2008). In addition, the organisms use the glucose molecules from starch as a nutrient for their cells.
The two main environmental factors that can affect the enzyme’s activity are temperature and pH. Each enzyme works best at a specific temperature and pH, known as the optimum. If the temperature or pH of the reaction is higher or lower than that of the optimum, the enzyme can break down (denaturation), which causes the active site to no longer fit the substrate molecule (Campbell and Reece 2008). The enzyme, alpha-amylase, is harvested commercially from germinating barley seeds (Vliet 2008). Amylase hydrolyzes starch molecules to glucose, which is then taken up by the developing seedling.
The reaction between alpha-amylase and starch was measured at specific temperatures and pH’s by using a spectrometer, an instrument that electronically measures the amount of a substance from its ability to absorb radiant energy (Vliet 2008). The alpha-amylase enzyme breaks down the starch within the solution and lowers the absorbance reading as time passes (Vliet 2008). The reaction rate of the alpha-amylase and starch reaction can be calculated using this data. In addition, the optimum temperature and pH for this reaction can be obtained.
The optimum temperature and pH for the reaction of alpha-amylase and starch was predicted to be a temperature of 50??C and a pH of 5. Materials and Methods There were two parts to this experiment: the testing of the affect of temperature on the absorbance of the starch solution and the testing of the affect of pH on the absorbance of the starch solution. I used a spectrophotometer to measure the absorbance. I turned on the spectrophotometer 15 minutes prior to the experiment and set it at a wavelength of 560 nm. For the temperature experiment, I made a blank by adding 5 ml of distilled water and 0. ml of iodine and potassium iodide (I2KI) indicator to a cuvette. I prepared a total of six blanks for each of the different temperatures that were tested. I used this blank cuvette to set the spectrophotometer to zero absorbance. For each of the six temperature experiments, I filled 11 more cuvettes with 0. 1 ml of I2KI. Then, I prepared six different water baths at the following temperatures: 15??C, 35??C, 45??C, 55??C, 65??C, and 70??C. I prepared six reaction flasks, and each of them contained 35 ml of starch solution and 35 ml of distilled water.
I placed one reaction flask in each of the six water baths. I allowed these reaction flasks to reach the appropriate temperature before I added the enzyme. Before I added the alpha-amylase enzyme, I took the time 0 reading by transferring 5 ml of the solution from each of the reaction flasks to a cuvette that contained I2KI indicator and mixed this solution. I read the absorbance of the starch-iodine complex on the spectrophotometer and recorded the absorbance reading. I added 1 ml of alpha-amylase solution to my six reaction flasks and mixed thoroughly. I immediately began timing the experiment.
For each subsequent timed reading, I transferred 5 ml of the solution from my reaction flasks to a cuvette that contained I2KI indicator and mixed. I read the absorbance of the starch-iodine complex on the spectrophotometer and recorded for each timed reading. I blanked the spectrophotometer between each reading. I did this for a total of 20 minutes. For the pH experiment, I made a blank by adding 5 ml of distilled water and 0. 1 ml of iodine and potassium iodide (I2KI) indicator to a cuvette. I prepared a total of six blanks for each of the different pHs that were tested.
I used this blank cuvette to set the spectrophotometer to zero absorbance. For each of the six pH experiments, I filled 11 more cuvettes with 0. 1 ml of I2KI. I prepared six different starch solutions by mixing 35 ml of stock starch solution with 35 ml of water buffered at the following pH’s: 4. 0, 4. 5, 5. 0, 5. 5, 6. 0, and 6. 5. Before I added the alpha-amylase enzyme, I took the time 0 reading by transferring 5 ml of the solution from each of the reaction flasks to a cuvette that contained I2KI indicator and mixed this solution.
I read the absorbance of the starch-iodine complex on the spectrophotometer and recorded the absorbance reading. I added 1 ml of alpha-amylase solution to my six reaction flasks and mixed thoroughly. I immediately began timing the experiment. For every two minutes (after I added the alpha-amylase), I transferred 5 ml of the solution into each of the 11 cuvettes for each of the buffered solutions. I read the absorbance of the starch-iodine complex on the spectrophotometer and recorded for each timed reading. I blank the spectrophotometer between each reading. I did this for a total of 20 minutes.
I recorded the temperature and pH absorbance readings in two separate tables. I constructed two graphs, one for the temperature absorbance readings and one for the pH absorbance readings. I also calculated the reaction rate once I plotted the raw data and drew the best fit curves for each set of data. I calculated the reaction rate by taking half the change in absorbance and dividing it by the temperature at which half the change in absorbance was reached (Vliet 2008). Then, I drew another two graphs, one for the reaction rate and specific temperatures and one for the reaction rate and specific pH’s.
Results The absorbance readings over time of the reactions at different temperatures and pHs can be found in Tables 1 and 2. I made a graph of absorbance verse time for the specific temperatures which can be found in Fig. 1. This graph shows that as time increases, the absorbance of the substrate starch decreases at different rates for each temperature. I made a graph of absorbance verse time for the pH which can be found in Fig. 2. In this graph, as the time increases, the absorbance of the substrate starch decreases at different rates for each pH.
In Tables 3 and 4, I recorded the calculations of the reaction rates that I obtained from each curve that I drew in Figs. 1 and 2. Figure 3 shows the reaction rate of alpha-enzyme with starch at different temperatures and indicates the optimum temperature. This graph shows that the reaction rate with the enzyme is highest when it reaches a temperature of 50??C. Figure 4 shows the reaction rate of alpha-enzyme with starch at different pH’s and shows the optimum pH is 5. This indicates that the reaction rate with the enzyme is highest when the pH is 5. Discussion
All of the results supported my prediction that the optimum temperature and pH for the reaction of alpha-amylase and starch should be 50??C and a pH of 5. The reaction rate of the starch solution with alpha-amylase was highest at a temperature of 50??C and a pH of 5. Thus, the collisions between the alpha-amylase enzymes and substrate starch most frequently occurred when the temperature of the solution reached 50??C. With a pH of 5, the solution was not too basic or too acidic in order for the enzyme to remain intact and react at a high rate with the starch substrate molecules.
In Fig. 1, the lines for the temperatures of 45??C and 55??C had the steepest decreasing slopes, which means that the substrate starch was absorbed the fastest during a certain amount of time when the solution was at these temperatures. In Fig. 2, the pH 5 line had the steepest decreasing slope, which means the substrate starch was absorbed the fastest when the solution had a pH of 5. The reaction of alpha-amylase enzyme with starch favored the environment with a 50??C temperature and a pH of 5.
With a temperature below or above 50??C and a pH below or above 5, the reaction will not occur as quickly because the enzyme can undergo denaturation. These experimental results can be useful to farmers that grow barley. The developing barley plants embryo synthesizes and releases alpha-amylase into the endosperm, the nutritive tissue within the seed (Vliet 2008). Amylase hydrolyzes starch molecules to glucose, which is then taken up by the developing seedling (Vliet 2008). Therefore, a farmer should grow barley seeds in soil that has a temperature of 50??C and a pH of 5.
In this favorable environment, the reaction between amylase and starch will occur faster, and this will increase the rate at which starch molecules break down into glucose molecules. Overall, the germination of seeds can occur quicker, and this can allow a farmer to grow barley faster. In addition, the experimental results lacked repetition. I only tested the reaction rate of alpha-amylase with starch at specific pHs and temperatures once. I should also test the reaction rate of alpha-amylase with starch at even smaller and more specific temperatures.
Instead of using increments of 15, 35, 45, 55, 65, and 70??C, I should change the increments to 47, 48, 49, 50, 51, 52 and 53??C. This can allow me to obtain a very specific temperature at which the enzyme favors the most. Literature Cited Campbell, N. A. and J. B. Reece. 2008. Biology, 8th ed. Benjamin Cummings Publishing Co. , San Francisco, CA. Johnson, G. B. and P. H. Raven. 1998. Biology: Principles and Explorations. Holt, Rinehart, and Winston Co. , Orlando, FL. Vliet, K. A. (ed. ). 2008. A Lab Manual for Integrated Principles of Biology: Part One BSC2010L. Pearson Custom Publishing, Boston, MA.