I.

Name:
(GROUP 05)
ORTONIO, Eunice Jireh F. (Leader)
LLANILLO, Piolo Angelo E.
MOPAL, Gerhard D.
NARVAZA, Donna Daniela A.
PABLICO, Jessica Lorenz M.
PAGUIA, Norhanna T.
PIZARRAS, Hannah Gale G.

II. Title of experiment and no.:

ENZYME, EXPERIMENT 3A

III. Objectives:
‣ Demonstrate the catalytic action of enzymes through different organic specimen.
‣ Recognize the specificity of enzymes to its substrate.
‣ Identify the products of each reaction though different color tests.
‣ Describe the role of enzyme in digestive processes.

IV. Theoretical Background:

Enzymes are an important protein in living organisms that are essential for the existence of life. Their role is to
speed up chemical reactions that are the foundation of bodily functions, including digestion, cell formation, and even waste
disposal. Without enzymes these chemical reactions would occur too slowly to support life. Thus, understanding enzymatic
reactions and how they affect chemical processes is crucial to better understanding how many of our bodily functions
happen.

An enzymatic reaction refers to a reaction in which an enzyme acts as a catalyst (Alberts et al, 2014). An enzyme
is a specialized protein that increases the rate of a specific chemical reaction by lowering the activation energy. Activation
energy is the energy a molecule requires to begin a chemical reaction (Alberts et al., 2014). An enzymatic reaction occurs
in two steps (Artioli, 2008). The enzyme first binds the substrate, a reactant, at its active site to form a substrate-enzyme
complex (Artioli, 2008). The substrate-enzyme complex then reacts (Artioli, 2008). The binding provides better chemical
conditions to activate the reaction and, in turn, lowers the activation energy (Artioli, 2008).

Amylase
Amylase is an enzyme that catalyses the hydrolysis of starch into sugars. Amylase is present in the saliva of humans
and some other mammals, where it begins the chemical process of digestion. Enzyme amylase is found in saliva which is
secreted by the salivary glands in the mouth palate. Amylase partially hydrolyses (breaks) starch or glycogen into glucose
and maltose. Salivary amylase acts at a temperature of 37°C and a pH of 6.6 (acidic).

Salivary amylase catalyzes the reaction, acting on starch as the substrate (Barrass, 1981). During the reaction, the
alpha-1,4 linkages between glucose units in starch are hydrolyzed (Sanderson & Walker, 1999) to form units of maltose, a
disaccharide and reducing sugar (Rostogi, 2005). This maltose becomes a source of energy for the body. The aforementioned
reaction occurs in the forward direction (meaning that the reactants, water and starch, collide to produce products) (Barrass,
1981).

In the experiment, we used a Benedict’s solution to determine the presence of maltose- the product of digestion of
salivary amylase and starch. A positive Benedict’s test confirms the presence of maltose, a reducing sugar (Kumar, 2007).
The initial solution for Benedict’s test is blue in colour. A precipitate ranging in colour from green, yellow, brown to red
then indicates the presence of maltose. If the solution remains the original blue colour of Benedict’s solution, the test for
presence of maltose is negative, meaning a reaction did not occur (Toole & Toole, 2004).

1 | 3A - Enzymes
Oxidase
Oxidase is an enzyme that catalyzes an oxidation-reduction reaction, especially one involving dioxygen (O2) as the
electron acceptor. In reactions involving donation of a hydrogen atom, oxygen is reduced to water (H2O) or hydrogen
peroxide (H2O2). One common example of this is the Catechol oxidase that is found widely in both plants and animals.
Catechol oxidase is also called polyphenoloxidase and in animals it is called tyrosinase. In this laboratory experiment, we
used a form of this enzyme that is found in potatoes. Potatoes are members of the family Solanaceae (so are tomatoes,
jimson weed, and deadly nightshade). Solanaceous plants originated in the new world, and the potato and its relatives were
first introduced to Europe in the 16th century. In the experiment, The catechol present in potatoes served as the substrate
(citeseerx.ist.psu.edu).

Catechol oxidase catalyzes the oxidation of catechol to benzoquinone and water. The reaction is shown below.
While catechol is colorless, benzoquinone has a brown color. The familiar browning of cut apples and potatoes to due to
the production of benzoquinone from catechol. The brown colored product of this reaction can be used to measure the
reaction rate. The browner the color of the solution indicates a higher enzymatic activity.

Protease
Proteolytic enzymes are a group of enzymes, the catalytic function of which is to hydrolyze the peptide bonds of
proteins. Proteases are commercially important enzymes, and it has been reported that approximately 60% of the total
worldwide market of enzymes is comprised of proteases .

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.In
the experiment, casein acts as a substrate. When the protease we are testing digests casein, the amino acids are liberated
along with other peptide fragments. Pepsin, which is used in the experiment, is a proteolytic enzyme of the stomach which
is normally responsible for less than 20% of the protein digestion that occurs in the gastrointestinal tract. When it interacts
with casein, it hydrolyzes the peptide bonds on it denaturing the protein (casein) causing it to produce coagulation or
precipitates (Jove.com). In the experiment, casein is the substrate while pepsin acts as an enzyme.

Rennin
Chymosin, known also as rennin, is a proteolytic enzyme related to pepsin that synthesized by chief cells in the
stomach of some animals. Its role in digestion is to curdle or coagulate milk in the stomach, a process of considerable
importance in the very young animal. If milk were not coagulated, it would rapidly flow through the stomach and miss the
opportunity for initial digestion of its proteins.

Rennin efficiently converts liquid milk to a semisolid like cottage cheese, allowing it to be retained for longer
periods in the stomach. Rennin secretion is maximal during the first few days after birth, and declines thereafter, replaced
in effect by secretion of pepsin as the major gastric protease. Rennin is secreted in the neonatal stomach of ruminants (cattle,
goats, camels), pigs, cats, and rats. Animals including humans, chimps, and horses have inactivating mutations in their
chymosin gene and do not secrete the enzyme.

2 | 3A - Enzymes
 Chymosin is secreted as an inactive proenzyme called prochymosin that, like pepsin, is activated on exposure to
acid. Chymosin or Rennin is also similar to pepsin in being most active in acidic environments, which makes sense
considering its mission. Rennin has an optimum pH of 3.4 for the proteolysis of bovine serum albumin1 and 3.8 for poly-
L-glutamic acid2. At pH values between 5 and 7 it will coagulate milk and slowly attack casein. It has maximum stability
at pH 5.4, while at values above 7 it loses activity rapidly. In the experiment, rennin served as the enzyme while casein on
the evaporated milk served as the substrate.

In the experiment, we used saturated ammonium oxalate on one of the test tubes. This chemical substance is known
to be an anticoagulant hence, an inhibitor in the process (Brittanica.com).

V. Data & Results

Table 1.1 AMYLASE (+)

With saliva and after a boiling water bath for
Without saliva
5 minutes.

After having a boiling water bath for 5

If without adding the saliva, the sample will minutes and adding the reagent, the sample
turn to a blue color. turned into a brownish-yellow color with an
orange precipitation that settled at the bottom

3 | 3A - Enzymes
 Table 1.2 OXIDASE (+)
Left side: Heated and Right side: Standard

(Left test tube) After having a water bath, the

The sample formed a dark brown precipitate
sample developed a lighter brown solution. It
at the bottom. The sample developed a red
had a light brown precipitate that settled at the
color after the reagents were added.
bottom.
Both of the samples developed a circular white precipitate at the bottom

Table 1.3 PROTEASE (+)

Left side: With Casein Right side: Without Casein

It developed a slight yellowish precipitation at It developed a very little feather-like

the bottom. precipitation at the bottom.

4 | 3A - Enzymes
 Table 1.4 RENNIN (+)

Left side: With Saturated Ammonium Oxidate Right side: Without Saturated Ammonium Oxidate
Right side: Without Saturated Ammonium Oxidate (after decant on a filter paper)

The sample remained the same The sample developed only a

Without Saturated Ammonium Oxidate that
and no precipitation was slight precipitation (white
developed a slight precipitation.
formed. precipitation).

VI. Data Analysis

In this experiment we experienced the action of some enzymes to its specific substrate and identify the products of
each reaction through different color test. Enzymes are known to be biological catalyst and one of its amazing property is
its high specificity, which means that it only acts to a specific substrate. The enzymes present in this experiment are Amylase
which catalyze the hydrolysis of starch, Oxidase catalyzes an oxidation-reduction reaction especially one involving
dioxygen as electron receptors, Protease which breaks down proteins and peptides and lastly Renin which is a protein
digesting enzyme that curdle or coagulate milk in stomach.

First on the enzyme amylase, there are two separate test tubes, one with saliva (amylase is found in saliva) and the
other without the saliva. The test tube without the saliva remained to be a color blue solution even after boiling. On the other
hand, the second test tube with saliva turned into a brownish-yellow solution with an orange precipitate. This indicates that
the first sample without the saliva remained color blue in solution because there is no activity present due to the absence of
enzyme amylase. If the amylase is inactivated or absent, it can no longer hydrolyze starch, so the blue color of the starch-
iodine complex will persist (Laney College, 2012). While the second test tube turned into a brownish solution with an
orange precipitate because this indicates that the sugar molecules began to break apart and release the iodine molecules due
to the presence of the enzyme amylase (UW Extension FYI, 2015). Therefore, we can say that the result of the experiment
is positive because a reaction occurred between the starch solution and the presence of enzyme amylase.

Second on the enzyme oxidase there two separate test tubes. The first test tube was placed on a water bath for 10
minutes while the second test tube was not placed in a water bath so we call it as our standard. The first test tube that was
placed on a water bath developed a lighter brown solution and a light brown precipitate that settled at the bottom, while the
standard sample developed a darker brown solution than the first test tube and its precipitation is dark brown. After all the

5 | 3A - Enzymes
reagents are added the samples turned brownish-red. Both samples have almost the same results aside from the level and
color of precipitation. The test tube that has been heated developed a brownish-red solution and a lighter-brown
precipitation, while the second test tube also developed a brownish-red solution with slightly darker-brown precipitation.
This is due to the factor that affected the enzymatic reaction which is the temperature. The test tube that has been heated in
a water bath may have denatured several proteins thus reducing the Enzyme Concentration level on the solution that resulted
to a lighter solution when it was heated and lighter color of precipitate after all the reagents has been added. According to
alevelnotes.com, Higher Enzyme Concentration increases the rate of reaction. This is because more enzyme molecules will
be colliding with substrate molecules, so more product will be formed. This is the reason as to why lower enzyme
concentration on one of the test tubes resulted to a lower enzymatic activity. Therefore, this experiment is positive because
both the samples performed an enzymatic reaction but only differed on the level of substrate concentration due to differing
temperature of both samples.

Next is the enzyme protease, on this part there are two separate test tubes, one with casein and the other without
casein. Note that the casein is our substrate and pepsin is our proteases because pepsin is a type of protease. The test tube
with casein developed a slight yellowish precipitation at the bottom while the test tube without casein developed only a very
little feather like precipitation at the bottom. This indicates that the first test tube reacted with the presence of casein and
developed a precipitation. This reaction is due to the presence of enzyme protease which broke down the proteins and
peptide bonds present in casein which explains the precipitation. On the other hand, the second test tube without casein only
developed a very little feather like precipitation because there is no presence of a substrate, which indicates that no reaction
occurred. For further explanation, an article of Khan Academy (2019) about enzyme review stated that increasing substrate
concentration also increases the rate of reaction to a certain point. In vice versa, this indicates that low substrate
concentration or an absence of substrate will decrease the of reaction to a certain point. Therefore, we can say that the result
of the experiment is positive because a reaction occurred between the enzyme protease and the casein in the first test tube
than the second test tube without the casein.

Lastly on the enzyme renin, there are two separate test tube the first test tube is with saturated ammonium oxalate
and the second test tube is without the saturated ammonium oxalate. The first test tube with the saturated ammonium oxalate
remains the same and no precipitation was formed while the second test tube without the saturated ammonium oxalate
developed a slight white precipitation. The first test tube with saturated ammonium oxalate did not perform any reaction
despite the presence of an enzyme and a substrate, this is due to the presence of an inhibitor which is the saturated ammonium
oxalate. Saturated ammonium oxalate is known as anticoagulant, which explains the absence of precipitation in the first test
tube because the inhibitor prevented the enzyme rennin to coagulate the milk. While the second test tube, without the
saturated ammonium oxalate developed a precipitation because the enzyme rennin coagulated the milk without any
inhibitors in the activity. Therefore, we can say that this experiment is positive because an activity occurred between the
enzyme rennin and the substrate. And also, the inhibitor saturated ammonium oxalate did decrease the activity of the enzyme
on the other test tube.

VII. Conclusion
This experiment aimed to involve various enzymatic activity that governs in the digestive processes in our body.
Amylase, Oxidase, Protease, and Rennin are four enzymes that are highlighted in the experiment. With these, we observed
the catalytic action of enzymes through different organic specimens provided. We recognize the specificity of enzymes to
its substrate and identify the products of each reaction through different color tests. We also identified the role of these
enzymes in the digestive processes.

Through different color tests, we were able to distinguish different enzymatic activity and its specificity. Amylase,
that is present in our saliva, is an enzyme that catalyses the hydrolysis of starch into sugars. This activity enables the starch
to be broken into maltose-the product of the activity which was evident in the change of color in our sample, from blue to
brownish- yellow solution. Oxidase, on the other hand, is an enzyme that catalyzes an oxidation-reduction reaction. Catechol
oxidase, which is found in potatoes was specifically used. The sample differed when the other test tube was exposed to heat
that may have denatured some proteins lowering the enzyme concentration of the sample thus also lowered the enzymatic
activity. Lastly, Rennin is a proteolytic enzyme related to pepsin that is synthesized by chief cells in the stomach of some

6 | 3A - Enzymes
animals. Its role in digestion is to curdle or coagulate milk in the stomach. One of the samples was added with saturated
ammonium oxalate, an anticoagulant which acted as an inhibitor that hindered the sample to produce precipitates.

Enzymes catalyze chemical reactions in various ways depending on what enzyme it is. Each of these enzymes are
specific to its substrates. Take the salivary amylase as an example. It reacts to its substrate, the starch which it catalyzes to
reduce to its disaccharide form, maltose. These enzymes are vital in one’s life as it interacts with our digestive processes,
chemical balance that drives different functions in our body and etc. With this experiment also, temperature and inhibitors
were explored which meddles with the normal enzymatic activity.

REFERENCES

Cupp-Enyard, C. (2008) Sigma’s Non-specific Protease Activity Assay - Casein as a Substrate. J. Vis. Exp. (19), e899,

doi:10.3791/899 (2008). Retrieved from: https://www.jove.com/video/899/sigma-s-non-specific-protease-

activity-assay-casein-as-a-substrate

Encyclopædia Brittannica. (n.d). Rennin Enzyme. Retrieved from: https://www.britannica.com/science/rennin

Khan Academy. (2019). Enzymes review. Retrieved from: https://www.khanacademy.org/science/high-school-

biology/hs-energy-and-transport/hs-enzymes/a/hs-enzymes-review

7 | 3A - Enzymes
Laney College. (2012). Experiment 10 Enzymes. Retrieved from:https://laney.edu/cheli-fossum/wp-

content/uploads/sites/210/2012/01/10-Enzymes.pdf

Schultz, D.L. (2006). 12 Biology 155 General Biology I Laboratory Supplement. 78 pp. . Retrieved from:

http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.566.9042&rep=rep1&type=pdf

UW Extension FYI. (2015). Digestive enzyme: Amylase. Retrieved from:

https://fyi.extension.wisc.edu/wi4hstem/files/2015/02/AnimalDigestion-Amylase.pdf

Writer Online Canada. (2013). Manipulation of Enzymes and Enzymatic Processes. Retrieved from:

http://writeonline.ca/media/documents/LabReport-AnnotatedFull.pdf

8 | 3A - Enzymes