Scientific Report
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Abstract
The experiment was performed to analyze the outcomes of temperature on the ability of fungal and bacterial amylase to breakdown starch to maltose. Therefore, the study will reveal the optimal temperature for amylase activity. The initial steps include preparing the materials and tools used during the experiment. A timetable for comparing temperature and function was generated. Test tubes and plates were properly labeled. Iodine was needed for every spot plate so that the fungal amylase, bacterial amylase, fungal amylase and the starch solution can be added to the iodine in a time and temperature dependent fashion. Observing the color change in the spot plates and comparing them with a color-coding scheme for starch hydrolysis allowed for determination of the optimal temperature for starch breakdown. Results depict extreme temperatures (both low and high) inhibit enzyme function causing scientists to conclude that enzymes require a particular temperature to achieve breakdown activity.
Introduction
Enzymes are tiny molecules made of proteins that perform catalytic functions in reactions throughout the body and in life. The mechanics of enzymes involve finding the substrate and accelerating the chemical reaction by affording a substitute pathway of reaction of subordinate activation energy (Bailey and Ollis, 1986). The particles that undergo the reaction process must bind to the active site of the enzyme to generate the desired products. Active sites are like exclusive to specific substrates because the morphology allows for a lock and key mechanism of action. Yet, the induced fit model permits active sites to modify their shape according to the substrate.
Enzymes are not consumed or changed during the reaction and they do not alter the equilibrium of the reactions they catalyze. High temperatures mean greater kinetic energy between the reacting molecules, which makes the occurrence of successful collision more likely thus increasing the rate of the reaction process. Human cells typically have the greatest enzymatic activity at around 38 degrees (Bailey and Ollis, 1986). This means that temperatures higher than this optimal temperature result in denaturing of overall enzyme structure due to the collapse of the intra-molecular and internal molecular bonds. The acidity and basicity also affect the ability of the enzyme to function because alterations in pH can compose and destruct the intermolecular bonds thus disturbing the shape and overall effectiveness of the enzyme (De Souza, 2010). Besides temperature and pH, the concentration of enzyme and substrate can disturb the catalytic activity as well.
The pancreas and salivary gland generate amylase in humans but plants and bacteria also produce amylase. Amylases are widespread enzymes found in human saliva which breakdown starch polymers into sugar components by process of hydrolysis in which water is added. Starch is an essential feature of human consumption that permits energy storage. Starch-converting enzymes like fungal amylase and bacterial amylase are used to produce beer, liquor, fructose, and maltodextrin (Villas-Boss, 2016). Both bacteria and fungi utilize amylases as a tool for feeding and breaking down nutrients. Amylases are significant in several manufacturing processes in food, paper, fermentation, textile and pharmaceutical productions (Aehle, 2004).
The source of the enzymes determines which bond the alpha-amylases will break. Presently, there are two main types of alpha-amylases regularly produced in the chemical industry via microbial fermentation process. Considering the specificity of attack on the sugar molecule, alpha-amylases can be categorized as liquefying or saccharifying.
The bacterial amylase used in the experiment is a liquefying alpha-amylase typically used in the textile industry to spontaneously break down alpha-1,4 bonds and easily rid of starch in material (1939). This amylase is also helpful in properly attacking sugar polymers to provide a smooth coating for paper.
The fungal amylase used in the experiment is categorized as saccharifying because it attacks the beta linkage resulting in a disaccharide. Saccharifying enzymes are more extensive than liquefying amylases because the attack causes two sugar subunits to break off the chain at a time (Prado, 2013). Fungal amylase is typically used to produce simple sugars like corn syrup.
Methods
Therefore, it is necessary to refer to the source of enzymes in comparing the products and kinetics of the two amylases in the experiment. The effect temperature has on reaction catalyzed by the amylases will be analyzed. The experiment was conducted using a liquefying amylase and a saccharifying amylase. A labeled paper with temperature and time was labeled for each spot plate. Four spot plates were used. The temperature values tested were 0, 25, 55, and 85 degrees Celsius and the observations were made at each time point 0,2,4,6,8 and 10 minutes. 4 test tubes were obtained and labeled with each temperature value and the enzyme source, bacterial. Another 4 test tubes were obtained and labeled with each temperature value and the enzyme source, fungal.
Obtain and label tubes that will receive starch. 5 mL of Starch solution was added to each test tube at a concentration of 1.5%. A milliliter of amylase was added to the tubes that do not contain starch so there were 8 tubes total. Next, the tines were placed in the proper location (ice bath or water bath) depending upon the labeled temperature and left for a 5 minute equilibration period. Afterwards the solutions equilibrated, 2 to 3 drops of starch was added to each tube without removing them from their respective temperature. The control was 0 minute time point because no starch was added but 2 to 3 drops of iodine was added. While keeping the test tubes in the water bath, 2 drops of starch from each tube were pipetted onto the primary row of the spot plate, which represented the 0 time increment.
A different pipette must be used for each tube to circumvent contamination. Then starch was pipetted for each temperature labeled tube that had the amylase and set an alarm for 2 minutes after addition of amylase. Iodine was again added to the next time increment row and the remaining rows. After a couple of minutes, the starch amylase solution was removed and added to the spot plate. Observations on color change are written down for both the fungal and bacterial amylase. After the ten minutes has passed, observations were recorded for temperature and time point of 100% hydrolysis for each source of amylase to compare physical results and numerical data.
Results
Following completion of the experiment, data was collected and analyzed. The results depict the rate of reaction for bacterial and fungal amylases are very similar. In fact 100 percent hydrolysis for both conditions occurred in 6 minutes even though the optimal temperature varied between both amylases. At temperature zero, there is the most starch present so no breakdown by either amylase because it’s too cold. At temperature 40, starch begins to breakdown as temperature increases and this is the optimal temperature for fungal amylase as you can see in graph 2. At temperature 55, the fungal amylase exhibits less productivity but the bacterial enzyme is working faster and more productively because the heat lowered the activation energy and reached its optimal temperature as you can see in graph 1. The area where the line peaks is representative of the optimal value. Bacterial amylase will have a higher optimal temperature because bacteria can thrive at extreme temperatures. Finally, both amylases begin to denature at 85 degrees as evident in the all black iodine test in figures 1 and 2. The graphs are a representation of how temperature affects the productivity of the enzymes and the optimal temperature at which breakdown occurs. During starch hydrolysis maltose is a byproduct of starch so they share an inverse relationship. There fore the amount of maltose produced over time is also an indication of the enzyme’s function. The data was calculated as amount of starch digested per minute. It is a good depiction of how the enzyme increases in function as it approaches optimal temperature but then decreases in function once it surpasses the optimal temperature. The fact that both enzymes broke down starch at equal rates at their optimal temperatures makes it difficult to say whether the bacterial or fungal amylase was more productive.
Bacterial Amylase: optimal temperature at 55, since there as a low concentration of starch, therefore enzyme efficiently broke down starch at this temperature. (Figure 1)
Chart 1
Graph 1
Fungal Amylase: 40 degrees is the optimal temperature since 100% hydrolysis occurs.
(Figure 2)
Chart 2
Graph 2
Discussion
After careful evaluation of the data collected in Chartss 1 and 2, the hypothesis offered in the introduction segment that enzymes require optimal temperature for catalytic function or it would be denatured or behave with reduced activity. The results in graphs 1 and 2 indicate cold extreme temperatures and hot extreme temperature impact amylase function. By analyzing the results based upon the picture in Figure 1, the optimal temperatures for bacterial and fungal amylases were found. Based upon the color scheme, a bright yellow color signifies optimal temperature for the amylase because the starch is completely broken down into small subunits. The dark blue color is indicative of the enzyme denaturing because it was incapable of digesting the starch.
In interpreting the results, It is important to remember the parameters taken into consideration throughout the experiment were temperature and time increments. The reaction between the starch and each alpha-amylase revealed a color change that can be compared to the iodine exam to conclude the optimal temperature for the bacterial amylase was 55 degrees, at 6 minute time point, while the optimum temperature for the fungal amylase was 4o degrees 6 minute time point as depicted in graph 1 and 2. Bacteria natural habitat includes several different ranges of temperatures because bacteria have been found in the artic oceans and hot springs so its not surprising that the optimal temperature for bacterial amylase was somewhere in the middle. One can predict human amylase will react very similarly to bacterial amylase because bacterial amylase is found in human saliva where the digestion activity begins. Normal body temperature is around 35 degrees so the optimal activity of human amylase would be around 35 degrees as well. The natural habitat for fungus is thermal conditions so it makes sense that the optimal temperature for fungal amylase activity is 40 degrees.
Literature Cited
Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, 2nd Ed., Chapter 3, McGraw-Hill, 1986.
De Souza, Paula Monteiro, and Pérola de Oliveira Magalhães. “Application of Microbial Α-Amylase in Industry – A Review.” Brazilian Journal of Microbiology41.4 (2010): 850–861. PMC. Web. 7 Mar. 2016.
Prado, Heloiza Alves Do, Aline Dos Reos, Erica Santos, and Raisa Sanches. "Fungal Amylase." Fungal Enzymes (2013). Web. 9 Mar. 2016.
Standard SKB method to determine enzyme activity: Cereal Chem., 16, 712, 1939.
Villas-Boas, Flávia, and Célia M.l. Franco. "Effect of Bacterial β-amylase and Fungal α-amylase on the Digestibility and Structural Characteristics of Potato and Arrowroot Starches." Food Hydrocolloids 52 (2016): 795-803. Web. 9 Mar. 2016.
