This experiment consisted of setting up a control group of starch in various temperature and then placing both fungal amylases and bacterial amylases in a mixture of starch and placing the solution of amylase and starch in various temperatures of water. After a certain amount of time- different amount of time needs to be used in order to have reliable results- iodine is added in a well on spot plates, then two drops of the mixture of amylase-starch is added from each temperature used, by adding iodine into the plates the mixture will show how much starch was hydrolyzed, this is used to calculate the amount of enzymatic activity each mixture had at the different set temperatures and at the different times the solution was extracted from.
The experiment was designed to find the optimal temperature in which both fungal amylase and bacterial amylase could function appropriately to produce an enzymatic reaction to a certain solution. The results concluded that bacterial amylase had an optimal temperature of around 60°C to 65°C. The fungal amylase on the other hand had an optimal temperature of around 40°C. These results show that the bacterial amylases have a higher optimal temperature than fungal amylases, meaning that bacterial amylases can react at higher temperature environments then the fungal amylases, that had a cooler optimal temperature that would denature at those high temperature conditions.
Before doing research on amylases, an explanation of enzymes is needed to fully understand each part of the experiment. An enzyme is a protein that helps speed up a chemical reaction (Raven, 2011). An enzyme is a catalyst, which is made by living cells; usually enzymes assist in developing the metabolic process of cells (Underkofler et al, 1958). The way in which enzymes speed up the chemical reaction is by lowering the activation energy, which the energy needed to start a reaction (Alberte et al, 2012). The way in which the chemical reactions occur is by having the substrates or the reactants bind to an active site of the enzyme- pockets of clefts (Raven, 2011), and form an enzyme-substrate complex, which is the form the to make by binding, this helps the substrate chemically bond and start the chain reactions needed in order to form the product or the end result (Alberte et al, 2012).
The experiment shows an enzyme catabolizing starch molecules, representing the enzymatic activity of the amylase. When starch is catabolized it means that the reaction occurred by breaking down the starch molecules to maltose to produce energy from them (Raven, 2011). As well, it shows the starch going through hydrolysis, which is a reaction that breaks a bond by adding water to the bond (Raven, 2011). For an enzyme to properly function there are certain factors that it cannot be affected by, including: pH, substrate concentration, salt concentration, temperature etc. (Alberte et al, 2012). Enzymes are not only used in developing the metabolic process but also in daily life use. Amylase is one enzyme that is used often in daily life, due to it being produced by the body to break down starch (Alberte et al, 2012). Amylase has mostly been found in microorganisms such as bacteria and fungal which the experiment focused on, although some amylases have been found in both plants and animals (Pandey et al, 2000).
Amylases as mentioned before have many different applications for daily life such as: in the use of food, pharmaceutical, textile etc. (Underkofler et al, 1958). The first use of an enzyme was in industries using fungal amylases as a pharmaceutical benefit to treat for digestive disorders in 1894 (Pandey et al, 2000) the preparation of the enzyme treatment was used by wheat bran koji culture adding the fungal amylase to it (Aiyer, 2005). In the food industry the use of amylases was used to convert starch into sugar and syrups (Aiyer, 2005). There has also been the use of fungal amylases in baking by enhancing the activity of flour with the fungal amylases (Underkofler et al, 1958). Bacterial amylases operate at higher temperatures than do fungal amylases. Fungal amylases react rapidly at lower temperatures; fungal amylases are used as an agent for alcohol fermentation for grain (Underkofler et al, 1958).
Fungal amylases is said to be denatured – change shape (Alberte et al, 2012), at high temperatures above 60° C and bacterial amylases on the other hand are stable and show little denaturing at temperatures up to 85°C 3 The question answered by the experiment is if the temperature is not within the range of the enzymes (fungal and bacterial amylase) optimal temperature (higher temperature) then will the enzymes denature and if the enzymes are placed in lower temperature from optimal the activity then will it slow down enough to stop all reaction, meaning each enzyme will not be operating efficiently. Knowing about a bacterial amylases and fungal amylases optimal temperatures are important for knowing which food products and industrial products it can be used on to conserve the product because then the producer knows about which products it can be incorporated into depending on the temperature it is manufactured at.
Initial Setup: Label each axis of the spot plates, across the top of the spot plates write each individual temperature (0° C, 40° C, 60° C, 95° C) and on the side write the time in minutes, starting with 0 in intervals of 2 until 10, as well distinguish the two different groups of amylase being used. Make sure that the setup looks like figure 1. Obtain four test tubes and label each with the different temperatures, enzyme source (B for Bacterial and F for Fungal) and a group number, total of 8 of labeled test tubes. Obtain another four test tubes and label those with a different temperature, enzyme source (B or F), group number and the letter S for starch solution, total of 8 of these labeled test tubes. Add 5 mL of 1.5 % starch solution into each of the test tubes labeled S. Experiment: Add 1 mL of Amylase into each of test tube that does not contain starch.
Make sure to conduct the experiment with one amylase type at a time. Place the four test tubes containing starch and the four test tubes containing amylase into the corresponding temperature baths. Allow all tubes to stand for 5 minutes in the respective temperatures. In the initial setup with the spot plates, add two to three drops of iodine into each well at the 0 minute row. At the end of the 5 minutes process, without removing the tubes from the water baths, add a few drops of the starch solution from each temperature treatment to the first row of the spot plate to time 0 minutes to each temperature. Make sure to use a separate transfer pipette for each temperature treatment. Label each of transfer pipettes with the correct temperature so that it can be reused for each time interval. Within each temperature, pour the rest of the starch into the tube containing the amylase. Set the timer for two minutes at the moment the amylase is added.
Then once again add two or three drops of iodine to each well at the 2-minute row. This will be repeated before adding each of starch amylase mixture into the spot plates. After 2 minutes, use the correct transfer pipette for each temperature to remove a few drops of the starch amylase mixture from each tube. Place two to three drops of the mixture in the second row on the spot plate under the corresponding temperature. Record the color change observed. After each additional 2-minute interval, repeat the two-steps done before. At the end of the 10 minutes, record the temperature and the time at which 100% hydrolysis occurred. Repeat the procedure using the other amylase type. Using a color-coding scheme like the one in figure 2 convert the results into quantitative data. Record the corresponding number (Alberte et al, 2012).
Figure 1- Spot Plate Setup
Figure 2- Starch hydrolysis
Graph 1 (graph at the top) represents the starch hydrolysis of each amylase at the different temperatures, representing the enzymatic activity of each amylase. Graph 2 (graph in the middle) represents the starch hydrolysis of the bacterial amylase at the different temperatures, representing the enzymatic activity of the bacterial amylase temperature at the different times it was transferred to the spot plates.
The higher the position in the graph the less enzymatic activity there is. Graph 3 (graph at the bottom) represents the starch hydrolysis of the bacterial amylase at the different temperatures, representing the enzymatic activity of the bacterial amylase temperature at the different times it was transferred to the spot plates. The higher the position in the graph the less enzymatic activity there is. Figure 3.a and b (left and right picture) represents the hydrolysis of the iodine with the amylase-starch mixture at the different times and temperatures in different color reactions. The only difference is fungal amylase is on the left and bacterial amylase is on the right.
The experiment was designed to determine the optimal temperature of both Bacterial and Fungal Amylase by observing the starch amylase mixture being catabolized. Various bath temperatures were used to place both the amylases as well as, starch in the solution of the amylases to observe the activity at the different temperatures. After a set time period the solutions were removed and two drops of each solution mixture was mixed with iodine that was placed in spot plates which would determine how much of the solution at the different temperatures show the starch being hydrolyzed. The hydrolysis of each solution was used to calculate the amount of enzymatic activity present at each temperature. The experiment concluded that the optimal temperature for Fungal Amylase was around 40° C and the Bacterial Amylase experiment concluded that the optimal temperature was around 60° C. Previous research and experiments showed that optimal temperature for Fungal Amylase was high temperatures above 60° C and bacterial amylases on the other hand are stable and show little denaturing at temperatures up to 85°C (Elkhalil et al, 2011).
These different conclusions could be due to the facts that the experiment was designed for only a certain set of varying temperatures that did not allow the researcher to observe the exact point at which the hydrolysis of starch lessens or stops. Graph 1 showed the optimal temperatures for each amylase represented in the lowest average starch hydrolysis for each amylase. The starch hydrolysis represented the enzyme activity because of the enzyme reacting with the starch breaking down the starch meaning that there should be less dark colored (according to the scale in figures 2 and 3 a. b.) iodine in the well. The average results concluded that average hydrolysis for the fungal amylase was in between 40 C and 60 C which would correlate with the previous experiments conducted showing that the fungal amylase could have a higher optimal temperature then this experiment concluded.
Although for the average of the bacterial amylase graph 1 showed the 60 C was the correct temperature for the starch to hydrolyze because of the numbers being low, which meant that when compared with figures 2 and 3.b. the lightest color meant that it was hydrolyzed the most. Graphs 2 and 3 represented the amount of time that each amylase mixture solution was catabolizing the starch. The results of the two graphs showed that at the early times, 2 minute interval, the solution was hydrolyzed the fastest and then at each other time interval it was a constant hydrolysis, catabolizing each solution less and less as the time progressed until the point in which there was nothing to catabolize. This concludes that the enzymes present in the amylases only are active for a certain period of time until the product is made or finishes and then it detaches from the substrate to attach to a new substrate and start the process once again.
The experiment rejects the null hypotheses concluding that there is a correlation between the temperature that the different type of amylases are placed in and the amount of enzymatic activity that will observed when the starch is hydrolyzed. The results concluded that at a certain high temperature, 95°C for Bacterial Amylase and 60° C and above for Fungal Amylase, the starch was not hydrolyzed meaning that the amylase-starch mixture, dark color like black (figure 3 a. b.) showed no enzymatic activity because the enzyme could not lower the activation energy of the reaction due to the temperature being to high, denaturing the enzymes structure not allowing the substrate to bind to the active site making it become unusable.
The results also concluded that the lower temperatures depending on which amylase was tested became unusable after that certain temperature, after 60°C in Bacterial and after 40°C in Fungal because the enzyme lowers the activation energy to a point were the reactions are responding too slow to function, making the whole reaction halt and partially hydrolyze the starch (represented by figure 3. a. b.) This shows that at the optimal temperatures of each amylase the enzyme lowers the activation energy enough to react appropriately and hydrolyze the starch with the iodine. The results overall showed that starch was not catabolized equally and efficiently in all the temperatures some were catabolized too much or too little, unless at the optimal temperature. Peers published studies results are the same as concluded by this experiment, there were some numbers that were different but there will always be variations due to errors, which are always accounted for. Some possible errors that could have occurred was the temperature on thermometer of the water bath read different at the end then what it read at the beginning of the experiment.
When observed the bath water of 60° C read to be 65° C and the bath water of 95°C read to be 92°C. Other errors that could have been observed would be too much iodine was placed in the wells and not enough amylase mixture was placed in the well to balance out the amount of iodine each well had, meaning that the hydrolysis of the starch would have been difficult because the iodine was overpowering the amount of amylase mixture was placed. Although there were errors in experiment the results of each different scenario will help for future food production conservation and industrial product conservations because the industries would be aware that the products that are being produced turn out slightly different from what was previously made because of the errors that could occur.
Although by knowing the optimal temperature in which each amylases operates efficiently, under certain products the industries can adjust the products a bit to mix with the amylase to be conserved and properly and efficiently be used without overusing too much of a certain product. The parameters that were important for the expected results were the varying bath temperatures each amylase was placed in because it would not give the actual optimal temperature range only giving the range for limited temperatures used.
If the temperatures were used in intervals of 10° C then the optimal temperature range would be better observed and more precise. Some of the artifacts that gave the results for the experiment were human error and the different readings of the temperatures of each water bath. This was observed with the optimal temperature range differing from the previous experiments done. Adding more bath water temperatures to get a precise optimal temperature as close as possible to other previous experiments can alter the experiment and diminish the artifacts.
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Alberte, Jose, Thomas Pitzer, and Kristy Calero. “Enzymes.” General Biology I Lab Manual. 2nd ed. Dubuque, IA: McGraw-Hill, 2012. 49-51.
Elkhalil, Elhadi A.I, and Fatima Y. Gaffar. “Biochemical Characterization of Thermophilic Amylase Enzyme
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