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The Abstract should be an

autonomous summary

of the entire report. It serves to help

readers determine how

relevant the report is to their own interests. This section is brief, only one paragraph, in which the author indicates

what was done, the reasoning behind it, the results and the conclusions.

It must highlight only the most important

elements of each major sections of the report (Introduction, Methods, Results, and Discussion).

The scientific report

can be summarized i

nto an abstract

with

four types of statements: purpose statements that are general

in

describing

the importance and/or goals of the research; methods statements that explain what was done and how it was done;

results statements that describe what informati

on or data was acquired; and

discussion/conclusion statements that

explain what the information or data probably means and what conclusions are drawn. Only the most important

aspects of the report should make it to the abstract.

This sectio

n should be

between 200 and 250

words in length

. This section should contain a clear summary of what

was demonstrated, how each part of the lab was carried out and how conclusions were reached. This section should

contain one or two purpose statements (wit

hout saying “The purpose of this experiment is…”), a complete summary

of each experiment (method statements) in a few sentences, and brief, accurate explanations of the results. The final

sentences should be the concluding statements.

Methods:
First, mark a piece a paper to keep track of the temperatures on the experiment you will be doing, label
0, 25, 55, and 85 degrees Celsius. On the side of the paper mark starting time followed by two minute
intervals until you get to 10 minutes. For each temperature, you will perform the same experiment;
therefore, doing the same thing four times with four different temperatures. Each temperature setting
requires 6 test tubes or 24 total. Mark all test tubes except four with the letter “S” these tubes will have
the iodine added at their respective times to collect the necessary data. Start the first unmarked tube at
0 minutes with 1 mL of bacterial amylase. After the amylase is set at the temperature, add 1.5 mL of the
starch solution and start timer. You will repeat this step every two minutes until you reach ten minutes
in the four unmarked tubes. After five minutes, place all tubes in their respective water bath
temperatures to begin the experimental reaction. In the tubes labeled with an “S” add 2-3 drops of
iodine starting with the 0 minute tube, then at 2 minutes add another 2-3 drops in the respective tube
and repeat at every two minute interval. After the ten minutes is up, take a sample and match it to the
tube that is not labelled with an “S”. When this complete, repeat all steps using fungal amylase opposed
to bacterial amylase. Create graphs with the collected data from both experiments and use the graph
data to compare to each other.
Bacterial Data:
0
2
4
6
8
10
25
55
85
0 Degrees Degrees
Degrees
Degrees
4.92
4.92
4.67
4.83
5
4.67
3.67
4.25
5
4.67
3.58
4.08
5
4.75
3.5
3.83
4.8
4.42
3.25
3.75
4.8
4.5
3.12
4
Fungal Data:
Fungal Starch Concentration over Temp
10
8
Minutes
0
2
4
6
8
10
25
55
85
0 Degrees Degrees
Degrees
Degrees
4.5
4.7
4.3
4.5
4.75
4.5
4.75
4.58
4.92
4.5
4.5
4.25
5
4.58
4.58
4.17
4.92
4.42
3.92
4.2
5
4.3
3.92
4
6
4
2
0
0
1
2
3
4
5
Starch Concentration
85 Degrees
55 Degrees
25 Degrees
0 Degrees
6
Results:
After creating the graphs it was concluded that the bacterial amylase supported the hypothesis.
However, after further inspection of the graphs it showed fungal amylase had a trend once the optimal
temperature had been reached there was no noticeable difference in the starch concentration. The
fungal graph demonstrated as the temperature got hotter the data increased, and the bacterial graph
demonstrated as the temperature got hotter the data decreased until it got to 85 degrees and suddenly
increased. Both proteins were denatured in each experiment.
1
Running head: ENZYME LAB
Effect of Temperature on Amylase Activity
Introduction
Enzymes are biological catalysts that alter the rate of biochemical processes. They lower
the activation energy to increase the rate of biochemical processes. They bind to specific sites on
1
ENZYME ASSAY
2
substrates. Enzymes control the rate of catalytic reactions in living organisms. Chemical catalysts
also increase the rate of chemical reactions by lower the activation energy. Commercial enzymes
such as papain, malt diastase, and bromelin ficin originate from plants, animals, and
microorganisms. The pancreas secretes exocrine enzymes such as amylase, lipase, trypsin, and
chymotrypsin to digest carbohydrates, lipids, and proteins respectively. The movement of food in
the gastrointestinal tract (GIT) stimulates the secretion of digestive enzymes to break down
complex food components into simple molecules that the body can utilize (Ge et al., 2017).
Some microorganisms grow in culture media containing complex carbohydrates such as
starch and inulin. They secrete exocrine enzymes to convert carbohydrates in the growth medium
to simple components that they can use as an energy source (the primary carbon source).
Isolation of pure colonies of bacteria that can ferment complex carbohydrates through substrates
containing such sugars. Secretion of specific enzymes by microbes is useful during biochemical
characterization. Most Bacillus species secrete extracellular amylase enzymes when cultured in
substrates containing starch as a survival mechanism (Arsalan & Younus, 2018).
Fluctuations in temperature, pH, enzyme concentration, substrate concentrations, and
presence of enzyme inhibitors affect enzyme activity. Enzymes are proteinous and therefore
work best at optimum temperature and pH. High temperatures coagulate the protein component
but low temperature inhibits their activity. They work best at either acidic, basic, or neutral pH.
An increase in enzyme concentration increases the rate of a biochemical reaction. An increase in
substrate concentration above the equilibrium does not increase the rate of biochemical reaction
but extends the time needed for complete hydrolyzation of the substrate. Enzyme inhibitors
compete for the active site with enzymes and therefore lower enzymatic activity. Simulations of
natural conditions such as the provision of optimum environmental conditions such as
temperature and pH when conducting in-vitro enzyme bioassays increase experimental success
rate (Tomaszewski, Cema, & Ziembińska-Buczyńska, 2017).
UV-VIS Spectrophotometer is referenced to eliminate zero error before recording the
absorbance of other bacteria and fungi tubes. Absorbance and concentration values of glucose
standards generate a standard curve for the determination of unknown concentrations. Earlier
reports indicate that most pathogenic bacteria thrive well at 37oC while the fungi thrive well at
25oC. The peak activity of fungal and bacterial amylase was expected at 25oC and 37oC
respectively. The control tube with the only substrate is expected to record the highest starch
concentration throughout the incubation period. This experiment investigated the impact of
temperature variations on bacterial and fungal amylase activity. The study also examined the
relationship between incubation time and starch hydrolysis rate. We believed that bacteria and
fungi secrete extracellular amylase enzymes. Iodine reacts with starch to form a blue-black
ENZYME ASSAY
3
compound. Ultrasonication of tubes during incubation increases the rate of carbohydrate
catabolism (Sethupathy & Sivashanmugam, 2019).
Discussion
This experiment explored the relationship between enzyme activity and temperature.
Consistent high levels of starch in the control tube throughout the incubation period due to lack
of amylase to hydrolyze the starch to glucose. The optimum temperature for bacterial amylase is
55oC because tubes incubated at that temperature had the lowest starch concentration. Tubes
incubated at 55oC for 10 minutes had the lowest starch concentration because the enzymes had
sufficient time to catabolize starch to glucose. Starch concentration remained high in all the tubes
at 0oC. This could be due to the inactivation of enzymes by the low temperature. Inactivated
enzymes could not hydrolyze the starch. The concentration of starch was also high in tubes
incubated at 85oC due to the enzyme denaturation. The enzyme activity was moderate at 25oC
because the temperature is within the range at which it works without peak performance.
The optimum temperature for fungal amylase activity is 25oC because all the tubes
incubated at that temperature had the lowest concentration of starch. Starch concentration
remained high in all the tubes incubated at 0oC due to enzyme inactivation by the low
temperature. Inactivated enzymes cannot hydrolyze starch solution. Starch concentration was
lowest in the tube incubated at 25oC for 10 minutes because the enzymes had sufficient time to
digest starch to glucose. The starch concentration was also high in tubes incubated at 85oC
because of enzyme denaturation by high temperature. Denaturation interferes with protein
structure and reduces their activity. Tubes incubated at 55oC also had moderate starch
concentration. This is a clear indication that catabolism occurred at a slow rate due to
unfavorable temperature to enzyme activity.
Incubation for an extended period provided sufficient time for starch hydrolysis. Fungal
and bacterial tubes incubated for 10 minutes recorded the lowest concentration of starch. This is
a clear indication that the enzymes bind to substrate and form an enzyme-substrate complex. The
release of an enzyme from the complex creates additional enzyme activity. Enzymes only speed
up the rate of biochemical reaction but they are not used in the reaction. The enzymes are reused
to catalyze the starch catabolism. The data generated in this bioassay supported my hypothesis.
There is a direct correlation between the optimum temperature for survival of fungi, bacteria, and
amylase enzyme from both microbes. The optimum temperature of fungal amylase was 25oC.
The inactivation and denaturation temperature for amylase was similar to values in other
scientific publications. The optimum temperature for survival of bacteria and performance of the
bacterial amylase enzyme used in this enzyme assay was similar. The optimum temperature was
close to 55oC in both. The inactivation and denaturation temperature for bacterial amylase
ENZYME ASSAY
4
happened at 0oC and 85oC respectively. Some bacteria and fungi secrete amylase enzymes to
hydrolyze starch to a form they can utilize. The enzymes work within a narrow range of
temperature because the proteinous component is sensitive to temperature changes. Extended
incubation provides sufficient time for catabolism of starch by the amylase enzyme. The data
generated in this bioassay supported my hypothesis.
References
ENZYME ASSAY
5
Arsalan, A., & Younus, H. (2018). Enzymes and nanoparticles: Modulation of enzymatic
activity via nanoparticles. International Journal of Biological Macromolecules, 118, 1833–1847.
https://doi.org/10.1016/j.ijbiomac.2018.07.030
Ge, T., Wei, X., Razavi, B. S., Zhu, Z., Hu, Y., Kuzyakov, Y., … Wu, J. (2017). Stability
and dynamics of enzyme activity patterns in the rice rhizosphere: Effects of plant growth and
temperature. Soil Biology and Biochemistry, 113(September 2018), 108–115.
https://doi.org/10.1016/j.soilbio.2017.06.005
Sethupathy, A., & Sivashanmugam, P. (2019). Investigation on ultrasonication mediated
biosurfactant disintegration method in sludge flocs for enhancing hydrolytic enzyme activity and
polyhydroxyalkanoates. Environmental Technology (United Kingdom), 40(27), 3547–3560.
https://doi.org/10.1080/09593330.2018.1481887
Tomaszewski, M., Cema, G., & Ziembińska-Buczyńska, A. (2017). Influence of
temperature and pH on the anammox process: A review and meta-analysis. Chemosphere, 182,
203–214. https://doi.org/10.1016/j.chemosphere.2017.05.003
Running head: ENZYME LAB ANNOTATED BIBLIOGRAPHY
1
Jesus W. Felipe
Tomaszewski, M., Cema, G., & Ziembińska-Buczyńska, A. (2017). Influence of temperature and
pH on the anammox process: a review and meta-analysis. Chemosphere, 182, 203-214.
Retrieved from: https://www.sciencedirect.com/science/article/pii/S0045653517306975
I will use this source to gather more data about the effects of temperature and pH on
enzymes. Fundamentally, this article briefly discusses the anammox process, and why it is a very
efficient and economic wastewater treatment technology, since its discovery back in 1995.
Additionally, from the article, the anammox is used as an enzyme and therefore, the authors in
the article tries to analyze how the anammox is affected by both temperature and pH. In this
regard, I will use this article when trying to analyze the effects of temperature and pH on
enzymes to make the appropriate conclusions.
ENZYME LAB ANNOTATED BIBLIOGRAPHY
2
Sethupathy, A., & Sivashanmugam, P. (2019). Investigation on ultrasonication mediated
biosurfactant disintegration method in sludge flocs for enhancing hydrolytic enzymes
activity and polyhydroxyalkanoates. Environmental technology, 40(27), 3547-3560.
Retrieved from: https://doi.org/10.1080/09593330.2018.1481887
For my lab, I will use this source to gather data on how the biological effects of
ultrasound on microbial growth and enzyme activity are used to lose cell bunches formed in the
process of microbial culture. In this regard, I will use this source to discuss how ultrasound as an
emerging trend in the world, can be used to promote the growth of microbial cells. In addition, I
will also be in a pole position to make a strong conclusion on the different factors that can affect
the enzyme activities. This article will therefore, very useful to me when writing the
introduction, and formulating the lab objectives.
Ge, T., Wei, X., Razavi, B. S., Zhu, Z., Hu, Y., Kuzyakov, Y., … & Wu, J. (2017). Stability and
dynamics of enzyme activity patterns in the rice rhizosphere: effects of plant growth and
temperature. Soil Biology and Biochemistry, 113, 108-115. Retrieved from:
https://doi.org/10.1016/j.soilbio.2017.06.005
This article gives a detailed analysis of both stability and dynamics of enzyme activity
pattens. In this case, I will use this article to help me come up with detailed background of
enzyme activity patterns. Additionally, I will also use this information to come up with an
appropriate procedure that I will follow during the experiment. Besides, it is through the
designed procedure that the materials to be used during the experiment will be determined.
Finally, I will also use this article when doing my analysis, to make a conclusion on how
enzymes can be affected by different factors.
Arsalan, A., & Younus, H. (2018). Enzymes and nanoparticles: Modulation of enzymatic activity
via nanoparticles. International journal of biological macromolecules, 118, 1833-1847.
Retrieved from: https://doi.org/10.1016/j.ijbiomac.2018.07.030
This article will be very paramount in helping me decide what kind of data I would be
required to collect. Notably, enzymes being specific in their substrates, this article will help me
develop the basic knowledge required to explain how enzyme catalysis increase the rate of
reaction. Therefore, I would say that this article will also be of importance when analyzing the
different factors that influence the catalysis by an enzyme.
ENZYME LAB ANNOTATED BIBLIOGRAPHY
3
References
Arsalan, A., & Younus, H. (2018). Enzymes and nanoparticles: Modulation of enzymatic activity
via nanoparticles. International journal of biological macromolecules, 118, 1833-1847.
Retrieved from: https://doi.org/10.1016/j.ijbiomac.2018.07.030
Ge, T., Wei, X., Razavi, B. S., Zhu, Z., Hu, Y., Kuzyakov, Y., … & Wu, J. (2017). Stability and
dynamics of enzyme activity patterns in the rice rhizosphere: effects of plant growth and
temperature. Soil Biology and Biochemistry, 113, 108-115. Retrieved from:
https://doi.org/10.1016/j.soilbio.2017.06.005
ENZYME LAB ANNOTATED BIBLIOGRAPHY
4
Sethupathy, A., & Sivashanmugam, P. (2019). Investigation on ultrasonication mediated
biosurfactant disintegration method in sludge flocs for enhancing hydrolytic enzymes
activity and polyhydroxyalkanoates. Environmental technology, 40(27), 3547-3560.
Retrieved from: https://doi.org/10.1080/09593330.2018.1481887
Tomaszewski, M., Cema, G., & Ziembińska-Buczyńska, A. (2017). Influence of temperature and
pH on the anammox process: a review and meta-analysis. Chemosphere, 182, 203-214.
Retrieved from: https://www.sciencedirect.com/science/article/pii/S0045653517306975

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