Enzymes report - Grade: A PDF

Preview

Summary

Gen Bio lab...

Description

Running head: REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 1

Temperature effect on bacterial and fungal amylase Erma Danielle Despierre Lab Partners: Dafne Pages Charles Batista Isabelle Diaz

Panther ID: 5871968 Section U37 Group 3 Florida International University

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 2 Abstract The function of an enzyme is to accelerate chemical reaction by decreasing the activation energy without being part of the reaction if not; metabolic processes would take longer, therefore, it would die. This research used visual monitoring to explore the ability of a gram-positive bacteria - Bacillus Licheniformis – and Aspergillus Oryzae- to create enzymes and digest starch at different temperatures, therefore, finding the optimal temperature for both amylases. Two spot plates were used- one for bacteria and the other one for fungal, and both temperature and time were labeled. They were then put into a water bath corresponding to their respective temperature and every 2 minutes, a mixture of starch-amylase was put into a well where two drops of iodine were placed seconds before. It was found that the optimal temperature for both fugal and Bacteria was between 25°C to 65°C. Anything bellow; the enzyme either worked slow or was not activated. Anything above denatured the enzymes. The two enzymes were almost alike except that fungal amylase took longer to show results at all temperatures compared to bacterial amylase. Enzymes are part of everyday life as it improved the standard of living from helping to create energy for the cells to the production of alcohol and other things. Keywords: enzymes, starch, optimal temperature.

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 3 Introduction According to Alberte, Pitzer and Calero in the general biology 1 lab manual (2012), a stable supply of energy, which is created by different chemical reactions, is required by all biological processes for them to perform necessary tasks such as reproduction and growth. Those chemical reactions are coordinated by a biological catalyst named enzymes. The function of an enzyme is to accelerate the chemical reaction by decreasing the activation energy without being part of the reaction if not, metabolic processes would take longer, therefore, it would die. As Copeland (2000) pointed out, enzymes make human and viruses’ life easier. Enzymes have enhanced everyday life; it can be used in the kitchen for baking but also in cleaning products. Alberte, Pitzer and Calero, (2012) also argued that life would be impossible if enzymes did not exist as the human body would shut down, damaged tissue wouldn’t be replaced by new ones and food wouldn’t convert into energy. Copeland (2000) in his book on enzymes pointed out how a previous researcher, such as Emil Fisher, came up with the “Lock and Key” model, which means that each enzyme with a specific shape has a specific site where they can bind called active site. Some enzymes have to modify their model to fit an active site, which is called “induced fit model.” Enzymes in the human body such as amylase break down starch or help the human body digest starch and protease breaks down proteins. In a research made by Li et al. (2007), it was noted that amylase is not only found in the human body but also fungi and gave them the ability to digest raw starch. Starch is found in plant cells as well in an animal. It is a way to store polysaccharide that can later be broken down to glucose then energy, therefore, starch synthesis allows the host cells to store a surplus of glucose. Starch is broken down by amylase, an enzyme that was also used in this experiment (Reece et al., 2015).

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 4 Enzyme functioning can be altered by temperature, PH or chemical that react with enzymes stated Reece et al. (2015) in Campbell biology. These local conditions can either inhibit enzymes reaction or stop them from happening. Temperature and PH are environmental factors that affect enzymes. Up to a certain temperature, enzymes work faster; and above that temperature, enzyme denature and stop working. In low temperatures, enzymes slow down but do not denature. All enzymes have an optimal temperature at which they function at their best. As amylase breaks down starch into smaller subunits or monomers, it is not visible to the naked eye. Iodine staining makes starch detectable as it turns the mixture from yellow or white to blueblack (Alberte et al, 2012). This research used visual monitoring to explore the ability of a gram-positive bacteria Bacillus Licheniformis – and Aspergillus Oryzae- a fungus that is mostly used in fermentationto digest starch. As Rey et al. (2004) explained, Bacillus Licheniformis is a close relative of Ecoli which is a bacterium found in the human intestine. It is a facultative anaerobe bacterium, which means that it can survive whether oxygen is present or not. He further explained how Bacillus Licheniformis produced amylases in the presence of starch and more likely to survive under environmental circumstances. Aspergillus Oryzae, on the other hand, is a fungus. As reported by Machida, Yamada, and Gomi, (2008), it is used widely in fermentation, fabrication of soy, sake (alcohol) as it can create a lot of enzymes including amylase. As this research focused on enzyme production and finding optimal temperature for the fungi and bacteria amylase, Bacillus Licheniformis and Aspergillus Oryzae were used and the temperature was manipulated. It is expected for Bacillus Licheniformis and Aspergillus Oryzae to stop creating enzymes at a high temperature such as 85°C and to perform properly from 25°C to 65°C.

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 5 Methods Two spot plates were used- one for bacteria and the other one for fungal, and both temperature and time were labeled. Time was label vertically as y-axis and temperature horizontally as x-axis (figure 1). Sixteen test tubes were used in total, four were used for bacterial at each temperature, noted as: B- for bacterial followed by the temperature and four were label: F- for fungal with the following temperature as well: 0, 25,55 and 85. 5mL of 1.5% of starch was added into eight different test tubes as each of them will be used at different temperatures and for different amylase. Eight pipettes were then labeled based on either B or F followed by the temperature. As fungal was the first one tested, each test tube – starch and fungi- was placed in a water bath corresponding to their temperature for 5 minutes allowing them to stabilize to that temperature. At time 0-minute, 2 drops of Iodine were added into the time 0 wells all across at each temperature. Starch only was taken from all the test tubes using a pipette and added to wells of the spot plates. After time 0 was filled, a timer for 2 minutes was set At the time mark 2 minutes, using the pipette correspondent to each temperature and amylase, starch was poured into the amylase without being removed from the water bath. 2 drops of iodine were dropped in row two in each temperature and the amylase-starch mixture was pipetted into the spot plates. Another 2-minute timer was set, and this procedure was repeated every 2 minutes until all spots were filled with the starch and amylase mixture. For time 10 minutes, all the starch left in the test tube was added to the amylase and it was pipetted to the last wells of the spot plates. Color changes were then noted based on a color chart. The same thing was repeated for Bacterial amylase, starting by putting them in the shower bath. (Alberte et al, 2012).

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 6 Results At 0°C, there was no significant difference between the means to the 10-minutes mark. Comparing 0 °C to 25°C, there is no significant difference. The means and standard deviation are still close to each other. There is a significant difference between 0°C and 55°C and the mean and standard deviation are also significantly different. The mean for each row was calculated by giving a numerical value to the representation in both figure 1 and figure 2 based on color. Number 1 was assigned to the lightest color or yellow and 5 to the darkest color or dark blue. The standard deviation was later calculated, which is also represented by ± followed by the number.

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 7 Tables and figures

Fungal Amylase 6

5

4

3

2

1

0

1

2

3 0degree

25 degree

4 55 degree

5

6

85 degree

Graph 1: This graph shows the relationship between the means at different temperatures for the fungal amylase. It also shows where there is a decrease in the graph, therefore, an increase in activity and where it is linear (no changes at all).

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 8

Bacterial Amylase 6

5

4

3

2

1

0

0

2

4 0 degree

6 25 degree

55 degree

8 85 degree

Graph 2: This graph shows the correlation between the means at different temperatures for the Bacterial amylase. It also shows decreases and increases in the graph where decrease indicates enzymatic activity and increases show less activity. When it is linear means there have been no changes at all.

10

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 9

Table 1 Fungal amylase Temperature 0 °C 25 °C 55 °C 85 °C Time 0 minute 3.90±1.34 3.90±1.34 4.60±0.42 5.00±0.00 2 minutes 4.00±1.17 4.00±1.17 4.30±0.27 5.00±0.00 4 minutes 4.20±0.76 4.20±0.76 4.60±0.42 5.00±0.00 6 minutes 4.30±0.57 4.30±0.84 4.10±0.65 5.00±0.00 8 minutes 4.00±0.35 4.20±0.57 4.10±0.65 5.00±0.00 10 minutes 4.30±0.45 4.40±0.42 4.20±0.67 5.00±0.00 Note: Aspergillus Oryzae Aspergillus Oryzae- This table shows the mean of the temperature of the fungal amylase starch iodine mixture at a different point in time. Time 0 minute represents the starch solution at different temperatures mixed with iodine. Every 2 minutes mark that follows, a sample of Fungi-amylase starch mixture was collected and dropped into a well of iodine (figure2). Each color was given a number based on a color-coding chart by Alberte et al, (2012).

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 10

Table 2 Bacteria Amylase Temperature Time 0 minute 2 minutes 4 minutes 6 minutes 8 minutes 10 minutes

0 °C

25 °C

55 °C

85 °C

4.30±0.27

4.40±0.42

4.50±0.35

4.90±0.22

4.30±0.27

3.80±0.45

3.10±0.74

5.00±0.00

4.00±0.35

3.70±0.57

3.20±0.45

5.00±0.00

4.50±0.35

3.50±0.50

3.00±0.35

5.00±0.00

4.40±0.22

3.50±0.71

2.70±0.45

4.90±0.22

4.50±0.00

3.50±0.50

2.40±0.42

4.90±0.22

Note: Bacillus Licheniformis - Bacillus Licheniformis -Time 0-minute shows numerical value for starch and iodine mixture. Every 2 minutes mark a sample of the bacteria-amylase starch mixture was collected and dropped into a well of iodine (figure1). Each color was given a number based on a color-coding chart by Alberte et al, (2012).

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 11

Figure 1: Bacillus Licheniformis - Bacterial specimen mixture (starch, bacteria amylase, iodine) at different temperatures.

Figure 2: Aspergillus Oryzae- Fungal specimen at different temperatures. In this picture, from left to right: 0 ° C ,25 ° C , 85 ° C

and 55 ° C .

Discussion.

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 12 The optimal temperature for the bacterial amylase: Bacillus Licheniformis as shown in figure 1 was determined to be between 25°C and a little above 55°C. Compared to time 0-minute where the bacteria amylase was not present, the bacteria starch-amylase mixture either got more or less hydrolyzed which was an indication of the amount of starch present at that specific temperature. The lighter the color was the less starch was present, the more hydrolyzed the mixture was. The darker it was, the more starch was present in the mixture and the more hydrolyzed was the mixture or the enzymes had denatured. At 0°C and 25°C, the mixture had the blue-black color which indicated that the enzyme was not working or was working to slow as the temperature was below what is considered optimal for the enzyme. At 85°C, the enzyme was also blue-black which suggested the denaturation of the enzyme as it went over the optimal temperature, therefore, it stopped working. At 55°C, Bacillus Licheniformis amylase mixture with starch and Iodine started to turn from dark blue to a lighter color which suggested that it was the optimal temperature for this bacterium. At a 10-minute mark, Bacillus Licheniformis showed a lighter color where it was suggested to be the optimal temperature and time to 100% hydrolysis. As Kaur (2012) explained, temperature plays a role in enzyme activity and using bacillus alcalophilus, it was found in their research that the optimal temperature was 60°C, while the enzyme remains active from 20°C to 90°C. The hypothesis was supported as Bacillus Licheniformis denatured and stopped producing enzymes at 85°C. Bacillus licheniformis did hydrolyze between 25°C to 55°C by showing a lighter color which is associated with the absence of starch. Aspergillus Oryzae – the fungi amylase correlation with temperature as shown in figure 2 was also determined to be between 25°C and 55°C. Taking time 0-minute as our negative control group and what the other temperature would be compared to, it was extremely darker compared

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 13 to 55°C. There were no significant differences between 0°C and 25°C; but at 55 ° C, there was a decrease in starch. The lighter the color was the less starch was present, the more hydrolyzed the mixture was. The darker it was, the more starch was present in the mixture and the more hydrolyzed was the mixture. At 85°C, the enzyme-amylase iodine mixture was blue-black which suggested the denaturation of the enzyme as it went over the optimal temperature, therefore, stopped working. Between 25°C and 55°C, Aspergillus Oryzae amylase mixture with starch and Iodine started to turn dark yellow to a lighter color which suggested that it was the optimal temperature for this fungus. At a 10-minute mark, Bacillus Licheniformis showed a lighter color where it was suggested to be the optimal temperature and time to 100% hydrolysis. According to Mohapatra, Banerjee, and Bapuji (1998), maximum amylase activity happened at 60°C. Between 40°C to 65°C the amylase activity increases, reaching its peak around 60°C to 65°C and started to decrease until denatured. This experiment failed to reject the hypothesis as the optimum temperature for the fungal amylase used was between 25°C and 55°C, and the column for 85°C was extremely black which suggested that the enzymes denatured. A way to improve this experiment would be to have more than one trial. When the amylase-starch mixture is transferred into a well, the bacteria or fungi would most likely adjust to the temperature of the wells which will reverse the enzymatic activity back to almost 30°C – dark blue color- therefore harder to see the differences. As shown in figure 2, the differences were barely visible as the picture was not taken right after the mixture was poured in the wells. In figure 2, 85°C was put before 55°C which can lead to a certain confusion. During the testing itself, some other errors were made. The water baths for 55°C and 85°C were over that temperature. 55°C was around 62°C and 85°C was 77°C. taking these errors into consideration,

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 14 the optimal temperature for fungi might be in the lower ends because at 55°C which was about 62°C the color was still too dark. Starch and amylase are both important in everyday life. Amylase breaks down starch polymers into smaller units: monomers – maltose; Starch molecule contains glucose which is one of the main ways the human body generates ATP. Just like the bacteria and the fungi, the human body produces enzymes and has an optimal temperature. Human enzymes can also denature and or stop working depending on body temperature. The human body have thousands of different type of enzymes and each of them execute a specific task at a specific set of condition where it can perform at it’s best. Enzymes are still subject of research and studies which means that there are more to find out (Alberte et al, 2012).

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 15 References Alberte, J., Pitzer, T., & Calero, K. (2012) Enzymes. In: General Biology I Lab Manual. McGraw Hill Education. pp. 49-61. Florida International University Copeland, R. A. (2000). Enzymes: a practical introduction to structure, mechanism, and data analysis. New York: J. Wiley. Kaur, P. (2012). Characterization and optimal production of alkaline α-amylase from Bacillus sp. DLB 9. African Journal of Microbiology Research, 6(11). doi: 10.5897/ajmr11.1139 Li, H., Chi, Z., Wang, X., Duan, X., Ma, L., & Gao, L. (2007). Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion. Enzyme and Microbial Technology, 40(5), 1006–1012. doi: 10.1016/j.enzmictec.2006.07.036 Machida, M., Yamada, O., & Gomi, K. (2008). Genomics of Aspergillus oryzae: Learning from the History of Koji Mold and Exploration of Its Future. DNA Research, 15(4), 173–183. doi: 10.1093/dnares/dsn020 Mohapatra, B., Banerjee, U., & Bapuji, M. (1998). Characterization of a fungal amylase from Mucor sp. associated with the marine sponge Spirastrella sp. Journal of Biotechnology, 60(1-2), 113–117. doi: 10.1016/s0168-1656(97)00197-1 Rey1, M. W., Beth, Brody-Karpin1, S. D., Zaretsky1, E. J., Tang1, M., Leon1, A. L. de, … Berka1, R. M. (2004, September 13). Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Reece, J. B., Meyers, N., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., … Campbell, N. A. (2015). Campbell biology. Melboune, Victoria: Pearson Australia. Souza, P. M. D., & Magalhães, P. D. O. E. (2010). Application of microbial α-amylase in

REACTION OF BACTERIAL AND FUNGAL AMYLASE IN THE PRESSENCE OF TEMPERATURE 16 industry - A review. Brazilian Journal of Microbiology, 41(4), 850–861. doi: 10.1590/s1517-83822010000400004...