Ateneo de Zamboanga University

College of Science and Information Technology

Natural Sciences Department
NURBIO Lab - E

LABORATORY REPORT #10

Group #: 5 Date Performed: September 17, 2019

Names: Basa, Joshua Kharl M. Date Submitted: September 24, 2019

Quijano, Ryle Renzo E.

Tabuco, Kyle Airon M.

Enzymes: Factors Affecting Enzyme Activity

I. Introduction

Enzymes are a type of protein that are found in all living things like plants, animals and
human beings. This enzymes are composed of proteins bust some contains metallic ions and
some other that are similar to a conjugated proteins. They are also called as biological
catalysts, expanding the pace of compound reactions without experiencing any lasting change
themselves.

Learning the basic enzyme hypothesis/theory is very significant in enzyme analysis all
together to comprehend the essential enzymatic mechanisms and to choose a strategy for
chemical examination. The conditions chose to measure the activity of an enzyme would not
be equivalent to those chosen to quantify the concentration of its substrate. A few factors
influence the rate at which enzymatic responses continue - temperature, pH, protein
concentration, substrate concentration, and the presence of any inhibitors or activators.

II. Results

A. Temperature

Color with Iodine

Time Interval, Minute 10oC 40oC 60oC
5 Brownish orange Light yellow with Dark brown
black spots
10 Brownish orange Light yellow with Dark yellow
black spots
15 Brownish orange Light yellow with Dark yellow
black spots
20 Brownish orange Light yellow with Dark yellow
black spots
25 Red orange Light yellow with Dark yellow
black spots
30 Red orange Light yellow with Dark yellow
black spots
 35 Red orange Light yellow with Golden yellow
black spots
40 Red orange Dark yellow with Golden yellow
black spots
45 Red orange Dark yellow with Golden yellow
black spots
50 Red orange Brown with black Golden yellow
spots
55 Red orange Brown with black Golden yellow
spots
60 Red orange Brown with black Golden yellow
spots

B. pH
BEFORE AFTER

Extend of Digestion Result with Biuret Test

1. Pepsin - 0.4% HCl 13 cm No change Violet color & positive result
2. Pepsin - 0.4% Na2CO3 13 cm No change Violet color & positive result
3. Pancreatin - 0.4% HCl 13 cm No change Violet color & positive result
4. Pancreatin - 0.4% Na2CO3 13 cm No change Violet color & positive result
Peptone – Blue color (negative result)

C. Coenzyme

Result with Benedict’s Test

1. 5% Sucrose – Distilled water
2. 5% Sucrose – Supernatant liquid
3. 5% Sucrose – Residue
4. 5% Sucrose – Supernatant liquid – Residue
Formed brick red precipitate – 56 seconds
Formed brick red precipitate – 28 seconds
Formed brick red precipitate – 42 seconds
Formed yellow precipitate – 1 minute & 23
seconds

III. Discussion

Effect of Temperature

From the experiment, the first 20 minutes of 10°C resulted into brownish orange and
afterwards it became red orange. In 40°C, the first 35 minutes resulted into light yellow with
black spots and afterwards, it became dark yellow with black spots to brown with black. This
is due to the increased temperature that made the rate of reaction and its kinetic energy also
increased. In 60°C, the first 30 minutes resulted into dark yellow and the second half resulted
into golden yellow. As observed, it became lighter compared to the 10°C and 40°C because
having too high temperature, the effect of bond breaking will become greater and greater, and
the rate of reaction will begin to decrease. All enzymes work within a range of temperature
specific to the organism. Increases in temperature generally lead to increases in reaction rates
and there is a limit to the increase because higher temperatures lead to a sharp decrease in
reaction rates. This is due to the denaturation (alteration) of protein structure resulting from
the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional
structure of the enzyme. The "optimum" temperature for human enzymes is usually between
35 °C and 40 °C. The average temperature for humans is 37 °C. Human enzymes start to
denature quickly at temperatures above 40 °C. Enzymes from thermophilic archaea found in
the hot springs are stable up to 100 °C. However, the idea of an "optimum" rate of an enzyme
reaction is misleading, as the rate observed at any temperature is the product of two rates,
the reaction rate and the denaturation rate. If a person will use an assay measuring activity
for one second, it would give high activity at high temperatures. However if the person will use
an assay measuring product formation over an hour, it would give low activity at these
temperatures.

Figure 1

Effect of pH

From the experiment, all mixtures got 13 centimeters and there’s no change after
observing it. All mixtures also got a positive result for Biuret’s test which is having a
formation of violet color. Same as with temperature, this is because that every enzyme
has a different pH optimum. In other words, enzyme activity increases with pH until the
optimum pH is reached then decreases again as pH continues to rise. Extreme pH levels
will cause irreversible denaturing. Most enzymes are sensitive to pH and have specific
ranges of activity. All have an optimum pH at which they work the best. The pH can stop
enzyme activity by denaturation (altering) the three dimensional shape of the enzyme by
breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8.
However, pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.
 Figure 2

Influence of Coenzymes

From the experiment, all formed a brick red precipitate in less than a minute except
from the 5% sucrose – supernatant liquid – residue that formed a yellow precipitate in 1
minute and 23 seconds. These are the outcomes because when Benedict’s solution and
simple carbohydrates are heated, the solution changes to orange red/ brick red. The color
of the precipitate determines the amount of reducing sugar where blue means there’s no
present simple carbohydrates in it and on the other hand, brick red precipitate is a sign
that the amount of reducing sugar is large. The reaction is caused by the reducing property
of simple carbohydrates where the copper (II) ions in the Benedict’s solution are reduced
to Copper (I) ions that caused the color to change. Coenzymes bind to the enzyme and
assist in enzyme activity. They can bind and react with many different enzymes, so they're
not specific to a particular enzyme. They help enzymes change starting elements
(substrates) into their final version (products). In doing this, the coenzyme can be changed
and often alternates between various forms. From the figure 3, at low substrate
concentration, the velocity of the reaction is directly proportional to the substrate level (part
A). In second phase (part B), the substrate concentration is not directly proportional to the
enzyme activity. In third and final phase (part C), the reaction is independent of the
substrate concentration.

Figure 3
IV. Answers to Questions

1. Why is the temperature of pasteurization at 60°C?

The temperature of pasteurization is at 60°C because germs and bacteria would

not be able to live and will eventually die at this temperature. Moreover, conventional
pasteurization processes often involve continuous heat-transfer mechanisms. The primary
aims of these processes are to destroy pathogenic organisms in liquid foods such as milk
and to extend the shelf-life of the product for a limited period of time.

2. Why are foods frozen at temperature below zero?

Most foods will maintain good quality longer when the freezer temperature is below
zero, around -10°F to -20°F. At temperatures between 0°F and 32°F, food deteriorates
more rapidly. Foods will not likely ever go bad or be unsafe to eat from a stay in a freezer
as long as the freezer temperature is at zero Fahrenheit. Also, when this temperature is
maintained, the movement of molecules are slowed down and so, freezing is enabled with
microbes moving up in a dormant stage.

3. In what test tube has enzyme action occurred? Why?

Enzyme action is usually demonstrated by the increased formation of the products

and decreased concentration of the substrate. STILL LACKING…

4. What is the optimum pH of pepsin?

Pepsin is a powerful enzyme that digests proteins such as those in meat, eggs,
seeds, or dairy products. The optimum pH of pepsin is from 1.5 to 2. The reason pepsin
functions best at pH 2 is because in the enzyme's active site the carboxylic acid group on
the amino acid must be in its protonated state, which means that it is bound to a hydrogen
atom. At low pH the carboxylic acid group is protonated, which allows it to catalyze the
chemical reaction of breaking chemical bonds.

5. What is the major protein–splitting enzyme in the pancreatin?

Pancreatin is a mixture of digestive enzymes produced by the pancreas, including

amylase, lipase and trypsin. It is also commonly known as pancreatic acid, although it is
not actually an acid or even a specific chemical. The major protein–splitting enzyme in the
pancreatin is Trypsin, also referred to as a proteolytic enzyme or proteinase, which takes
part in the digestion of food proteins and other biological processes. Trypsin is produced
by the pancreas in an inactive form called trypsinogen. After which, the trypsinogen finds
its way to the small intestine through the common bile duct and is converted to active
trypsin.

6. What is the cofactor found in yeast?

The cofactor found in the yeast is transketolase in which it contains two atoms of
calcium per molecule of protein. A removal of an atom serves as a decrease of an
enzymatic activity, then the removal of the second atom does not affect anything unless a
metal has been added.
V. References

Adam-Day, S. (2018). Factors affecting Enzyme Activity: Temperature. Retrieved from

https://alevelnotes.com/notes/biology/biological-molecules/enzymes/factors-
affecting-enzyme-activity

Aryal, S. (2019, August 15). Benedict’s Test- Principle, Composition, Preparation,

Procedure and Result Interpretation. Retrieved from

https://microbiologyinfo.com/benedicts-test-principle-composition-preparation-
procedure-and-result-
interpretation/?fbclid=IwAR2HRcBFELCL4ty4ya_Zb3HLFgDoWb-
f5HfXkFfhVgQlk2Kqwq-6rp0z3kk

Coenzyme: Definition & Function. (n.d.). Retrieved from

https://study.com/academy/lesson/coenzyme-definition-function-quiz.html

Effects of pH. (n.d.). Retrieved from

http://www.worthington-biochem.com/introbiochem/effectspH.html

Frothingham, S. (n.d.). Trypsin Function. Retrieved from

https://www.healthline.com/health/trypsin-function

Health Benefits And Uses Of Pancreatin. (n.d.). Retrieved from

https://www.xtend-life.com/blogs/supplement-ingredients/pancreatin

Kavimandan, B. (n.d.). Enzymes – Factors Affecting Enzyme Activity. Retrieved from

https://www.kcesmjcollege.in/ICT/Biochemistry/Enzymes-
Factors%20affecting%20enzyme%20activity.pdf?fbclid=IwAR0KLzH3bx2AT8Hn
ZsgWtRp_r-xoVtkrYJ7n5qjN_YC5XCLxxBFFhdsypGU

Practical of Chemistry 2 (Biochemistry): AC 0102. (n.d.). Retrieved from

http://bu.edu.eg/portal/uploads/Agriculture/Agricultural%20Biochemistry/1117/crs
-
13490/Practical%20Biochemistry.doc.pdf?fbclid=IwAR1ccmXZqnF17vhzOVPlXD
ZrCaP1uZC5EeGJaj0akVsDjOQWREPe3AqRKIw

Refrigerator and Freezer Storage. (n.d.). Retrieved from

https://food.unl.edu/refrigerator-and-freezer-storage

The Effects of Temperature and pH on Enzyme Activity. (2018). Retrieved from

https://www.hygrozyme.com/resources/temperature-enzyme-activity/
Updegraff, E. (2018, June 25). What Is the Optimum pH for Human Stomach Enzyme
Activity? Retrieved from

https://sciencing.com/happens-pepsin-mixes-food-stomach-8206.html

Why is the temperature of pasteurization at 60 degree C? (n.d.). Retrieved from

https://www.answers.com/Q/Why_is_the_temperature_of_pasteurization_at_60_
degree_C

Jooste, P. & Swart, G. (2003). Pasteurization: Other Pasteurization Processes. Retrieved

from

https://www.sciencedirect.com/topics/food-science/pasteurization