Activity 2: Enzyme Action

Vladimir Rellon

Jaypee Marfil

Hazel Kareen Villacanas

Mary Celman Guaza

I. Abstract

We observe the rate of reaction of an enzyme in the presence product in this

experiment. We observed the mechanism of enzyme cellobiase along with its rate of
activity. The experiment consists of two activities, preparing the materials and
observation of enzyme action on each fruits. The second activity talks about the
breakdown of the artificial substrate known as p-nitrophenyl through both the
presence and absence of the cellulase enzyme. The data obtained from the last
activity suggests that the higher the concentration of the enzyme, the faster the
reaction time is. With the results obtained from this experiment, the overall conclusion
is that as temperature rises, the rate of reaction also increases whereas the pH level
of an enzyme is at a lower rate in order to for the enzyme to work in an optimal
environment.

II. Introduction

Enzymes are any complex chemical produced by living cells that is a biochemical
catalyst. These are substances that speeds up chemical reactions without being used up
in the process. They do this by lowering the activation energy, thus greatly reducing the
amount of energy needed in the said reaction. To have an even better understanding
to the concept of enzymes said experiment is introduced. Series of experiments are
conducted. The presence of pH and heat greatly affects the activity of enzymes, as there
are certain temperatures and acidity needed to have the optimum results. Pineapples
are known to have protein-digesting enzyme, Bromelain. We explore the rate of reaction
of an enzyme and its substrate by studying the enzyme cellobiase and its substrate
cellobiose. In order for an enzyme to begin work, the process must begin with the collision
of two molecules producing enough energy in order to overcome the energy barrier
present in the reaction. Once an enzyme comes into a reaction after the introduction of
the reactant molecules, the enzyme and substrate have to bind in order for the enzyme
to catalyze the reaction. The site where the enzyme and substrate bind is called the
active site.This site is often called the lock and key model due to the fact that each
enzyme is substrate specific.

For this lab, the enzyme used to catalyze the reaction is the enzyme known as cellobiase.
Cellobiase breaks down the component known as cellulose, a molecule composed of
glucose found in plant cell walls. The presence of cellobiase in the last step of cellulose
breakdown is essential for the survival for many fungi and bacteria. This process is the
main source of glucose and energy for these organisms.

Hypothesis formulated by our group was that the rate of the enzyme would increase
once reaction has begun and will gradually decrease as the amount of substrate
needed decreases. Enzyme should be in a high temperature, best results in optimum
temperature, low pH and optimal amounts of enzyme and substrate must be present in
order to achieve fast and effective reaction.

III. Methods

A. Preparation of Gelatin and Fruit Juices

We prepared the gelatin solution by heating six grams of gelatin in a 200 mL distilled
water (We used beaker and double up our ingredients due to the size of the beaker)
until dissolved. Gently mixing the solution, we carefully observed the heated mixture
to assure that no boiling occurred. After observing that the gelatin powder has
dissolved completely, we cool the solution in room temperature. We prepared the
fruits and the canned pineapple and pureed them in a blender. After that we strained
the puree in a cheesecloth (separate cheesecloth is used to avoid contaminations)
to separate the pulp from the juice.

B. Enzyme Action
We prepared 5 test tubes and labeled each as test tube A (Pineapple juice), test
tube B (Orange juice), test tube C (Apple juice), test tube D (Canned Pineapple juice)
and left another one empty and labeled it E (for pure gelatin solution). We added 3
mL of pineapple juice in A, 3 mL of orange juice in B, 3 mL of apple juice in C, and 3
mL of canned pineapple juice in D. After that we added 10 mL gelatin solution to
each tube (gelatin solution must be semi-solid now) and mixed each solution well. We
placed the tubes in an ice bath at the same time for 10-15 minutes. We removed the
tubes and noted the degree of gelatinization on each test tubes.
C. Effect of Temperature
We labeled five test tubes A, B, C, D and E and pour 3mL of fresh pineapple juice into
the tubes. We placed the test tube E in a rack and assign that as room temperature.
We placed the other tubes in a water bath and remove the tubes when test tube A
reaches 40°C, test tube B reaches 60°C, test tube C reaches 80°C, test tube D reaches
100°C. We added 10mL of gelatin solution to each tube and mixed the solutions well.
We place the tubes in an ice bath for 10-15 minutes and remove the tubes from the
ice bath and note the degree of gelatinization.

D. Effect of pH
We labeled three test tubes A, B and C and pour 3mL of fresh pineapple juice into the
tubes and add the following accordingly: test tube A with 3mL HCl, test tube B with
3mL of NaOH, test tube C with 3mL of distilled water and pour 10mL gelatin solution
 into each tube and mixed it well. We place the tubes in an ice bath for 10-15 minutes
and remove the tubes from the ice bath and note the degree of gelatinization.

IV. Results

The results of the overall experiment were in accordance with the hypothesis that our
group established. It was hypothesized and valid based on the data that if the
temperature and concentration increased, so would the rate of the reaction, this all
resulting in a positive relationship. With the pH, it was predicted and valid based on the
data established that a smaller pH level is an optimum condition for the enzyme to fully
function in a reaction.

V. Conclusion

Objectives established since the beginning of the experiments was to understand

more about enzymes. Enzymes catalyzes reactions in order to reduce needed activation
energy and thus increase efficiency of basic biological reactions within our bodies. They
goal of this experiment is to see what factors affect the rate of reaction of an enzyme.
These factors, as we discovered, were temperature, pH, the concentration of the enzyme
and the concentration of the substrate. According to the hypothesis earlier, the rate of
reaction would increase with both temperature and pH is in its optimum state along with
the concentration of substrate and enzyme. Based on the data gathered, the hypothesis
is correct. For pH levels, the hypothesis was that as pH levels increase, the rate of the
reaction decreased. The hypothesis was proven correct after the data obtained. As the
pH level increases, the rate of the reaction decreases thus stating that an enzyme
functions most suitably in a reaction with a low pH level. With these factors in mind, an
enzyme is the basic component of all biological reactions and processes.