Abstract
Amylase from Bacillus Subtilis and Aspergillus oryzae were studied as representative candidates for bacterial and fungal enzymes, respectively. The activities of these two enzymes were studies comparatively over a period of 10 minutes and at temperatures of 0, 40, 60 and 95°C. The experiment was conducted to find the most effective temperatures for bacterial amylase and fungal amylase to catalyze starch hydrolysis. The data suggest that fungal enzyme was relatively more active, particularly in the first 6 minutes, than the bacterial counterpart at 0°C.
On the other hand, the fungal enzyme activity deteriorated more rapidly than that of the bacterial enzyme at higher temperatures of 40°C and 60°C. In contrast, the bacterial enzyme demonstrated higher activity at 40°C. As expected, there was minimal enzyme activity observed at 95°C, but inexplicably some minimal residual activity is notable with the fungal enzyme. No enzyme activity is notable for either enzyme at 0 time point. Starch hydrolysis was maximal in the first 2minutes and remained plateau from 0°C to 60°C for the fungal enzyme, implying that the fungal enzyme operates rather efficiently at lower temperatures. The bacterial enzyme activity showed clear temperature dependence from 0°C to 60°C. Both the enzymes were practically inactive at 95°C. Bohager (2006).
Introduction
Enzymes are proteins in nature and always help in catalyzing chemical reactions. Enzymes are very specific in nature and therefore only catalyses reactions involving certain substrates. This implies that such enzymes can only speed up a few reactions from among the many possibilities. It is prudent to note temperatures affects enzyme function by altering its shape which eventually changes its function because the substrate is not able to fit into the active site of the enzyme due to the altered shape. Therefore, changes in temperatures affect the enzymatic reaction. For instance, when temperatures are too high, enzymes becomes inactive or are denatured.
All chemical reactions by enzymatic activity require energy, and proteins that make up an enzyme are able to lower the activation energy in order to induce or accelerate the reaction vastly (Low et al 1973). The area of importance on an enzyme is the active site, which is where the chemical reactions take place producing final products. Substrates must be able to connect onto the active site of enzymes forming an enzyme-substrate (ES) complex in order to initialize a chemical reaction.
In order for an enzyme to work at peak capacity, it must be able to perform under optimal conditions. One of the key regulators of all enzymatic activity is temperature. All organisms metabolize optimally under a narrow range of the temperature setting in which it is set in. It is based on the genetically inherited protein tolerances of the enzymes that control and regulate the cell’s ability to perform all normal cell activities.
Temperature regulates the movement of substrates because of kinetic energy. At lower or colder temperatures, substrate movement slows down causing less interaction with an enzyme’s active site. This creates a static environment, which inhibits enzymatic activity. As the temperature increases past the optimum range, proteins start to become denatured which change the conformation or shape of the enzyme preventing the substrate from binding to its active site. Since the biological processes of all life depend on optimal temperature conditions, it must be considered greatly when studying certain organisms in order to better understand them.
The enzyme for the purpose of this lab report is the amylase; amylase enzyme is mainly made up of the pancreatic juice and the saliva which are vital for the breakdown of the long-chain carbohydrates like starch into smaller units. However, the primary function of the enzyme amylase is to breakdown starches into food so that they can be used by the body.
In order to better understand both organisms, the study compares temperature tolerances specifically on the hydrolysis of starch using their respective amylases. Both species of amylases have an optimal temperature range. Bohager (2006).
Fungi species generally prefer cooler, damper areas and A. oryzae prefer to grow on carbon-rich substances. Bacteria in general have a wide range of temperature tolerances, but B. lichenformis is specifically a thermopile, which have a higher temperature tolerance than most bacteria. When comparing the two, the fungi will demonstrate a lower temperature tolerance for starch hydrolysis and the bacteria will be able to hydrolyze the starch at higher temperatures. In addition to measuring temperature changes, reaction time will also be evaluated to compare differences between the two species.
At colder temperatures fungi will also demonstrate a faster reaction time than bacteria because of its high affinity for the carbon molecules in starch. As the temperature increases, the bacteria will demonstrate a faster reaction time as well as a higher rate of hydrolysis since the species optimally operates at 37°C.
This paper therefore fundamentally aims at comparing the effects of temperature on the two species of amylase: bacterial amylase and the fungal amylase. The study will examine how the two species of amylase react to environmental changes in temperature.
METHODS
Materials such as soluble starch spot plates, bacterial amylase from Bacillus subtilis, and fungal amylase from Aspergillus oryzae and iodine reagents were obtained from various commercial sources of high quality or analytical grade.
The activity of amylase enzyme was measured in a spot plate set up and was based on the starch-iodine test according to Xiao et al (Xiao, Storms, & Tsang, 2006). The assay mixtures of bacterial, fungal amylase and soluble starch (1.5%, w/v) were pre-incubated at different temperatures (0, 45, 60, 95°C) for 5 min to attain equilibrium. The reactions were initiated by mixing the contents of enzyme mixture with soluble starch. The activity was followed for 10 minutes at 2 min intervals (0,2,4,6,8, & 10) by removing an aliquot into a spot plate well labelled appropriately (ID: enzyme source, Temp & Time). There were essentially eight sets of reactions, four for bacterial enzyme (one for each temperature mentioned above) and four for fungal enzyme (again, one for each temperature mentioned above). The spot plates were positioned with paper napkins underneath for easy scoring of the activities. The plates were top labelled with temperatures and on the left side with times.
Each set of reactions was initiated individually and the aliquots at the indicated times were transferred into spot plate well containing 1/2 drops of iodine solution. The change in color in the spot plate wells was recorded. The process was repeated for all 8 sets of reactions.
RESULTS
For the purposes of verifying the results, the iodine solution was used in each test trial. Iodine as we all understand always turns blue black in the presence of starch. Before adding the starch solution to the amylase solution, a few drops were added to the iodine at each tested temperature so as to illustrate the visual indication of the presence of starch with zero hydrolysis conducted. As a control, it served as a reference point to make comparisons against the tested trials with the starch-amylase solution. As an indicator, it demonstrated the visual indication of starch hydrolysis into maltose. Bohager (2006).
Using data collected, the results of the study revealed the range of optimum enzyme activity and compared differences in temperature variation over a fixed period of time between the two species of amylase. The results are expected to correlate with each respective species’ natural habitat temperature environments. This will not only help explain their survival, but assist in replicating their natural environment in a laboratory setting in order to further study these species.
Figure 1: illustration of the presence of starch 1 represents the least amount of starch (maximum starch hydrolysis) and 5 represents the most of amount starch (minimum starch hydrolysis).
Figure 2: results of fungal amylase when subjected to different temperature range
Figure 3: results for bacterial amylase when subjected to different temperature range
The results were then analysed by a group of students after which the results obtained from different groups were tabulated as shown below.
Figure 4: analysis for the effect of temperature on fungi and bacteria amylase
The graphical presentation of the results was done to illustrate various points regarding amylase activity of fungal and bacterial sources. According to the results collected by different groups, the fungal enzyme shows that fungal enzyme was relatively more active, particularly in the first 6 minutes, than the bacterial amylase at 0°C as can be seen in graph 1. On the other hand, the fungal enzyme activity deteriorated more rapidly than that of the bacterial enzyme at higher temperatures of 40°C and 60°C that is according to the information available in graph 2 and 3. As expected, there was minimal enzyme activity observed at 95°C, but inexplicably some minimal residual activity is notable with the fungal enzyme (Graph 4). Gerald, Jim and Breithaupt (2003).
Graph 5 and 6 depict that no enzyme activity is notable for either enzyme at 0 time point. Starch hydrolysis was maximal in the first 2minutes and remained plateau from 0°C to 60°C for the fungal enzyme (Graph 7 and 8). The bacterial enzyme activity showed clear temperature dependence from 0°C to 60°C. Both the enzymes were inactive at 95°C.
Figure 5: Graph 1: Data points represent the average of 6 trials performed at 0°C.
Figure 6: Graph 2: Data points represent the average of 6 trials performed at 40°C.
Discussion
The data collected illustrates differences in amylase activity for the both species of amylase which were subjected to different temperature range under by different groups of students. The student who was subdivided into six different groups performed their experiment after which comparison was made. In order to accurately find out the effect of temperature on the two species of amylase, a total of 36 samples were used among six test trials to collect the data and class average values were calculated among the six test groups. Variances and standard deviations were also taken into consideration while doing the calculations.
Before the amylase and starch solutions were mixed, the control was tested and functioned as designed. The wells turned blue-black when the starch was added to the iodine. This was expected since iodine turns a blue-black color in the presence of starch illustrating no enzymatic activity.
As expected, the fungal enzyme demonstrated a lower and faster reaction time at 0°C. This can be seen by the steep slope on Graph 1 at the 2 min mark as it nears a colour of 2 for hydrolysis. The bacterial enzyme has a flatter slope as it near the color of 2 at the 10 min mark. This implies that despite the fact that the chemical reactions of both the enzymes are lower at temperature zero, the rate of reaction of the bacterial amylase is slightly higher at that zero temperature compared to the chemical reactions catalysed by the fungal amylase.
At the tested temperature of 40°C, the bacterial amylase shows a steeper slope illustrating faster reaction time as well as maintaining maximum hydrolysis at the 1 level. The fungal enzyme, in contrast, was only able to maintain a hydrolysis between 2 and 3. These results are consistent with the prediction made earlier that the bacterial species’ optimal enzyme activity is at 37°C. Therefore, it can be said that the optimum temperature for the bacterial amylase enzyme is between 370C +-30C. On the other hand, since the fungal enzyme was only able to maintain hydrolysis between two to three, it is clear evidence that the optimum temperature for the fungal amylase is slightly lower compared to that of bacterial amylase. Therefore temperatures at 400C denatures the fungal amylase hence altering its active site thereby slowing down the reaction.
The data from 95°C temperature showed surprising results. As illustrated in Graph 4, the fungal enzyme 95°C curve line shows a slope beginning at the 2 min mark at a value of 5.00 for color and ending at 3.92 at the 10 min mark. The bacterial enzyme was only able to hydrolyze the starch at a level of 4.83. These results are unexpected and inconsistent with initial predictions. It does however demonstrate that even though the bacterial species is a thermophile, there is a narrower range of optimum enzymatic activity as compared to the fungi species. Xiao Storms & Tsang (2006).
Comparison to the previous study
In the previous experiments done and which was almost the same as this experiments, the optimum temperature for the amylase was determined by varying the temperature from 10 to 800C. The enzyme was found to show maximum activity at 300C .Actually, this result is almost the same to the one obtained in this experiment of about 370C. The temperature optimum for the activity of α-amylase was thermostable enzyme and the thermostabilities are affected by many factors such as the presence of calcium, substrate and the other stabilizers. (Vihinen et al., 1989).
References
Kumar, A. (2004). Environment and Health. U.S.A: APH Publishing.
Gerald, D. Jim, A. Breithaupt, E. (2003). AQA Modular Science for GCSE, Book 1. U.S.A: Nelson Thornes.
Bohager, T. (2006). Enzymes : what the experts know! : your journey to health and longevity starts here. Prescott, Ariz.: One World Press.
Xiao, Z., Storms, R., & Tsang, A. (2006). A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Anal Biochem, 351(1), 146-148.
Low, P. S., Bada, J. L., & Somero, G. N. (1973). Temperature adaptation of enzymes: roles of the free energy, the enthalpy, and the entropy of activation. Proc Natl Acad Sci U S A, 70(2), 430-432. Radzicka, A., & Wolfenden, R. (1995). A proficient enzyme. Science, 267(5194), 90-93.
Vihinen M. Mantsala, P. microbial amylolytic enzymes. (1989). Crit. Rev. Biochem. Mol.Biol, 24:329-418.
