Laboratory Report No.

4
Enzyme Hydrolysis of Starch
 
 
Esconde, Yvonne Keithlene J.  BS Nursing 1A
Grp. 3 / Thurs (7:00–7:30 PM)      Sept. 6, 2018 
Rating: ________
 
I. Objectives
1. To identify the factors affecting enzyme catalysis.
2. To demonstrate enzyme-catalyzed hydrolysis of starch using amylase.
 
II. Introduction
Enzymes are synthesized in a living cell and speed up reactions efficiently. It decreased the
energy of activation. The effectiveness of enzymes as catalysts is demonstrated by the very high
reaction rates at physiological temperatures.
In the following experiments, the highly specific nature of the enzyme is shown. Enzymes are
extracted from plants and biological materials by different methods. Amylase is an enzyme
found in human saliva and pancreas. It is the digestive enzyme that is needed to break down
starch molecules. Amylase must be kept at certain conditions to function at its optimum level.
This experiment will explore the effect of pH, temperature, and disinfectant on the activity of
amylase to hydrolyze the starch.
Starch is mainly composed of two components, amylase (linear polymer of glucose), which
makes up 20-30% of the structure; and amylopectin (branched polymer of glucose).
Of the two components of starch, amylopectin presents the great challenge to hydrolytic enzyme
systems. This is due to the residues involved in a-1,6-glycosidic branch points which constitute
about 4 – 6% of the glucose present. Most hydrolytic enzymes are specific for a-1,4-glucosidic
links yet the a-1,6-glucosidic links must also be cleaved for complete hydrolysis of amylopectin
to glucose.
III. Materials
A. Equipment
• (1) Triple Beam • Spot Plate
Balance
• (2) 100-mL Beaker • Hot Plate
• (1) Thermometer • (6) 10-mL Test Tubes
B. Reagents
• Lysol Solution • NaHCO3
• 2% Starch • pH 4 Buffer Solution
• pH 10 Buffer Solution • pH 7 Buffer Solution
• (1) 25-mL Graduated • Amylase
Cylinder
• 0.2 M Citric Acid • 0.2 M KH2PO4
• 0.01 M NaOH • Iodine Solution
• 0.5% NaCl • Starch Solution
IIII. Methodology (Schematic Diagram)
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
V. Data and Results
Effect of pH
pH Change
in color
of iodine
at
different
time
interval
(minute)
1 2 3 4 5 6 7 8 9 10
4 Brown Brown Brown Brown Dark Dark Dark Dark Black Black
Brown Brown Brown Brown
7 Slightly Slightly Light Light Blue Blue Deep Deep Brownish Brown
Blue Blue Blue Blue Blue Blue Blue
10 Black Black Black Black Black Black Black Black Black Black
 
Effect of Temperature
Temperatur Change
e (°C) in color
of
iodine
at
differen
t time
interval
(minute
)
1 2 3 4 5 6 7 8 9 10
0 Brown Brown Brow Brow Dark Dark Dark Dark Black Black
n n Brow Brow Brow Brow
n n n n
37 Slightly Slightl Light Light Blue Blue Deep Deep Brow Brow
Blue y Blue Blue Blue Blue Blue n n
70 Black Black Black Black Black Black Black Black Black Black
 
Effect of Disinfectant
Disinfectan Change
t in color
of
iodine
at
differen
t time
interval
(minute
 )
1 2 3 4 5 6 7 8 9 10
Lysol Slightly Slightl Brow Brow Brow Brow Brow Brow Brow Blac
Blue y Blue n n n n n n n k
 
VI. Discussion
 
When put into the spot plate with a drop of iodine, the columns for pH 4 achieved a
brown color for the first 4 minutes, dark brown for the next 4, and then eventually
became black in the last 2 minutes of the trial. For pH 7, A bluish hue retained
throughout the 10 minutes except for the final one. pH 10’s reactions all remained
black.
The temperature tests yielded similar results, with 0°C getting a brown reaction for the
first 4 minutes, and getting darker down the column. 37°C achieved the same with pH
7, and 70°C with pH 10.
For the Lysol, meanwhile, a slightly blue color was observed for the first 2 minutes,
brown for the 3 – 9 minutes, and then black for the final spot.
 
VII. Conclusion
 
The group was able to observe the different factors affecting the enzyme activities and
reactions rates well during the experiment. It was concluded that the optimum
temperature for the enzymatic activity of salivary amylase is 37°C, and the optimum
pH is in its neutral state, or 7. It was observed that beyond these ranges, whether higher
or lower, the enzymes denature and the reaction rates suffer as a consequence. This is
backed by the fact that the tests with different temperatures and pH levels yielded a
variety of color reactions save for the 37°C and pH 7 ones, wherein they achieved
consistent hues.
 
VIII. Assessment (Q&A)
1. Do all enzymes in nature work best at pH 7 and 37°C? Explain.
 
- No. While enzymes in humans do work best in the environment stated above, there are some
enzymes that work better in higher or lower temperatures, depending on the climate the host has
adapted to (Santhosh, 2018). Though, as a general principle, temperature is directly proportional
to reaction rates and enzyme activities, but very high temperatures can denature the enzymes.
The same is true for pH. The optimum pH is different for every enzyme, as it changes their
shape, which determines their function and functionality, and could ultimately impair them in
certain levels (Chaplin, 2014).
 
2. Explain why heat, cold, and disinfectants prevent microbial growth.
 
- All bacteria have their own optimum environmental surroundings and temperatures in which
they thrive the most (Lumen, n.d.).  
- Heat denatures the proteins of the bacteria. It breaks down the hydrogen bond holding together
the bacterium’s shape and ultimately kills it (Lumen, n.d.).
- Freezing temperatures put bacteria in a dormant state as it slows down processes within the
microorganism. Repeated cycles of freezing and thawing can actually kill the bacterium through
the denaturing of their proteins. Also, freezing causes water to expand and form ice crystals,
ultimately rupturing the microorganisms (Bisht, 2011).
- Disinfectants are chemicals used to kill microorganisms, treated as an alternative to antiseptics
that can prevent growth and reproduction but never wholly destroy them (Global Safe
Technologies Australia, n.d.). How disinfectants prevent microbial growth depends on the type
used. For example. Alcohol can cause the clumping of cells; the cell membrane will lose its
structure and collapse, killing the bacterium. This is commonly known as clumping. Other
processes include coagulating, cross-linking, and oxidizing (Thompson, 2012).
 
3. What are the optimum conditions of the a-amylase enzymes as regard to temperature and
pressure?
 
- Optimum temperature ranges from 32 – 37°C (Santhosh, 2018), while its optimum pressure
was not tested for, or ever tackled, during the experiment.
IX. References

Bisht, S. C. (2011). Effect of low temperature on bacterial growth. Biotech articles. Retrieved

fromhttps://www.biotecharticles.com/Biology-Article/Effect-of-Low-Temperature-on-Bacterial-
Growth-721.html
Bisswanger, H. (2014). Enzyme assays. Perspectives in science, 1 (1-6), pp. 41 – 55. Retrieved
fromhttps://www.sciencedirect.com/science/article/pii/S2213020914000068
Britannica. (2018). Enzyme. Biochemistry. Retrieved
from https://www.britannica.com/science/enzyme
Castro, J. (2014). How do enzymes work? Live science. Retrieved
from https://www.livescience.com/45145-how-do-enzymes-work.html
Chaplin, M. (2014). Effect of temperature and pressure. Enzyme technology. Retrieved
fromhttp://www1.lsbu.ac.uk/water/enztech/temperature.html
Global Safe Technologies Australia. (n.d.). What are disinfectants? Retrieved
fromhttps://www.globalsafe.com.au/what_are_disinfectants.php
Khan Academy. (n.d.). Enzymes and the active site. Biology. Retrieved
fromhttps://www.khanacademy.org/science/biology/energy-and-enzymes/introduction-to-
enzymes/a/enzymes-and-the-active-site
Lumen. (n.d.). Temperature and microbial growth. Culturing microorganisms. Retrieved
fromhttps://courses.lumenlearning.com/boundless-microbiology/chapter/temperature-and-
microbial-growth/
Santhosh, L. (2018). The effect of temperature on enzyme activity and biology. Sciencing.
Retrieved from https://sciencing.com/effects-temperature-enzyme-activity-biology-6049.html
Thompson, K. M. (2012). The science of disinfectants. Cleaning and maintenance management.
Retrieved from https://www.cmmonline.com/articles/the-science-of-disinfectants

 
X. Certification / Conforme
 
 
 
Esconde, Yvonne Keithlene J. 
Signature over printed name
 
September 13, 2018
Date Submitted: