How Does Temperature Affect The Reaction Between Bacterial And Fungal Amylase
Abstract
Enzymes are biological catalysts that speed up the rate of chemical reactions by lowering the reactants’ activation energy. The goal of this lab was conducted to determine the optimal temperature for bacterial and fungal Amylases and evaluate how temperature affects the catabolic rate of enzymes. Enzyme reaction rate was measured using an Iodine test in which drops of starch solution with either fungal or bacterial Amylase exposed to different temperatures were mixed with Iodine. Iodine is a dark blue color in the presence of starch and turns light yellow in its absence. Bacterial Amylase had an optimal temperature of 55°C, meaning that starch was broken down the fastest at this temperature. Fungal Amylase showed a slightly lower optimal temperature of 40°C. Bacterial and fungal Amylase at lower temperatures had a slower reaction rate because starch and Amylase molecules moved slower. The reaction rate of both enzymes decreasing after 55°C could be due because the enzymes may have begun to denature, losing its potency to break down starch. In order for each …show more content…
enzyme to perform at its highest reaction rate it must be exposed to its optimal temperature.
Introduction
All living things require energy to drive chemical reactions in their body. An organism’s metabolism is all of the chemical reactions that take place within the cells. In order for organisms to start chemical reactions they often require the presence of enzymes. Enzymes are biological catalysts that speed up the rate of a chemical reaction by lowering the activation energy. Reactants called substrates bind to an enzyme’s active site resulting in the enzyme-substrate complex. After the enzyme-substrate complex is formed the enzyme goes through a form change in order to accommodate the substrate (Alberte et al., 2012). Enzymes generally work by inflicting stress on the substrate’s chemical bonds, which then lowers the amount of energy needed to break the bonds so new products can form (Raven et al., 2014). Missing enzymes can lead to many complications for an organism including death. An enzyme’s function and reaction rate can be affected by factors like temperature, pH, the amount of substrate present, salinity, inhibitors, and activators (Raven et al., 2014). Enzymes are most potent at their optimal temperature; most enzymes have an optimal temperature of forty degrees Celsius and a pH range of six to eight (Pendey et al., 2012). If enzymes are placed in temperatures that are too high they will denature and no longer function, while if the temperature is too low then molecules will be moving at a much slower rate making it less likely for substrates to bind to the enzymes active site and slowing down the rate of the chemical reaction (Alberte et al., 2012). When an enzyme is denatured, it becomes damaged and can no longer function. The enzyme Amylase breaks down polymers of starch into monomers of Maltose, Maltriose and short oligosaccharides (Abe et al., 1988). Starch is a polysaccharide that plants use for storage of Glucose and as a source of energy. B. licheniformis and A. oryzae Amylases can both be used to catabolize starch. This experiment will assess the optimal temperature for B. licheniformis and A. oryzae Amylase by measuring the reaction rate of each enzyme at different temperatures. If B. licheniformis Amylase is placed in the presence of starch and water at different temperatures the higher temperatures will show higher reaction rates for the breakdown of starch. If the temperature exceeds the optimal temperature for B. licheniformis amylase the enzyme will begin to denature and the reaction rate will slow down and eventually stop the product frm forming. B. licheniformis Amylase at temperatures on the lower end of the spectrum will have a lower reaction rate because the starch substrates will be less likely to collide with the enzyme. If the temperature is extremely low B. licheniformis Amylase and the starch substrate will not come in contact with each other and no prod- uct will be formed. A. oryzae Amylase is expected to behave in the same way as B. licheniformis Amylase, although I predict that both enzymes will have slightly different optimal temperatures because they have evolved to adapt in different temperature environments.
Method
The lab was done according to how General Biology 1 Lab Manual indicated the students to do. A spot plate was placed on top of a large wide napkin, Temperature (Celsius) was written across the napkin along with 0, 40, 55, and 75-85 degrees at each column of wells. On the side of the napkin Time (minutes) was written along with 0, 2, 4, 6, 8, and 10 at each row of wells. Four test tubes were obtained and labeled with each different temperature, the enzyme source (B for B. licheniformis Amylase and F for A. oryzae), and group number. Four additional test tubes were obtained and labeled with each different temperature, enzyme source, group number, and the letter S (indicating starch solution). Five mL of starch solution was added to each test tube labeled with the letter S and 1 mL of Amylase was added to each test tube that did not contain starch. The bacterial amylase solution was poured into its designated test tube and all four test tubes were placed in their designated temperatures. After waiting five minutes three Iodine drops were dropped into each well of the zero minutes row. Next the starch solution was transferred into the Amylase containing tubes and the timer was set for two minutes. After two minutes 3 drops of the solution from each test tube containing amylase and starch, and from each temperature treatment was transferred to its designated well in the spot plates. The change in color of the solution was noted and the observations were recorded. Three drops from the starch solution and bacterial amylase test tube was then transferred every two minutes for ten minutes to its corresponding well. Each time the solution was transferred the pipette with its label was used. The procedure above was repeated for the test tubes (Alberte et al., 2012).Results
The control for both the Bacillus lichenformis and Aspergillus oryzae amylase sample is the amount of starch catabolized at 0 minutes because it is compared to the amount of starch broken down at each time interval. The difference in reaction rate between 40°C and 55°C was not big. The amount of starch hydrolyzed by Bacillus licheniformis and Aspergillus oryzae amylase respectively at each temperature (0°C, 40°C, 55°C, and 75°-85°C) as time elapses. Neither of the Bacillus licheniformis or Aspergillus oryzae amylase samples hydrolyzed the complete starch solution in ten minutes.
The experiment was conducted using the plates and Iodine to indicate starch hydrolysis. The lighter the solution turns the more starch has been catabolized by the Bacillus licheniformis or Aspergillus oryzae enzyme into monomers of maltose and oligosaccharides. The results indicate that Bacillus lichenformis amylase broke down starch the fastest at 55°C because the spot plates were lighter throughout that column. The results also indicate that Aspergillus oryzae amylase hydrolyzed starch the fastest at 40°C because the spot plates were lightest throughout that column. The Iodine test used in this experiment shows the amount of starch that has not been catabolized. The more starch that is present in the solution the darker it will appear. In contrast, the less starch that is present in the solution the lighter it will be. Scores range from 1 being the lightest to 5 being the darkest.
Discussion
The results of the experiment indicate that Bacillus licheniformis and Aspergillus oryzae amylase’s reaction rate are affected by temperature. Experimental data also shows that both bacterial and fungal amylase have an optimal temperature under which they catalyze chemical reactions the fastest. The bacterial amylase showed the slowest reaction rate at 0°C because the enzyme and starch molecules move slower at lower temperatures. At 40°C bacterial amylase’s reaction rate rose due to the increasing movement of molecules raising the chances of starch’s attachment to the enzyme’s active site and allowing starch hydrolysis to commence. At 55°C the bacterial amylase showed the highest reaction rate because the random movement of starch and enzyme molecules rose. At 75°-85°C no starch was catabolized because the high temperature denatured the enzyme, since the enzyme lost its formation the substrate could not bond to the deformed active site. The reaction rates are the fastest during the first two time intervals because the starch molecules are directly binding to the enzymes.
Aspergillus oryzae amylase showed reaction rate patterns similar to Bacillus licheniformis.
The lowest reaction rate of fungal amylase was at 0°C which is accounted by the slow movement of molecules that decreases enzyme and substrate collision. The difference in optimal temperature between the fungal and bacterial amylase may be due to the different environments that each enzyme is naturally found in. Bacillus licheniformis is found in a wider range of environments so its enzymes have to survive a higher range of temperatures. Where as Aspergillus oryzae is found in lower temperature habitats preventing its enzymes from tolerating a wider range of temperatures (Hideki, 1982). At 75°-85 °C no starch was hydrolyzed because the fungal enzyme denatured. If both bacterial and fungal enzymes were allowed to react with starch for a longer period all of the starch solution would have been
catabolized.
Experimental data indicates a significant difference in reaction rates between fungal and bacterial amylase at different temperatures as was predicted by the hypothesis. The data also supports the hypothesis that neither enzyme would catabolize starch at 75°-85°C because the enzyme denatures and loses its ability to function, and that the bacterial and fungal enzyme would have two different optimal temperatures that vary slightly in reaction rate. The experiment demonstrated the importance of enzymes to living organisms as biological catalysts for chemical reactions. If amylase is absent in the starch solution the activation energy needed to break the glycoside linkage between starch monomers cannot be reduced and starch hydrolysis does not commence (Raven et al., 2014). An organism’s entire metabolism depends on enzymes lowering activation energy in order for chemical reactions to proceed.
Lipase and cellulose enzymes are among the many enzymes used in detergents. Protease and phytase is found in animal feed, xylanase is used in paper manu- facturing, pectinase in wine, and hydrolase in gas and oil (Charnock et al., 2007). Enzymes are also heavily used in food industries. For instance papain is used to tenderize meat, chymosin in milk processing, and glucoamylase in corn syrup and brewing (Houston, 2010). The pharmaceutical industry also integrates enzymes in medications. For example the proteolytic enzyme bromelain is used in the treatment of arthritis, ulcerative colitis, and chronic obstructive pulmonary disease. Bromelain also reduces pain, swelling, aids in digestion, and prevents blood clots (Pavan et al., 2012).
Factors that may have led to experimental errors are fluctuating water bath temperatures, crowding of students around the water baths and spot plates, contaminated pipettes, and inconsistent readings of solution colors. For example, a 3 for one student may have been considered a 2 by someone else. Using spot plates also increased the chances of samples being dropped in the wrong spot plate. Future experiments can be improved by using bigger facilities that accommodate a large number of students. Future experimental design can consist of a wider range of temperatures and different types of enzymes to gain further insight on the effect of temperature on enzyme activity.
Enzymes are biological catalysts that speed up the rate of chemical reactions by lowering the reactants’ activation energy. The goal of this lab was conducted to determine the optimal temperature for bacterial and fungal Amylases and evaluate how temperature affects the catabolic rate of enzymes. Enzyme reaction rate was measured using an Iodine test in which drops of starch solution with either fungal or bacterial Amylase exposed to different temperatures were mixed with Iodine. Iodine is a dark blue color in the presence of starch and turns light yellow in its absence. Bacterial Amylase had an optimal temperature of 55°C, meaning that starch was broken down the fastest at this temperature. Fungal Amylase showed a slightly lower optimal temperature of 40°C. Bacterial and fungal Amylase at lower temperatures had a slower reaction rate because starch and Amylase molecules moved slower. The reaction rate of both enzymes decreasing after 55°C could be due because the enzymes may have begun to denature, losing its potency to break down starch. In order for each …show more content…
enzyme to perform at its highest reaction rate it must be exposed to its optimal temperature.
Introduction
All living things require energy to drive chemical reactions in their body. An organism’s metabolism is all of the chemical reactions that take place within the cells. In order for organisms to start chemical reactions they often require the presence of enzymes. Enzymes are biological catalysts that speed up the rate of a chemical reaction by lowering the activation energy. Reactants called substrates bind to an enzyme’s active site resulting in the enzyme-substrate complex. After the enzyme-substrate complex is formed the enzyme goes through a form change in order to accommodate the substrate (Alberte et al., 2012). Enzymes generally work by inflicting stress on the substrate’s chemical bonds, which then lowers the amount of energy needed to break the bonds so new products can form (Raven et al., 2014). Missing enzymes can lead to many complications for an organism including death. An enzyme’s function and reaction rate can be affected by factors like temperature, pH, the amount of substrate present, salinity, inhibitors, and activators (Raven et al., 2014). Enzymes are most potent at their optimal temperature; most enzymes have an optimal temperature of forty degrees Celsius and a pH range of six to eight (Pendey et al., 2012). If enzymes are placed in temperatures that are too high they will denature and no longer function, while if the temperature is too low then molecules will be moving at a much slower rate making it less likely for substrates to bind to the enzymes active site and slowing down the rate of the chemical reaction (Alberte et al., 2012). When an enzyme is denatured, it becomes damaged and can no longer function. The enzyme Amylase breaks down polymers of starch into monomers of Maltose, Maltriose and short oligosaccharides (Abe et al., 1988). Starch is a polysaccharide that plants use for storage of Glucose and as a source of energy. B. licheniformis and A. oryzae Amylases can both be used to catabolize starch. This experiment will assess the optimal temperature for B. licheniformis and A. oryzae Amylase by measuring the reaction rate of each enzyme at different temperatures. If B. licheniformis Amylase is placed in the presence of starch and water at different temperatures the higher temperatures will show higher reaction rates for the breakdown of starch. If the temperature exceeds the optimal temperature for B. licheniformis amylase the enzyme will begin to denature and the reaction rate will slow down and eventually stop the product frm forming. B. licheniformis Amylase at temperatures on the lower end of the spectrum will have a lower reaction rate because the starch substrates will be less likely to collide with the enzyme. If the temperature is extremely low B. licheniformis Amylase and the starch substrate will not come in contact with each other and no prod- uct will be formed. A. oryzae Amylase is expected to behave in the same way as B. licheniformis Amylase, although I predict that both enzymes will have slightly different optimal temperatures because they have evolved to adapt in different temperature environments.
Method
The lab was done according to how General Biology 1 Lab Manual indicated the students to do. A spot plate was placed on top of a large wide napkin, Temperature (Celsius) was written across the napkin along with 0, 40, 55, and 75-85 degrees at each column of wells. On the side of the napkin Time (minutes) was written along with 0, 2, 4, 6, 8, and 10 at each row of wells. Four test tubes were obtained and labeled with each different temperature, the enzyme source (B for B. licheniformis Amylase and F for A. oryzae), and group number. Four additional test tubes were obtained and labeled with each different temperature, enzyme source, group number, and the letter S (indicating starch solution). Five mL of starch solution was added to each test tube labeled with the letter S and 1 mL of Amylase was added to each test tube that did not contain starch. The bacterial amylase solution was poured into its designated test tube and all four test tubes were placed in their designated temperatures. After waiting five minutes three Iodine drops were dropped into each well of the zero minutes row. Next the starch solution was transferred into the Amylase containing tubes and the timer was set for two minutes. After two minutes 3 drops of the solution from each test tube containing amylase and starch, and from each temperature treatment was transferred to its designated well in the spot plates. The change in color of the solution was noted and the observations were recorded. Three drops from the starch solution and bacterial amylase test tube was then transferred every two minutes for ten minutes to its corresponding well. Each time the solution was transferred the pipette with its label was used. The procedure above was repeated for the test tubes (Alberte et al., 2012).Results
The control for both the Bacillus lichenformis and Aspergillus oryzae amylase sample is the amount of starch catabolized at 0 minutes because it is compared to the amount of starch broken down at each time interval. The difference in reaction rate between 40°C and 55°C was not big. The amount of starch hydrolyzed by Bacillus licheniformis and Aspergillus oryzae amylase respectively at each temperature (0°C, 40°C, 55°C, and 75°-85°C) as time elapses. Neither of the Bacillus licheniformis or Aspergillus oryzae amylase samples hydrolyzed the complete starch solution in ten minutes.
The experiment was conducted using the plates and Iodine to indicate starch hydrolysis. The lighter the solution turns the more starch has been catabolized by the Bacillus licheniformis or Aspergillus oryzae enzyme into monomers of maltose and oligosaccharides. The results indicate that Bacillus lichenformis amylase broke down starch the fastest at 55°C because the spot plates were lighter throughout that column. The results also indicate that Aspergillus oryzae amylase hydrolyzed starch the fastest at 40°C because the spot plates were lightest throughout that column. The Iodine test used in this experiment shows the amount of starch that has not been catabolized. The more starch that is present in the solution the darker it will appear. In contrast, the less starch that is present in the solution the lighter it will be. Scores range from 1 being the lightest to 5 being the darkest.
Discussion
The results of the experiment indicate that Bacillus licheniformis and Aspergillus oryzae amylase’s reaction rate are affected by temperature. Experimental data also shows that both bacterial and fungal amylase have an optimal temperature under which they catalyze chemical reactions the fastest. The bacterial amylase showed the slowest reaction rate at 0°C because the enzyme and starch molecules move slower at lower temperatures. At 40°C bacterial amylase’s reaction rate rose due to the increasing movement of molecules raising the chances of starch’s attachment to the enzyme’s active site and allowing starch hydrolysis to commence. At 55°C the bacterial amylase showed the highest reaction rate because the random movement of starch and enzyme molecules rose. At 75°-85°C no starch was catabolized because the high temperature denatured the enzyme, since the enzyme lost its formation the substrate could not bond to the deformed active site. The reaction rates are the fastest during the first two time intervals because the starch molecules are directly binding to the enzymes.
Aspergillus oryzae amylase showed reaction rate patterns similar to Bacillus licheniformis.
The lowest reaction rate of fungal amylase was at 0°C which is accounted by the slow movement of molecules that decreases enzyme and substrate collision. The difference in optimal temperature between the fungal and bacterial amylase may be due to the different environments that each enzyme is naturally found in. Bacillus licheniformis is found in a wider range of environments so its enzymes have to survive a higher range of temperatures. Where as Aspergillus oryzae is found in lower temperature habitats preventing its enzymes from tolerating a wider range of temperatures (Hideki, 1982). At 75°-85 °C no starch was hydrolyzed because the fungal enzyme denatured. If both bacterial and fungal enzymes were allowed to react with starch for a longer period all of the starch solution would have been
catabolized.
Experimental data indicates a significant difference in reaction rates between fungal and bacterial amylase at different temperatures as was predicted by the hypothesis. The data also supports the hypothesis that neither enzyme would catabolize starch at 75°-85°C because the enzyme denatures and loses its ability to function, and that the bacterial and fungal enzyme would have two different optimal temperatures that vary slightly in reaction rate. The experiment demonstrated the importance of enzymes to living organisms as biological catalysts for chemical reactions. If amylase is absent in the starch solution the activation energy needed to break the glycoside linkage between starch monomers cannot be reduced and starch hydrolysis does not commence (Raven et al., 2014). An organism’s entire metabolism depends on enzymes lowering activation energy in order for chemical reactions to proceed.
Lipase and cellulose enzymes are among the many enzymes used in detergents. Protease and phytase is found in animal feed, xylanase is used in paper manu- facturing, pectinase in wine, and hydrolase in gas and oil (Charnock et al., 2007). Enzymes are also heavily used in food industries. For instance papain is used to tenderize meat, chymosin in milk processing, and glucoamylase in corn syrup and brewing (Houston, 2010). The pharmaceutical industry also integrates enzymes in medications. For example the proteolytic enzyme bromelain is used in the treatment of arthritis, ulcerative colitis, and chronic obstructive pulmonary disease. Bromelain also reduces pain, swelling, aids in digestion, and prevents blood clots (Pavan et al., 2012).
Factors that may have led to experimental errors are fluctuating water bath temperatures, crowding of students around the water baths and spot plates, contaminated pipettes, and inconsistent readings of solution colors. For example, a 3 for one student may have been considered a 2 by someone else. Using spot plates also increased the chances of samples being dropped in the wrong spot plate. Future experiments can be improved by using bigger facilities that accommodate a large number of students. Future experimental design can consist of a wider range of temperatures and different types of enzymes to gain further insight on the effect of temperature on enzyme activity.