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GROUP MEMBERS: Fernandez, Ashley Anne; Bello, Crhistal Jane; Dorimon,

Jonathan; Simbajon, Dennis; Zita, Jesselle Ann
DATE: October 14, 2024 TEACHER: Joevennel P. Escare
SUBJECT: Chem4 (Biochemistry) Laboratory

EXPERIMENT NO.: 4
Enzymes

OBJECTIVES:
 Understand the catalytic role of enzymes in biological processes.
 Demonstrate practical applications of enzyme reactions
 Examine factors influencing enzyme activity

PRE-LABORATORY DISCUSSION:
1. What is the effect of pH on enzyme activity?
a) Enzymes have an optimal pH at which they function best.
b) Enzymes are not affected by pH.
c) Enzymes are most active at very high pH.
d) Enzymes are most active at very low pH.

2. What is the name of the molecule that an enzyme acts upon?

a) Catalyst
b) Substrate
c) Product
d) Activator

3. What is the effect of temperature on enzyme activity?

a) Enzyme activity increases with increasing temperature.
b) Enzyme activity decreases with increasing temperature.
c) Enzyme activity remains constant with increasing temperature.
d) Enzyme activity first increases then decreases with increasing temperature.

4. Which of the following is NOT an example of an enzyme?

a) Amylase
b) Catalase
c) Invertase
d) Glucose

5. Which of the following enzymes is responsible for breaking down starch?

a) Pepsin
b) Amylase
c) Lipase
d) Protease
DISCUSSION/THEORIES INVOLVED:
An enzyme is a protein biomolecule that acts as a biocatalyst by regulating
the rate of various metabolic reactions without itself being altered in the process.
The name ‘enzyme’ literally means ‘in yeast’, and this was referred to denote
one of the most important reactions involved in the production of ethyl alcohol and
carbon dioxide through the agency of an enzyme zymase, present in yeast.
Enzymes are biological catalysts that catalyze more than 5000 different
biochemical reactions taking place in all living organisms. However, these are
different from other catalysts which are chemical and can last indefinitely. Enzymes
are proteins that are prone to damage and inactivation. Enzymes are also highly
specific and usually act on a specific substrate of specific reactions.

Amylase
It is an enzyme that occurs naturally in the saliva of some mammals and
humans that aids in the process of digestion. It accelerates the breakdown or
hydrolysis of starch into simple sugars. The pancreas and the salivary glands mainly
synthesize amylase to hydrolyze dietary starch into disaccharides and trisaccharides
that are converted into glucose and used as energy.
Amylase was one of the first enzymes to be discovered in the 1800s. It was
initially named diastaste but later renamed amylase in the late 20th century.

Classification of Amylase
There are three types of amylases known: alpha, beta and gamma. All three
are found in different organisms and catalyse different sites of the starch molecule.

ɑ-AMYLASES are

calcium metalloenzymes that cleave random α-1,4 glycosidic bonds to yield either
maltose and maltotriose from amylose chains or glucose, maltose and dextrin from
amylopectin chains.
In humans, amylase secreted from the pancreas and salivary gland is ɑ-

work more quickly than the 𝛃-amylases. Their optimum pH is 6.7 – 7.0. They are a
amylases. Because they can break random bonds in the starch chain, they tend to

member of the glycosidic hydrolase family 13.

 The 𝛃-AMYLASES are found in microbes and plants. They hydrolyse the
second α-1,4 glycosidic bond in the starch molecule and yield two maltose molecules
at a time. They are a member of the glycosidic hydrolase family 14.

gives a sweet taste to the fruits. Their optimum pH is 4.0 – 5.0. 𝛃-amylases are
At the time of ripening of fruits, the starch is hydrolysed into maltose which

found in seeds in an inactive form prior to germination.

𝛄-AMYLASES are found in plants and animals. They cleave the last α-1,4
glycosidic bond and the α-1,6 glycosidic bond in the starch molecule to yield glucose
molecules. Their optimum pH is 3. They are a member of the glycosidic hydrolase
family 15.

The enzyme ɑ-amylase finds a great deal of use in brewing liquor and beer
that is made from starch. It is achieved through a process of fermentation, where the
yeast consumes sugar and yields alcohol. In breadmaking, yeasts, which already
secrete amylase, break down the starch in the flour into carbon dioxide and ethanol,
giving rise to the bread and also adding flavour. In molecular biology, amylase can
be used as a method of selection for antibiotic gene resistance.
The blood serum amylase level is tested for various diagnostic purposes. A
higher concentration of amylase (hyperamylasemia) can be indicative of acute
pancreatitis, strangulation, peptic ulcer, ileus or mumps.
(Ravendraan, 2001)

Catalase
As the name suggests, catalase is an enzyme bringing about the catalysis of
a reaction through which hydrogen peroxide is decomposed into oxygen and water.
Catalase is responsible for preventing the accumulation of cellular organelles and
safeguards these organelles and tissues from any destruction by the peroxide as
peroxide is continuously synthesized by a number of metabolic reactions. Catalase is
mainly found in the liver of mammals.
Commonly found in almost all living entities enabled with oxygen exposure,
catalase is pivotal in preventing cells from oxidative damage by the ROS (reactive
oxygen species). This antioxidant enzyme is found in all aerobic entities and
catalyzes in an energy-efficient manner in the cells uncovered to environmental
stress.
In higher plants, they are situated in all the major sites of hydrogen peroxide
production in the cellular components such as chloroplast, mitochondria, cytosol and
peroxisomes. A factor which indicates its versatility in the plant system is the
existence of catalase isozymes in multiple molecular forms.
For the sustenance of life on earth, oxygen is pivotal. When the body uses
oxygen, it constantly generates free radicals. These free radicals are atoms or
molecules that are known to be chemically unstable. In addition to this, they also
lead to the instability of other atoms or molecules present in the body which
subsequently causes damage to cell membranes, proteins and even the DNA
structure. The phenomena can also result in permanent cellular and tissue damage
causing joint and heart diseases, infections, depressed immunity systems, mental
decline and so on.
This is where catalase pitches in, it has a role to play in fighting against the
threat that free radicals pose to the body. It converts toxic superoxide radicals into
hydrogen peroxides which later decomposes it into oxygen and water.
(Ravendraan, 2024)

Catecholase
Catechol is oxidized by catechol oxidase in the presence of oxygen to form
benzoquinone, which, on exposure to air, forms melanin. This enzyme is also known
by other names, such as tyrosinase, diphenol oxidase and polyphenol oxidase.
Potatoes, apples and bananas contain catechol oxidase which acts on colorless
catechol and converts it to brown-colored melanin. The browning that occurs when
you cut and expose these items to air is a result of this reaction.
Catechol oxidase may be extracted from bananas or potatoes. Mash a
banana with twice the volume of water in a mortar and pestle. Alternatively, blend a
banana with water to obtain the catechol oxidase extract. Filter through butter muslin
and store in the refrigerator. If you use potatoes, peel and chunk them, then blend at
a high speed using 700 ml of cold, distilled water. Filter this potato juice through
cheesecloth and refrigerate.
(Stewart, 2004)

Invertase
It is an enzyme, when added to sucrose (table sugar) or foods that include
sucrose, invertase splits the sugar into its component parts of glucose and fructose.
It is commonly called "invert sugar" or "inverted sugar syrup." Inverted sugar is
frequently used in commercial baking and candy recipes because it keeps baked
goods moist for longer periods of time.
Chemists during the 1800s were studying the effect of yeast on sugar and
realized that before the sugar began fermenting, it changed form. After much
research, the chemists isolated the enzyme that caused this: invertase. By the year
1900, the process for deriving invertase from yeast was commonly used. Over the
course of the next 20 plus years, chemists found many uses for invertase, most
importantly in candy-making.

Invertase Uses
When invertase is added to sugar candy recipes, like fondant candy fillings, it
gradually liquefies the fondant. This is one way of producing the liquid center in
candies like cherry cordials. The reaction takes a few days to occur, so there is a
waiting period when making liquid centers with invertase. This enzyme also makes
fondant appear smoother.
Although it sounds like something made in a lab, invertase is a part of many
different natural processes. Besides bees, we actually have our own supply of
invertase as part of our saliva.
(Labau, 2022)

Papain
Papain is a proteolytic enzyme extracted from the raw fruit of the papaya
plant. Proteolytic enzymes help break proteins down into smaller protein fragments
called peptides and amino acids. This is why papain is a popular ingredient in meat
tenderizer.
You can get papain from eating raw papaya. Papain is also available in
topical, chewable, and capsule forms. You can purchase papain-only supplements or
supplements that pair papain with other enzymes, such as bromelain.
 The extraction process of papain involves collecting the latex from the unripe
papaya fruit and subjecting it to various purification steps to obtain a refined and
concentrated enzyme. The resulting papain is a yellowish-white powder that is highly
soluble in water. It possesses a wide pH range for its activity, with an optimal range
of 6.0 to 7.0. It is important to note that the potency and activity of papain can vary
depending on factors such as the source, extraction method, and processing
conditions.
Papain exhibits several remarkable properties and functions that contribute to
its effectiveness and versatility. It is a highly efficient proteolytic enzyme, meaning it
has the ability to break down proteins into their constituent amino acids. This
property makes papain an excellent aid in digestion, as it helps to break down
complex proteins in the stomach, facilitating better nutrient absorption.
(Mcdermott, 2024)

Pectinase
Pectinases are a group of enzymes that breaks down pectin, a polysaccharide
found in plant cell walls, through hydrolysis, transelimination and deesterification
reactions. Commonly referred to as pectic enzymes, they include pectolyase,
pectozyme, and polygalacturonase. It is useful because pectin is the jelly-like matrix
which helps cement plant cells together and in which other cell wall components,
such as cellulose fibrils, are embedded. Therefore, pectinase enzymes are
commonly used in processes involving the degradation of plant materials, such as
speeding up the extraction of fruit juice from fruit, including apples and sapota.
Pectinases have also been used in wine production since the 1960s. The function of
pectinase in brewing is twofold, first it helps break down the plant (typically fruit)
material and so helps the extraction of flavors from the mash. Secondly the presence
of pectin in finished wine causes a haze or slight cloudiness. Pectinase is used to
break this down and so clear the wine.
Pectinases can be extracted from fungi such as Aspergillus niger. The fungus
produces these enzymes to break down the middle lamella in plants so that it can
extract nutrients from the plant tissues and insert fungal hyphae. If pectinase is
boiled it is denatured (unfolded) making it harder to connect with the pectin at the
active site, and produce as much juice.
(International Journal of Biological Macromolecules, 2024)

Pepsin
Pepsin is the principal acid protease of the stomach. It is generally recognized
as the first enzyme to be discovered (in the eighteenth century) and was named by
T. Schwann in 1825. Pig pepsin was the second enzyme, after urease, to be
crystallized.
Pepsin is a crucial digestive enzyme that plays a significant role in breaking
down proteins in the stomach. It is produced by the chief cells in the gastric glands in
an inactive form called pepsinogen. The activation of pepsinogen occurs in the acidic
environment of the stomach (typically at a pH of 1.5 to 2), where it is converted into
active pepsin. This acidic condition not only activates the enzyme but also facilitates
the cleaving of peptide bonds in proteins, transforming them into smaller peptides
that can be further digested in the intestines.
(Levy, 2018)
Rennin
The renin-angiotensin system began in 1898 with the studies made by
Tigerstedt and Bergman, who reported the pressor effect of renal extracts; they
named the renal substance renin based on its origin.
Renin is an enzyme made by special cells in your kidneys. It’s part of the
renin-angiotensin-aldosterone system — a chain reaction designed to regulate your
blood pressure. Specifically, renin controls the production of aldosterone, a hormone
made by your adrenal glands. Blood pressure regulation is the main function of
renin. It works together with angiotensin and aldosterone to manage the levels of
sodium and potassium in your body.
(Trerattanavong, 2023)

MATERIALS/CHEMICALS AND APPARATUS NEEDED:

Amylase
Apparatus/Materials Reagents/Chemicals
1. Wax pencil 1.Starch-agar plates (0.2% soluble starch,
2% agar)
2. Sharp razor blade 2. Corn grain
3. Saliva
4. Distilled water (in wash bottle)

Catalase
Apparatus/Materials Reagents/Chemicals
1. Test tubes (one for each material 1. Hydrogen peroxide (H2O2) (3% solution)
to be tested plus extra for control)

2. Assorted living tissue: sliced raw potato,

ground meat, liver, yeast cells, ground
young leaves
3. Assorted non-living material: piece of
baked potato or cooked liver, etc. (Use
caution with rocks or
sand, some will “bubble.”)

Catecholase
Apparatus/Materials Reagents/Chemicals
1. Electric blender 1. 0.1% Catechol solution (in dropper bottle
with dark glass)
2. Test tube rack 2. Distilled water (150 ml plus full dropper
bottle)
3. Test tubes (2)
4. Dropper bottle for potato extract
5. Beaker (250 ml)
6. Cheese cloth
8. Balance
9. Potatoes

Invertase
Apparatus/Materials Reagents/Chemicals
1. Hot plate with water bath 1. Sucrose (0.25 M solution)
2. Stirring rods (2) 2. Glucose (0.25 M solution)
3. Beaker (50 ml) 3. Assorted non-living material: piece of
baked potato or cooked liver, etc. (Use
caution with rocks or
sand, some will “bubble.”)
4. Test tubes (5) 3. Benedicts solution (in dropper bottle)
5. Test tube rack 4. Distilled water (100 ml)
6. Pipet or syringe to measure 3 ml 5. Yeast
Tes-Tape (tape from drug store
used to test for glucose in urine
samples)
7. Balance or teaspoon

Papain
Apparatus/Materials Reagents/Chemicals
1. Beaker (150 ml) 1. Gelatin (Knox, Jell-O)
2. Balance or teaspoon 2. Distilled water (100 ml)
3. Stirring rods (3) 3. Meat tenderizer
4. Test tubes (2) 3. Benedicts solution (in dropper bottle)
5. Test tube rack 4. Distilled water (100 ml)
6. Beaker of ice water 5. Yeast
7. Hot plate

Pectinase
Apparatus/Materials Reagents/Chemicals
 1. Funnels (2) 1. Apple sauce
2. Graduated cylinders, 100-ml (2) 2. Pectinase (available from several
biological supply companies or businesses
providing cider and/or
wine making supplies)
3. Beakers, 50-ml (2) 3. Distilled water (10 ml)
4. Pipets or syringe, to measure 0.5
ml liquid (2)
5. Spatulas or spoons (2)
6. Wax pencil
7. Cheesecloth (two squares to fit
funnel)

Pepsin
Apparatus/Materials Reagents/Chemicals
1. Test tubes (6) 1. Egg white (hard-boiled)
2. Test tube rack 2. 1.0% Pepsin
3. Metric ruler 3. Pepsin (1.0%) in 0.4% hydrochloric acid
4. Knife 4. 0.4% Hydrochloric acid
5. Pepsin (1.0%) in 0.5% sodium
bicarbonate
6. 0.5% Sodium bicarbonate
7. Distilled water

Rennin
Apparatus/Materials Reagents/Chemicals
1. Beaker (100 ml) 1. Junket
2. Stirring rods (3) 2. Milk (do not use UHT long shelf -life type)
3. Test tubes (2) 3. Distilled Water (50ml)
4. Test tube rack
(Miller, 1992)

PROCEDURE:
Amylase
1. Prepare starch-agar plates (do not have to be sterile if used within a day or
two). Allow to solidify and cool.
 2. Use a wax pencil to label the bottom of the plate: “soaked seeds”, “boiled
seeds”, “dry seeds”, etc. (You might want to include a few drops of saliva from
your mouth for comparison.)
3. Use a sharp razor blade to cut the corn grain longitudinally and place, cut
surface down, onto the agar surface. (You may wish to dissect out the
embryo.) Be sure to space corn grains at least 2 cm from each other.
4. Incubate for 30 minutes.
5. Remove corn and rinse plate gently with distilled water.
6. Flood plate with iodine solution, swish around as color develops, rinse with
distilled water, record results. (Any clear areas of agar can be removed and
tested for sugars.)

Catalase
1. Fill each labelled test tube approximately 1/3 full with fresh hydrogen
peroxide.
2. Add a small amount of material to be tested.
3. Note whether or not bubbles are produced.

Catecholase
1. Prepare catecholase by thoroughly blending potatoes (15 g/100 ml of water)
in distilled water. Filter through cheese cloth. Put filtrate in dropper bottle.
2. Fill each of two test tubes 1/4 full (3 ml) of distilled water.
3. Add 10 drops of catechol to each tube.
4. Add 10 drops of potato extract to one tube, label C.
5. Add 10 drops of distilled water to the unlabelled test tube. Mix.
6. Note and record color of each tube at 1-minute intervals for 5–10 minutes.

Invertase
1. Prepare yeast by mixing 1 teaspoon (3 g) dry yeast with 20 ml of distilled
water. Let stand for 20 minutes.
2. Fill each of two test tubes 1/3 full with sucrose solution.
3. Add 3 ml yeast suspension to one tube (label 1). Mix.
4. Add 3 ml distilled water to the other tube. Mix.
After 10 minutes test each test tube plus the yeast suspension with a strip of
Tes-Tape. (Benedict's test for reducing sugars can also be used here since
sucrose will give a negative Benedict's test and glucose/fructose will give a
positive test (yellow-orange-red) depending on the amount of reducing sugar
present. Remove about 2 ml of the solution, place in another test tube, add 10
drops of Benedict's solution and place in a boiling water bath for about 3
minutes. Prepare a glucose solution for comparison.)

Papain
1. Prepare a gelatin solution by heating 1 teaspoon (3.0 g) of gelatin in 100 ml
distilled water until dissolved. (Gently mix, do not boil.) Cool to room
temperature.
2. Pour meat tenderizer into one of the two test tubes until it fills approximately
0.5 cm of the tube. Label this tube as P. Do not put meat tenderizer in the
other tube.
3. Fill each test tube 1/3 full (5 ml) with the gelatin solution. Mix gently.
 4. Place tubes in ice water for 10 minutes.
5. Remove from ice bath and note the degree of gelatinization.

Pectinase
Cheesecloth (two squares to fit funnel)
1. Place approximately 25 ml of apple sauce into each of the two beakers
labelled “no enzyme” and “pectinase”.
2. Add 0.5 ml distilled water to the “no enzyme” beaker and 0.5 ml pectinase to
the “pectinase” beaker.
3. Stir the contents of each beaker thoroughly (using separate spatulas).
4. Let stand 10 minutes or longer.
5. Place cheesecloth in a funnel and place the funnel into the graduated
cylinder.
6. With the aid of the spatula, pour contents of each beaker into a separate
funnel and collect filtrate.
7. Record the amount of juice collected in each cylinder.

Pepsin
1. Label test tubes A to F.
2. Fill each tube 1/3 full (5 ml) with the corresponding solution.
3. Drop a small (2 mm) cube of egg white into each tube.
4. Incubate at room temperature for approximately 12 hours. (The speed of this
reaction can be increased by using very thin strips of egg white and/or
incubating at 30°C. Students might want to compare the action of papain or
bromelin under similar conditions.)
5. Examine tubes for the presence of the egg white.

Rennin

1. Prepare Tubes: In one tube, add an equal volume of the Junket solution and
label it "R." Mix thoroughly.
2. Control Tube: In another tube, add an equal volume of distilled water and mix.
3. Incubation: If using cold milk, allow the tubes to sit for 30 minutes before
checking results. If the milk has been warmed to room temperature, examine
4. Results: The tube labeled "R," containing the enzyme from the Junket
solution, will begin to clot, while the control tube with distilled water will remain
liquid.
(Miller, 1992)

DATA AND RESULTS:

POST-LAB DISCUSSIONS:
1. What is the purpose of using iodine solution in this experiment?
a) To sterilize the agar plates.
b) To detect the presence of starch.
c) To measure the pH of the agar.
d) To provide nutrients for the seeds.
2. Why is it important to use a control group in this experiment?
a) To ensure that all seeds are the same size.
b) To establish a baseline for comparison.
c) To make the experiment more complex.
d) To increase the accuracy of the results.

3. Which of the following factors might affect the rate of enzyme activity?
a) Temperature
b) pH
c) Enzyme concentration
d) All of the above

4. Which of the following best describe the difference between "soaked seeds" and
"dry seeds" in this experiment.
a) Soaked seeds have been exposed to water, while dry seeds have not.
b) Soaked seeds are larger than dry seeds.
c) Soaked seeds are more likely to germinate than dry seeds.
d) Both a and c

5. What are some real-world applications of enzymes?

a) Food processing
b) Medicine
c) Biotechnology
d) All of the above

CONCLUSION:
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REFERENCE/S:
International Journal of Biological Macromolecules, 2024. (Available at
Enzyme-responsive controlled-release materials for food preservation and crop
protection - A review)
 LaBau E. 2022. What Is Invertase? (Available at
https://www.thespruceeats.com/what-is-invertase-520344)
Levy, M. 2018. Pepsin: Signs You Need More of This Digestive Enzyme &
How to Get It in Your Diet (Available at https://draxe.com/nutrition/pepsin/)
McDermott, E. 2024. 5 Ways to Use Papain (Available at
https://www.healthline.com/health/food-nutrition/papain)
Miller, S. B. 1992. Simple enzyme experiments. Pages 153-161, in Tested
studies for laboratory teaching, Volume 6 (C.A. Goldman, S.E. Andrews, P.L. Hauta,
and R. Ketchum, Editors). Proceedings of the 6th Workshop/Conference of the
Association for Biology Laboratory Education (ABLE), 161 pages.
Ravendraan, B. 2001. Amylase: Definition, Classification and Uses (Available
at https://byjus.com/neet/amylase/#:~:text=The%20pancreas%20and%20the
%20salivary%20glands%20mainly%20synthesise,later%20renamed%20amylase
%20in%20the%20late%2020th%20century.)
Ravendraan, B. 2001. Catalase Enzyme (Available at
https://byjus.com/neet/catalase-enzyme/)
Smith, D. J. Stroup, W. M. Surver, and C. K. Wagner. 1983. General biology:
A laboratory manual for Biology 105. Contemporary Publishing Co., Raleigh, North
Carolina, 265 pages. (See pages 95–104 for a detailed study of catecholase.)
Stewart, D. 2004. The Effects of pH on Catechol Oxidase (Available at
https://sciencing.com/hydrogen-peroxide-experiments-8462947.html)
Trerattanavong, K. 2023. Biochemistry, Renin (Available at
https://www.ncbi.nlm.nih.gov/books/NBK556056/)