The Hydrolysis of Alpha-Amylase - Lab Report Example

The Hydrolysis of Alpha Amylase Optimal temperature is the temperature at which bacterial fungal amylases work best. This experiment was made up of setting up a starch control group placing bacterial and fungal amylases in starch and placing them in various temperatures. Two drops of iodine was added to the mixture containing either of the amylases after a certain amount of time. This experiment was to determine the optimal temperature in which both bacterial and fungal amylases would function to produce a suitable enzymatic action. First, the equipment to be used during the laboratory experiment was set up. Test tubes were labeled, and spot plates were placed in temperature against time table created. For each temperature, iodine was placed in each row of the spot plates and the bacterial and fungal amylase and starch mixture solutions contained in the test tubes were added to the spots depending on time and temperatures. Different times are used in order to obtain reliable results. The optimal temperature was determined by observing the color change in the spot plates and comparing it with the color coding scheme (Alberte, Pitzer and Calero 340). The results indicated that bacterial amylase had an optimal temperature of 600C while fungal amylase had an optimal temperature of 400C. This means that fungal amylase has a lower optimal temperature compare to bacterial amylase. This means that bacterial amylases would work at a higher temperature than fungal amylases. The fungal amylases are likely to denature at the high temperatures. In addition, conclusions also suggested that change in temperature affects the working of enzyme amylase. At extreme high or low temperatures enzymes were denatured or inactive therefore inability to function best. This reduced their ability to break down starch (Cordeiro, Martins and Luciano, 2002). Introduction Substrates such as starch undergo reactions by binding to specific active sites of enzymes such as amylase to form different simple products. Active sites are specific sites that enzyme and substrate molecule combine at specific sites complementary to each other through lock and key theory and induced fit theory of enzyme action. For instance, starch is broken down by amylase to form maltose which can be easily absorbed in the body. The shape of the enzyme changes rapidly to allow the substrate to bind. Amylase is composed in saliva and pancreatic juices to break down long chains of carbohydrates such as starch into smaller molecules like maltose and sucrose. Generally enzymes play an important role in complex processes such as digestion and are used in industrial processes. Most enzymes are obtained from bacteria and fungi. An enzyme is a complex protein whose function is to speed up chemical reactions through catalysis. Enzymes are biological catalysts that increase the rate of chemical reactions. In chemical reactions enzymes are not consumed therefore can be re-used. Minute quantities of enzymes are enough to catalyze a reaction (Bisena, 201). Enzyme activity is affected by variety of factors such as pH, temperature, substrate concentration, inhibitors, co-factors, and co-enzymes. Each enzyme has an optimal pH where it operates at peak efficiency in speeding the rate of a chemical reaction. A suitable pH ranges from 6-8. The temperature at which enzymes work effectively is referred to as optimal. The rate of a reaction increases with temperature until it reaches an optimum temperature and becomes constant. Low temperatures inactivate enzymes while high temperatures denature the enzymes therefore preventing them from binding to the active sites of their respective substrates. Enzymes are sensitive to temperature and pH due to their protein nature (Morton, Perry and Perry 267). If the amount of enzyme is maintained but the amount of substrate slowly increased, the rate of the reaction will increase until it reaches maximum and becomes constant. This indicates that increase in substrate concentration increases the rate of reaction to a given point. At low substrate concentrations, there are more enzyme molecules than substrate molecules. This increases the rate of reaction because active sites are available. An increase in substrate concentration will speed up a reaction. Inhibitors are substances that alter the active sites hence interfere with the activity of enzymes. Inhibitors can either be competitive or non-competitive. Competitive inhibitors bind to the active sites of enzyme while non-competitive inhibitors bind to enzymes whether they have been to the substrate or not therefore reducing the function of enzymes. Most enzymes require activation by other compounds referred to as co-factors and co-enzymes. They are non-protein substances which activate the enzymes. Co-enzymes are organic, mainly vitamins while co-factors are usually metallic ions (Dashmanb, 132). Starch is a polysaccharide made of long chains of simple units of simple sugars joined together by glycosidic bonds. It is a source of energy and produced by most green plants during photosynthesis in the presence of light energy and carbon dioxide. In the human diet, it is mainly consumed in potatoes, rice and maize. Starch that is pure is insoluble in both cold water and alcohol. In the presence of an enzyme such as amylase, starch is broken down into simple sugars. The chemical test for starch is addition of iodine which turns black in the presence of starch. Methods The experiment should be done using bacterial and fungal amylase. In the presence of starch, iodine turns blue-black. The iodine test aids to monitor catalysis of starch. In setting up the experiment, under the spot plates, a paper should be placed and labeled the temperature values 0, 40, 60 and 95 °C respectively and indicated the times 0, 2, 4, 6, and 10 minutes. Four test tubes should be obtained and labelled with a different temperature, type of enzyme and the number of the group. This step should be repeated while starch solution labelled S. In the last step, pipette 5ml of 1.5% and add into the test tubes labelled S. In order to test the effect of temperature in amylase activity, add 1ml of amylase into each of the test tubes that do not contain starch and place all the test tubes , four containing amylase and four containing amylase into their respective temperatures and equilibrate for 5 minutes. Add 2-3 drops of iodine to the first row of the plate indicated as 0 minutes. After a period of 5 minutes, after the test tubes are equilibrated, move a few drops of starch solution from each temperature to the row where iodine had been previously added. Pour the starch solution into the tube containing amylase without taking it put of bath and set a timer for two minutes. In the second row add 2-3 drops of iodine to the second row and after 2 minutes have elapsed, transfer a few drops of the starch amylase mixture using a pipette from each tube to the 2 minutes corresponding to each temperature. After each additional 2 minutes, add 2 drops of iodine and a few drops from the starch amylase mixture. After 10 minutes the temperature and the time at which 100% hydrolysis occurred should be noted. The procedure should be repeated using the other amylase type and using color-coding scheme convert results into numerical values (Alberte, Pitzer and Calero 177). Results Bacterial Amylase Temp (OC) 0 40 60 95 Color # Color # Color # Color # Time (min) 0 Black 1 Black 1 Black 1 Black 1 2 Black 1 Black 1 Brown 2 Black 1 4 Black 1 Brown 2 Brown 2 Black 1 6 Black 1 Brown 2 Deep yellow 3 Black 1 8 Black 1 Brown 2 Brown 2 Black 1 10 Black 1 Brown 1 Brown 2 Black 1 Table 1: Product concentrations at various temperatures against time of bacterial amylase Fungal Amylase Temp (OC) 0 40 60 95 Color # Color # Color # Color # Time (min) 0 Black 1 Black 1 Black 1 Black 1 2 Black 1 Brown 2 Black 1 Black 1 4 Black 1 Brown 2 Brown 2 Black 1 6 Black 1 Dark yellow 3 Brown 2 Black 1 8 Black 1 Brown 2 Brown 2 Black 1 10 Black 1 Brown 2 Brown 2 Black 1 Table 2: Product concentrations at various temperatures against time of fungal amylase Fungal Amylase Temp (°C) 0 40 60 95 Time (min) Group 1 1 3 1 1 Group 2 5 4 3 3 Group 3 3 3 3 1 0 minutes Mean +- SD 3+-1.633 3.333+- 0.4714 2.333+- 0.9428 1.6667+-0.9428 Group 1 2 3 2 1 Group 2 2 4 4 2 Group 3 2 2 2 1 2 minutes Mean +_ SD 2+-0 3+- 0.8165 2.667+-0.9428 1.333+-0.4714 Group 1 2 3 2 1 Group 2 2 4 4 2 Group 3 2 2 2 1 4 minutes Mean +- SD 2+-0 3+-0.8165 2.667+-0.9428 1.333+-0.4714 Group 1 2 3 2 1 Group 2 2 4 4 2 Group 3 2 2 2 1 6 minutes Mean +- SD 2+-0 3+-0.8165 2.667+-0.9428 1.333+-0.4714 Group 1 2 3 2 1 Group 2 2 4 4 1 Group 3 2 2 2 1 8 minutes Mean+_ SD 2+-0 3+-0.8165 2.667+-0.9428 1+-0 Group 1 2 3 2 1 Group 2 2 4 4 1 Group 3 2 2 2 1 10 minutes Mean+_SD 2+-0 3+-0.8165 2.667+-0.9428 1+-0 OPTIMAL TEMP 40 TIME TO 100% 6 minutes Table 3: Class data of various temperatures against time of fungal amylase Bacterial Amylase Temp (oC) 0 40 60 95 Time (min) Group 1 1 1 1 1 Group 2 1 1 1 1 Group 3 1 1 1 1 0 minutes Mean +- SD 1+- 0 1+- 0 1+- 0 1+- 0 Group 1 1 1 1 1 Group 2 1 1 3 1 Group 3 2 2 1 1 2 minutes Mean +_ SD 1.333+-0.4714 1.333+-0.4714 1.667+- 0.9428 1+- 0 Group 1 1 1 1 1 Group 2 1 3 3 1 Group 3 1 1 1 1 4 minutes Mean +- SD 1+- 0 1.667+- 0.9428 1.667+- 0.9428 1+- 0 Group 1 1 1 1 2 Group 2 1 3 3 1 Group 3 1 1 1 1 6 minutes Mean +- SD 1+- 0 1.667+- 0.9428 1.667+- 0.9428 1.333+-0.4714 Group 1 1 1 1 2 Group 2 1 3 3 1 Group 3 1 1 1 1 8 minutes Mean+_ SD 1+- 0 1.667+-0.9428 1.667+-0.9428 1.333+-0.4714 Group 1 1 1 1 2 Group 2 1 3 3 1 Group 3 1 1 1 1 10 minutes Mean+_SD 1+- 0 1.667+- 0.9428 1.667+- 0.9428 1.333+-0.4714 OPTIMAL TEMP 60 TIME TO 100% 6 minutes Table 4: Class data at various temperatures against time of bacterial amylase Discussion The purpose of this experiment is to determine the optimal temperature at which fungal and bacterial amylase works best. Theoretically, fungal amylase works at an optimal temperature of 40°C while bacterial amylase works best at 60°C. This states the hypothesis. The mean and the standard deviation for each temperature and time showed that the results collected by each group for the bacterial enzyme results were accurate since there was a little variation between the group data. However, the standard deviation in fungal amylase is significant. The data presented in the tables above indicate that when temperature is not optimal, it reduces the working of enzymes. High and low temperatures have varying effect on the ability of amylases to break down the starch. In case the iodine turned black it indicated that starch was present and amylase could not break down the starch. However if the color was yellow or brown, it mean that starch had been broken down and comparing the color to the color coding scheme would determine the optimal temperature. Iodine helps to determine the amount of starch that was hydrolyzed therefore used to calculate the amount of enzymatic activity each mixture had at the different set of temperatures and time. Different times were used to obtain reliable results (Seager and Slabaugh 308). It is evident that starch catabolism is not equally efficient across all temperatures. Variation in temperature has different effects on working of enzymes. The optimal temperature for bacterial amylase was at 60o C at the 6th minute when the color was brown whereas fungal amylase broke down starch at an optimal temperature of 40 °C at the same time. This means that the amylase was able to break down the starch that was present in the solution. When the color was black in case of 0°C and 95 °C, it meant that the enzyme was inactive due to low temperatures or the enzymes were denatured due to extreme high temperatures therefore inability to breakdown starch. It can be concluded that the optimal temperature for fungal amylase was at 60 ° C. Consequently, fungal and bacterial amylases breakdown starch at different temperatures and rates. Bacterial amylases break starch at a slower rate compared to fungal amylases. This means that fungal amylase has a lower optimal temperature compare to bacterial amylase. This means that bacterial amylases would work at a higher temperature than fungal amylases. The fungal amylases are likely to denature at the high temperatures (Seager and Slabaugh 311). In the experiment the sources of errors included inaccurate measurement of reagents due to personal carelessness and uncertainty in determination of the product concentration using color. This can be reduced by repeating the experiment severally and using average values. Bacterial amylase Graph 1: Product concentration against time of bacterial amylase As depicted in the graph above, the product concentration was highest at the sixth minute. The graph also indicates that the optimal temperature for bacterial amylase was at 60°C. Fungal amylase Graph 2: Temperature against time of fungal amylase The graph above indicates that the product concentration was highest at the sixth minute. The graph also indicates that the optimal temperature for fungal amylase was at 40°C. The rate of an enzyme catalyzed reaction increase until it reaches the optimal temperature, decreases rapidly after the optimal temperature and stops after an enzyme is denatured. As the temperature increases, the kinetic motion of all molecules increases, with more frequent and stronger collisions. Since enzymes are protein in nature, the kinetic motions of the amino acid chains of the enzymes increase along with the strength and frequency of collisions between enzymes and surrounding molecules. The disturbances may be strong enough to the enzymes. The hydrogen bonds and other forces that maintain its three dimensional structure break making the enzyme unfold and lose its function. Above the optimal temperature the rate of enzyme activity reduces. As the temperature continues to rise, the unfolding causes the reaction rate to level off at a peak. Further increases cause such extensive unfolding that the reaction rate decreases rapidly to zero. The rate of an enzyme catalyzed reaction peaks at a temperature at which kinetic motion is greatest but no significant unfolding of the enzyme has occurred. At low temperatures the reaction is slow because molecules of enzymes and substrates have little kinetic energy and rarely collide (Seager and Slabaugh 320). In conclusion, temperature affects enzyme activity as illustrated in the experiment. Enzyme amylases work best at an optimum temperature and are unable to work at extreme low and high temperatures therefore inability to break down compounds into simple products. (Johnson and Case 289) Works Cited Alberte, J, T Pitzer and K Calero. General Biology I Lab Manual. Florida International University: McGraw-Hill, 2012. Print. Bisena, Prakash Singh. Laboratory Protocols in Applied Life Sciences. CRC Press, 2014. Print. Cordeiro, Carlos Alberto Martins, Meire Lelis Leal Martins, and Angelica Barbara Luciano."Production and Properties of a - Amylase from Thermophilic Bacillus SP." Brazilian Journal of Microbiology, 33.1 (2002): n. pag. Web. 22 June 2014 Dashmanb. Laboratory Manual/Human Nutr 2. CRC Press, 1996. Print Johnson, Ted and Christine L. Case. Laboratory experiments in mycrobiology. Redwood City, Calif: Benjamins Cummings Publications, 2004. Print. Morton, David, James W Perry and Joy B Perry. Labaratory manual for human biology. Belmont, CA: Brooks/Cole, 2012. Print. Seager, Spencer L and Michael R Slabaugh. Organic and biochemisry for today. Belmont, CA: Brooks/Cole Cengage learning , 2013. Print. Read More